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Peer-Reviewed Publications

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Creative Biolabs has provided high-quality antibody discovery, engineering and manufacturing services to numerous customers. A long list of papers has been published in peer-reviewed journals and a lot of patents have been approved or in process which cited custom products or services from Creative Biolabs. Below are some selected patents and articles to give you an idea of how we assist life science research in academia and industry.

For your convenience, we have grouped these patents and articles into the following categories:

SECTION 1: Phage Display Services and Products[Top]

Phage display is a laboratory technique for the study of protein-protein, protein-peptide, and protein-DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Creative Biolabs is a leading service provider that focuses on phage display technology. Our services include phage display library construction and screening.

  • Trieu, Vuong, Xiping Liu, and Neil Desai. "Sparc binding antibodies and uses thereof." EP 2563393 A1. Mar 6, 2013 EP2563393 B1. Jun 8, 2016 U.S. US9096660.Patents Application 13/643,609. Apr 25, 2013 WO 2011137114 A1. Nov 3, 2011 Abstract
    The invention provides SPARC binding antibodies that target disease sites, in particular, tumors and uses thereof to diagnose and treat diseases, in particular, cancerous tumors.
  • Vuong Trieu, Xiping Liu, Neil Desai. “Peripheral blood SPARC antibodies and uses thereof.” EP2598164 A4. Apr 9, 2014 CN 103221062. Jul 24, 2013 WO2011153431. Apr 11, 2013 WO2011153431 A2. DEC 8, 2011 US 9096660 B2. Aug 4, 2015 Abstract
    The invention provides SPARC binding antibodies that have high affinity to SPARC, particularly plasma SPARC, and methods of using such antibodies in treating conditions including cancer.
  • Vuong Trieu, Xiping Liu, Neil Desai. “Peripheral blood sparc binding antibodies and uses thereof.” CA 2801184. Dec 8, 2011 US20120052007 A1. Mar 1, 2012 Abstract
    The invention provides SPARC binding antibodies that have high affinity to SPARC, particularly plasma SPARC, and methods of using such antibodies in treating conditions including cancer.

These patents describes Creative Biolabs’ scientists panned SPARC against the premade human Fab phage display library HuFabL of Creative Biolabs for the client.

  • John T. Unger, Rolf Hilfiker. “Conformational epitope initiated signal amplification.” U.S. Patents No. 13.263.367. Feb 16, 2012 WO 2010118300. Oct 14, 2010 Abstract
    This invention relates to a method to generate a signal used to detect the presence or quantity of a biomarker in a sample. The signal generating reaction is initiated when the biomarker under assay interacts with a specific binding partner. The interaction produces a structural change in the binding partner that is recognized by additional binding partners capable of generating a signal. The reaction produces a localized cluster of signaling molecules that can be detected above background. The signaling cluster is detectable within minutes when interrogated in a chamber of specific dimensions. The presence of the signaling clusters is a qualitative indication of the presence of the analyte, while the number of signaling clusters detected is a direct quantification of the number of biomarker molecules in the sample. The reaction can be formatted to detect proteins, nucleic acids, cells or other informative biomarkers.

These patents describe Creative Biolabs’ scientists isolated mouse anti-C1Q binding site and mouse anti-FcgR1 binding site Fabs from a phage display library made in Creative Biolabs’ Lab, and expressed them as soluble proteins.

  • Tay, Moon YF, et al. "Identification of Dengue-Specific Human Antibody Fragments Using Phage Display." Dengue. Springer New York, 2014. 161-173 Abstract
    High-affinity antibodies are valuable tools for dengue research. A method for the selection of dengue-specific, human antibody fragments using naïve repertoires displayed on M13 filamentous bacteriophage is described. Naïve repertoires are unbiased, thus enabling the identification of antibodies to dengue structural and nonstructural proteins from the same library. Dengue-specific clones are enriched by binding to an immobilized dengue antigen, followed by washing, elution, and amplification of phage for subsequent rounds of selection. Dengue virus has four antigenically related serotypes, and the serotype of the antigen can be kept constant or alternated during the selection process depending on whether serotype-specific or cross-reactive antibodies are required. After the selection process, clones are screened, and specific clones are identified by phage ELISA and Western blot.

The paper describes Creative Biolabs is a custom service company from where you can get naïve human Fab phage library generated as client demand.

  • Franco, Alessandra. "Glycopeptides and methods of making and using them." WO 2009108807. Sep 3, 2009 EP2250189. Jul 4, 2012 EP2250189 A1. Nov 17, 2012 U.S. Patents Application 12,918,155. Feb 24, 2011 CA 2716622. Sep 3, 2009 US 9156906 B2. Oct 13, 2015 Abstract
    In one embodiment, the invention provides glycopeptides (or carbohydrate-peptide conjugates) comprising TACAs that direct against (e.g., bind specifically to) cytotoxic T lymphocytes (CTLs) or helper T cells for, e.g., CTL- or T-helper-based immunotherapy of carcinomas, and methods for making and using the glycopeptides of the invention. In one embodiment, the invention provides novel glycopeptides comprising tumor-derived carbohydrate or tumor-derived epitopes that specifically bind to major histocompatibility (MHC) class I molecules on cytotoxic T lymphocytes (CTLs) or MHC molecules on helper T cells, and methods for using same, e.g., as a vaccine, including a pan-cancer vaccine.

These patents describe the client used a premade phage display libraries having synthetic peptides from Creative Biolabs and accomplished their study.

  • Stagliano, Nancy E., et al. "Modified antibody compositions, methods of making and using thereof." U.S. Patents No. 14,038,232. Jan 23, 2014 U.S. Patents No. 13,784,407. Nov 21, 2013 U.S. Patents No. 8,563,269. 22 Oct. 2013 U.S. Patents No. 8,513,390. 20 Aug. 2013 EP2385955. Feb 13, 2013 CN 102482347. May 30, 2012 EP 2385955. Nov 16, 2011 WO2010081173. Nov 4, 2010 U.S. Patents No. 12,686,344. Jul 29, 2010 CA2749339. Jul 15, 2010 Abstract
    The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolysed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolysing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.

These patents describe Creative Biolabs supplied a M13 bacteriophage capable of binding human CTLA for the client.

  • Fukunaga, Keisuke, and Masumi Taki. "Practical tips for construction of custom peptide libraries and affinity selection by using commercially available phage display cloning systems." Journal of nucleic acids 2012 (2012) Abstract
    Phage display technology is undoubtedly a powerful tool for affinity selection of target-specific peptide. Commercially available premade phage libraries allow us to take screening in the easiest way. On the other hand, construction of a custom phage library seems to be inaccessible, because several practical tips are absent in instructions. This paper focuses on what should be born in mind for beginners using commercially available cloning kits (Ph.D. with type 3 vector and T7Select systems for M13 and T7 phage, respectively). In the M13 system, Pro or a basic amino acid (especially, Arg) should be avoided at the N-terminus of peptide fused to gp3. In both systems, peptides containing odd number(s) of Cys should be designed with caution. Also, DNA sequencing of a constructed library before biopanning is highly recommended for finding unexpected bias.

The paper describes Creative Biolabs accepts service contract from any commercial premade library, a custom-constructed one in the company, or a hand-made one.

  • Torres, Lorena, et al. “Functional genomics of the horn fly, Haematobiairritans (Linnaeus, 1758)." BMC genomics 12.1 (2011): 105. Abstract
    The horn fly, Haematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of pastured cattle. Horn flies infestations reduce cattle weight gain and milk production. Additionally, horn flies are mechanical vectors of different pathogens that cause disease in cattle. The aim of this study was to conduct a functional genomics study in female horn flies using Expressed Sequence Tags (EST) analysis and RNA interference (RNAi).

    A cDNA library was made from whole abdominal tissues collected from partially fed adult female horn flies. High quality horn fly ESTs (2,160) were sequenced and assembled into 992 unigenes (178 contigs and 814 singlets) representing molecular functions such as serine proteases, cell metabolism, mitochondrial function, transcription and translation, transport, chromatin structure, vitellogenesis, cytoskeleton, DNA replication, cell response to stress and infection, cell proliferation and cell-cell interactions, intracellular trafficking and secretion, and development. Functional analyses were conducted using RNAi for the first time in horn flies. Gene knockdown by RNAi resulted in higher horn fly mortality (protease inhibitor functional group), reduced oviposition (vitellogenin, ferritin and vATPase groups) or both (immune response and 5'-NUC groups) when compared to controls. Silencing of ubiquitination ESTs did not affect horn fly mortality and oviposition while gene knockdown in the ferritin and vATPse functional groups reduced mortality when compared to controls.

    These results advanced the molecular characterization of this important ectoparasite and suggested candidate protective antigens for the development of vaccines for the control of horn fly infestations.

The paper describes the cDNA library was synthesized using the SMART™ cDNA Library Construction Kit at Creative Biolabs and a total of 2,462 ESTs were 5' sequenced in Creative Biolabs.

  • Soto, Carissa M., and Banahalli R. Ratna. "Virus hybrids as nanomaterials for biotechnology." Current opinion in biotechnology 21.4 (2010): 426-438. Abstract
    The current review describes advances in the field of bionanotechnology in which viruses are used to fabricate nanomaterials. Viruses are introduced as protein cages, scaffolds, and templates for the production of biohybrid nanostructured materials where organic and inorganic molecules are incorporated in a precise and a controlled fashion. Genetic engineering enables the insertion or replacement of selected amino acids on virus capsids for uses from bioconjugation to crystal growth. The variety of nanomaterials generated in rod-like and spherical viruses is highlighted for tobacco mosaic virus (TMV), M13 bacteriophage, cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Functional biohybrid nanomaterials find applications in biosensing, memory devices, nanocircuits, light-harvesting systems, and nanobatteries.

The paper describes the author used custom-made phage libraries from Creative Biolabs for material science applications.

  • Kim, Dae Young, et al. "Mutational approaches to improve the biophysical properties of human single-domain antibodies." Biochimicaet Biophysica Acta (BBA)-Proteins and Proteomics (2014). Abstract
    Monoclonal antibodies are a remarkably successful class of therapeutics used to treat a wide range of indications. There has been growing interest in smaller antibody fragments such as Fabs, scFvs and domain antibodies in recent years. In particular, the development of human VH and VL single-domain antibody therapeutics, as stand-alone affinity reagents or as "warheads" for larger molecules, are favored over other sources of antibodies due to their perceived lack of immunogenicity in humans. However, unlike camelid heavy-chain antibody variable domains (VHHs) which almost unanimously resist aggregation and are highly stable, human VHs and VLs are prone to aggregation and exhibit poor solubility. Approaches to reduce VH and VL aggregation and increase solubility are therefore very active areas of research within the antibody engineering community. Here we extensively chronicle the various mutational approaches that have been applied to human VHs and VLs to improve their biophysical properties such as expression yield, thermal stability, reversible unfolding and aggregation resistance. In addition, we describe stages of the VH and VL development process where these mutations could best be implemented. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

The paper describes Creative Biolabs is a company focused on the development of VH- and/or VL-based therapeutics and affinity reagents.

  • Jim De Yoreo (2013). Biomimetic crystallization techniques in vitro. In Research Methods in Biomineralization Science(pp308). Amsterdam, Netherlands: Elsevier. Abstract
    This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in biomineralization science, and includes sections on such topics as determining solution chemistry, structure and nucleation; probing structure and dynamics at surfaces; and interfaces mapping biomineral and morphology and ultrastructure.

This book describes Creative Biolabs is custom phage display library supplier for immunized, or naïve, and semisynthetic/synthetic antibody libraries, phage display peptide libraries, cDNA phage display libraries.

  • Doshi, Rupak, et al. "In vitro nanobody discovery for integral membrane protein targets." Scientific reports 4 (2014). Abstract
    Nanobodies (Nbs) or single-domain antibodies are among the smallest and most stable binder scaffolds known. In vitro display is a powerful antibody discovery technique used worldwide. We describe the first adaptation of in vitro mRNA/cDNA display for the rapid, automatable discovery of Nbs against desired targets, and use it to discover the first ever reported nanobody against the human full-length glucose transporter, GLUT-1. We envision our streamlined method as a bench-top platform technology, in combination with various molecular evolution techniques, for expedited Nb discovery.

The author’s work used Creative Biolabs’ Human Single Domain Antibody Library (HuSdL®), which was constructed, based on the camelized human VH3 with elongated and randomized HCDR3 sequences, and has a calculated diversity of 1.5 × 109 as per the manufacturer.

  • Zhao, Aizhi, et al. "Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy." Critical reviews in biotechnology 0 (2014): 1-14. Abstract
    Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.

Creative Biolabs provided several types of HuScL libraries for the author.

  • Lin, Dong. "New Generation Affibody Molecule Phage Library for Highly Stringent Binder Selections." (2014). Abstract
    Affibody is a new class of engineered affinity protein, and has great properties compared with antibody. The aim of the project is to construct a new generation Affibody molecule phage library in which the bound phages are cleaved by trypsin in the selections against target molecules. First, a new phagemid vector pAffi-100-Tryp was created for new library preparation. Second, in comparative test selections with low-pH acid and trypsin cleavage elution, the functional display was ensured. Then, a new generation Affibody molecule library was constructed for highly stringent binder selections. The size of the library is approximately 4.7×10^9 cfu. In the selection against rhEphrin-B3, the bound phages were able to be cleaved by trypsin, and re-infect. After four rounds selection, candidate binders were selected for subcloning. According to the sequencing results, there were similar patterns among those candidate binders. Also, only Ile was found in Position 31.

Creative Biolabs provided the helper phage KM13 (1.0 × 1011 pfu/mL) for the author.

  • Omidfar, Kobra, and Maryam Daneshpour. "Advances in phage display technology for drug discovery." Expert opinion on drug discovery 0 (2015): 1-19. Abstract
    Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery.

This author mentioned that the most widely used commercial phage display libraries are from Creative Biolabs, which contains 107-10 variants.

  • Farhan M. Khan, Dalhousie University. “Construction and Characterization of a Single-Chain Variable Fragment Antibody Library against Fusobacterium nucleatum.”Department of Microbiology and Immunology (2012). Abstract
    The accumulation of oral bacteria forming dental plaque, or biofilm, promotes the development of dental caries and periodontal diseases. These biofilms are formed through a sequential process of bacterial accretion. Early colonizing bacteria are mainly commensal streptococci and Actinomyces species. Pathogenic late colonizers consist largely of Gram-negative anaerobes. A key bridge organism capable of coaggregating with both the early and late colonizers is Fusobacterium nucleatum. Despite being the focus of research for over 20 years, very few F. nucleatum adhesins are known due to the lack of an effective tool to identify these adhesins. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help to elucidate the mechanism of coaggregation between F. nucleatum and other oral bacteria. Therefore, a scFv M13 phage display library, consisting of 4 u 108 clones, was created using RNA from the spleen of a mouse immunized with F. nucleatum. The library was enriched by biopanning 6 times against F. nucleatum whole cells. Individual clones of the enriched library were analyzed by ELISA to identify scFvs specific for F. nucleatum. All 292 individual clones tested in ELISA reacted strongly to F. nucleatum. Sixty two of the 292 clones inhibited F. nucleatum interaction with Streptococcus sanguinis in coaggregation assays. The 62 scFvs were further grouped into 5 categories based on their reactivity with the F. nucleatum outer membrane proteins in western immunoblotting. Analysis of 11 representative clones from the 5 categories revealed differences in coaggregation inhibition with S. sanguinis, recognition of outer membrane proteins and BstOI restriction pattern. DNA sequencing of the 11 representative clones revealed 6 unique scFvs and of them, 3 strongly inhibited interaction with 5 Streptococcus species. Mass spectrometry identified the protein recognized by the function-blocking scFvs as RadD, a previously identified F. nucleatum adhesin involved in coaggregation with S. sanguinis. The construction, characterization and use of this anti-F. nucleatum scFv library to identify an adhesin involved in coaggregation, demonstrates the utility of this library and its potential for future use in the discovery of adhesins and cognate receptors.

Creative Biolabs constructed a Single-chain Variable Fragment Antibody Library against Fusobacterium Nucleatum for the author.

  • James William WEST, Jason Gary SAGERT, Paul H. Bessette, Henry Bernard LOWMAN, Nancy E. Stagliano, Olga Vasiljeva, Elizabeth-Edna Mary MENENDEZ. “Anti-jagged 1/jagged 2 cross-reactive antibodies, activatable anti-jagged antibodies and methods of use thereof.” CA2876904A1. Dec 27. 2013CN104661677A. May 27. 2015EP2863948A2. Apr 29. 2015US9127053. Sep 8. 2015US20140010810. Jan 9. 2014WO2013192550A2. Dec 27. 2013WO 2013192550 A3. Apr 27. 2014 Abstract
    This invention relates generally to the generation of antibodies, e.g., monoclonal antibodies including fully human monoclonal antibodies, that recognize Jagged 1 and/or Jagged 2, to antibodies, e.g., monoclonal antibodies including fully human antibodies that recognize Jagged 1 and/or Jagged 2, and nucleic acid molecules that encode antibodies, e.g., nucleic acid molecules that encode monoclonal antibodies including fully human cross-reactive antibodies that recognize both Jagged 1 and Jagged 2, and to methods of making the anti- Jagged antibodies and methods of using the anti- Jagged antibodies as therapeutics, prophylactics, and diagnostics. The invention also relates generally to activatable antibodies that include a masking moiety (MM), a cleavable moiety (CM), and an antibody (AB) that specifically bind to Jagged 1 and Jagged 2, and to methods of making and using these activatable anti- Jagged antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

scFvs used in these patents were selected from a fully human scFv library displayed on bacteriophage, from Creative Biolabs, and the scFv phage selection was conducted under contract with Creative Biolabs.

  • Melidoni, Anna N., Michael R. Dyson, and John McCafferty. "Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation." Embryonic Stem Cell Protocols (2016): 111-132. Abstract
    Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

Creative Biolabs used antibody phage display libraries to complete the antibody populations for ES-ICER expression.

  • Ma, Lin, et al. "Generation and characterization of a human nanobody against VEGFR-2." Acta Pharmacologica Sinica (2016). Abstract
    Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library.

    To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1-4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1-4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs).

    In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1-1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs.

    The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics.

Creative Biolabs provided the authors the HuSdL® phage display human single domain antibody library.

SECTION 2: Hybridoma Services[Top]

Creative Biolabs provides a full range of services in custom polyclonal and monoclonal antibody production from gene expression or peptide synthesis to antibody purification and labeling. We have the capacity of producing antibodies in either research quantities or in large scale for industrial applications. We do epitope prediction, peptide synthesize, protein expression, polyclonal or monoclonal antibody production as well as antibody purification from cell culture, ascites fluid or whole serum. We can also generate mono-specific polyclonal antibodies by ligand affinity chromatography.

  • Jani, Priyam H., et al. "Transgenic expression of Dspp partially rescued the long bone defects of Dmp1-null mice." Matrix Biology 52 (2016): 95-112. Abstract
    Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1(-/-)) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1(-/-) mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as "Dmp1(-/-);Dspp-Tg mice"). We characterized the long bones of the Dmp1(-/-);Dspp-Tg mice at different ages and compared them with those from Dmp1(-/-) and Dmp1(+/-) (normal control) mice. Our analyses showed that the long bones of Dmp1(-/-);Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1(-/-) mice. The long bones of Dmp1(-/-);Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1(-/-) mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1(-/-) mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that DSPP and DMP1 may function synergistically within the complex milieus of bone matrices.

The paper describes that five BALB/C mice were immunized with the full-length human recombinant FGF23 using a Bac-to-Bac baculovirus expression system and the fusion cells showing positive reaction to FGF23 were screened with the Omni Hybridoma Platform and cloned in Creative Biolabs.

  • Maragos, Chris M., Lan Li, and Donghai Chen. "Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol." Food and Agricultural Immunology 23.1 (2012): 51-67. Abstract
    Deoxynivalenol (DON) is a mycotoxin produced by certain fungi that infest cereal grains. A hybridoma cell line producing a monoclonal antibody (Mab) was used as the starting point in the development of a recombinant single chain variable fragment antibody (scFv) recognising DON. The scFv and Mab were characterised using two immunoassay formats: competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and biolayer interferometry (BLI). Using CD-ELISA the IC50s for DON were 36.1 and 13.8 ng/ml for assays based on the scFv and Mab, respectively. The cross-reactivity to DON analogs was very similar for the scFv and the Mab. The real-time binding of the antibodies to an immobilised DON-protein conjugate was also monitored. In competitive BLI assays the IC50s using the scFv and Mab were 68.3 and 15.8 ng/ml, respectively. The results suggest that sensitivity of assays, but not selectivity, was affected by removal of the constant regions of the Mab.

The paper describes a study from which a hybridoma cell line was cultured and validated in Creative Biolabs’ Lab. Creative Biolabs’ scientists synthesized first-strand cDNA, then constructed the Fab and scFv with phagemid pDisplay-4™ and pDisplay-2™ respectively, and expressed the soluble Fab and scFv exactly as the study designed.

  • Wu, Yongzhong, et al. "Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines." The Journal of Immunology 190.4 (2013): 1859-1872. Abstract
    Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

The paper describes that Creative Biolabs’ scientist developed anti-human Duox mAb, S-12, using hybridoma technique.

  • Borges, Sahra, et al. "Pharmacologic reversion of epigenetic silencing of the PRKD1 promoter blocks breast tumor cell invasion and metastasis." Breast Cancer Res 15 (2013): R66. Abstract
    DNA methylation-induced silencing of genes encoding tumor suppressors is common in many types of cancer, but little is known about how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes protein kinase D1 (PKD1), a serine/threonine kinase that is expressed in cells of the normal mammary gland, where it maintains the epithelial phenotype by preventing epithelial-to-mesenchymal transition.

    The status of PRKD1 promoter methylation was analyzed by reduced representation bisulfite deep sequencing, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR in invasive and noninvasive breast cancer lines, as well as in humans in 34 cases of "normal" tissue, 22 cases of ductal carcinoma in situ, 22 cases of estrogen receptor positive, HER2-negative (ER+/HER2-) invasive lobular carcinoma, 43 cases of ER+/HER2- invasive ductal carcinoma (IDC), 93 cases of HER2+ IDC and 96 cases of triple-negative IDC. A reexpression strategy using the DNA methyltransferase inhibitor decitabine was used in vitro in MDA-MB-231 cells as well as in vivo in a tumor xenograft model and measured by RT-PCR, immunoblotting and immunohistochemistry. The effect of PKD1 reexpression on cell invasion was analyzed in vitro by transwell invasion assay. Tumor growth and metastasis were monitored in vivo using the IVIS Spectrum Pre-clinical In Vivo Imaging System.

    Herein we show that the gene promoter of PRKD1 is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, we show that reversion of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion.

    Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells.

The paper describes the mouse monoclonal antibody specific for PKD1 was raised by Creative Biolabs against a 21-amino acid peptide in the N-terminal of human PKD1, which is not present in PKD2 and PKD3.

  • Woods, Alisa G., et al. "Identification of tumor differentiation factor (TDF) in select CNS neurons." Brain Structure and Function (2013): 1-10. Abstract
    Identification of central nervous system (CNS) molecules elucidates normal and pathological brain function. Tumor differentiation factor (TDF) is a recently-found protein secreted by the pituitary into the blood. TDF mRNA was detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. However, TDF has an unclear function. It is not known whether TDF is expressed only by pituitary or by other brain regions. It is also not known precisely where TDF is expressed in the brain or which cells produce TDF. Database searching revealed that this molecule shares no homology with any known protein. Therefore, we investigated the distribution of TDF in the rat brain using immunohistochemistry (IHC) and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions. Double-staining for TDF and glial fibrillary acidic protein (GFAP), an astrocyte marker, showed no co-localization. Double-staining for TDF with NeuN, a neuronal marker, showed co-localization. Not all NeuN positive cells were positive for TDF. Western blotting (WB) using NG108 neuroblastoma and GS9L astrocytoma cell lysate revealed TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized in neurons, not in astrocytes. This is the first report of any cellular localization of TDF. TDF may have specific roles as a pituitary-derived hormone and in the CNS, and appears to be produced by distinct CNS neurons, not astroglia.

The paper describes Creative Biolabs generated the antibodies specific for three peptides from the ORF of TDF protein and used in both IHC and WB to confirm anti-TDF Ab results.

  • Antony, Smitha, et al. "Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody." Free Radical Biology and Medicine 65 (2013): 497-508. Abstract
    Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.

The paper describes expression of human Nox5, immunization of mice and Nox5 monoclonal antibody production by hybridoma technique were carried out by Creative Biolabs. Finally, 8 positive clones were selected and evaluated further. Isotyping of 8 clones identified 7 of them as IgG clones.

  • Wang X, Wang S, Lu Y, et al. “FAM20C plays an essential role in the formation of murine teeth.” Journal of Biological Chemistry, 2012, 287(43): 35934-35942. Abstract
    FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.

The paper describes the generation of FAM20C monoclonal antibodies with the Omni-Hybridoma platform from Creative Biolabs. Three positive clones (clones 3, 5, and 6) with the IgG isotype were further screened using IHC and Western immunoblotting analyses.

  • Parra, Gabriel I., et al. "Multiple antigenic sites are involved in blocking the interaction of GII. 4 norovirus capsid with ABH histo-blood group antigens." Journal of virology 86.13 (2012): 7414-7426. Abstract
    Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes.

The paper describes anti-VLPs mAb production was carried out at Creative Biolabs using hybridoma technique.

  • Wu, Yongzhong, et al. "Up-regulation and sustained activation of Stat1 are essential for interferon-γ (IFN-γ)-induced dual oxidase 2 (Duox2) and dual oxidase A2 (DuoxA2) expression in human pancreatic cancer cell lines." Journal of Biological Chemistry 286.14 (2011): 12245-12256. Abstract
    Dual oxidase 2 is a member of the NADPH oxidase (Nox) gene family that plays a critical role in the biosynthesis of thyroid hormone as well as in the inflammatory response of the upper airway mucosa and in wound healing, presumably through its ability to generate reactive oxygen species, including H2O2. The recently discovered overexpression of Duox2 in gastrointestinal malignancies, as well as our limited understanding of the regulation of Duox2 expression, led us to examine the effect of cytokines and growth factors on Duox2 in human tumor cells. We found that exposure of human pancreatic cancer cells to IFN-γ (but not other agents) produced a profound up-regulation of the expression of Duox2, and its cognate maturation factor DuoxA2, but not other members of the Nox family. Furthermore, increased Duox2/DuoxA2 expression was closely associated with a significant increase in the production of both intracellular reactive oxygen species and extracellular H2O2. Examination of IFN-γ-mediated signaling events demonstrated that in addition to the canonical Jak-Stat1 pathway, IFN-γ activated the p38-MAPK pathway in pancreatic cancer cells, and both played an important role in the induction of Duox2 by IFN-γ. Duox2 up-regulation following IFN-γ exposure is also directly associated with the binding of Stat1 to elements of the Duox2 promoter. Our findings suggest that the pro-inflammatory cytokine IFN-γ initiates a Duox2-mediated reactive oxygen cascade in human pancreatic cancer cells; reactive oxygen species production in this setting could contribute to the pathophysiologic characteristics of these tumors.

The paper describes mouse anti-human Duox2 monoclonal antibody S-40 was developed by Creative Biolabs by using hybridoma technique.

  • Jia, Lijun, et al. "Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target." Clinical Cancer Research 16.3 (2010): 814-824. Abstract
    Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity.

    SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins.

    SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life.

    The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target.

The paper describes to precisely determine the expression status of SAG in human tumor tissues, scientists of Creative Biolabs raised an antibody against purified human SAG RING domain (AA44-113) fused with glutathione S-transferase, and performed immunostaining analysis using the SAG mAb specificity against SAG.

  • Tanese, Keiji, Elizabeth A. Grimm, and Suhendan Ekmekcioglu. "The role of melanoma tumor‐derived nitric oxide in the tumor inflammatory microenvironment: Its impact on the chemokine expression profile, including suppression of CXCL10." International journal of cancer 131.4 (2012): 891-901. Abstract
    Melanoma appears to be heterogeneous in terms of its molecular biology, etiology and epidemiology. We previously reported that the expression of inducible nitric-oxide synthase (iNOS) in melanoma tumor cells is strongly correlated with poor patient survival. Therefore, we hypothesized that nitric oxide (NO) produced by iNOS promotes the melanoma inflammatory tumor microenvironment associated with poor outcome. To understand the role of NO and iNOS in the melanoma inflammatory tumor microenvironment, polymerase chain reaction arrays of inflammatory and autoimmunity genes were performed on a series of stage III melanoma lymph node metastasis samples to compare the gene expression profiles of iNOS-expressing and nonexpressing tumor samples. The results indicate that expression of CXC chemokine ligand 10 (CXCL10) was inversely correlated with iNOS expression, and the high CXCL10-expressing cases had more favorable prognoses than the low CXCL10-expressing cases. Functional studies revealed that treating iNOS-negative/CXCL10-positive melanoma cell lines with a NO donor suppressed the expression of CXCL10. Furthermore, scavenging NO from iNOS-expressing cell lines significantly affected the chemokine expression profile. Culture supernatants from NO scavenger-treated melanoma cells promoted the migration of plasmacytoid dendritic cells, which was diminished when the cells were treated with a CXCL10-neutralizing antibody. CXCL10 has been reported to be an antitumorigenic chemokine. Our study suggests that the production of NO by iNOS inhibits the expression of CXCL10 in melanoma cells and leads to a protumorigenic tumor microenvironment. Inhibiting NO induces an antitumorigenic environment, and thus, iNOS should be considered to be an important therapeutic target in melanoma.

The paper describes a mouse anti-iNOS monoclonal antibody created at Creative Biolabs used to analysis the CXCL10 and CXCL9 expression by immunocytochemistry.

  • Roy, Urmi, et al. "Structural investigation of tumor differentiation factor." Biotechnology and applied biochemistry 59.6 (2012): 445-450. Abstract
    Tumor differentiation factor (TDF) is a 17 kDa protein produced by the pituitary and secreted into the bloodstream, with no definitive function and incomplete characterization. TDF has the following four cysteine (Cys) residues: Cys17, Cys70, Cys97, and Cys98. To understand the function of TDF, we (1) overexpressed and characterized recombinant TDF (rTDF); (2) investigated native, secreted TDF; and (3) assessed potential disulfide connectivities using molecular modeling. Our results from Western blotting (WB) experiments suggest that rTDF is mostly expressed as insoluble, monomeric, and dimeric forms. Mass spectrometry analysis of the overexpressed rTDF identified a peptide that is a part of TDF protein. WB of the native, secreted TDF detected it as a 50 kDa band. In addition, investigation of TDF by molecular modeling suggests that the Cys residues may form disulfide bridges between Cys17-Cys98 and Cys70-Cys17.

The paper describes anti-TDF antibodies were custom-made by Creative Biolabs using TDF peptide P1 (TDF-P1) as an immunogen. Creative Biolabs’ scientists synthesized TDF-P1 peptide and coupled them to KLH, then used for immunization of rabbits. The correct sequence of TDF-P1 was confirmed by MS and the specificity of the antibody was confirmed by pre-incubation of anti-TDF antibodies with its peptide antigen in an inhibition assay.

SECTION 3: Antibody Engineering and Antibody Sequencing[Top]

Creative Biolabs is a recognized service provider in converting small gene-engineered scFv or Fab human/mouse antibodies derived from phage display antibody library screening or mouse/rat hybridoma cell lines into full-size recombinant human or mouse IgGs with Fc fragments of various functions, e.g. ADCC, or non-ADCC. Creative Biolabs has established a solid platform for industrial scale production of recombinant antibodies in various expression systems.

Creative Biolabs has developed the proprietary Database Assisted Shotgun Sequencing (DASS) technology, which based on our next generation antibody sequencing platform. Purified monoclonal antibodies in multivalent forms can be sequenced with 100% coverage and 100% accuracy.

  • Bruce Hammerberg, and Sitka Eguiluz-Hernandez. “Therapeutic anti-IgE monoclonal antibody single chain variable fragment (scFv) safety and immunomodulatory effects after one-time injection in four dogs.” Veterinary Dermatology (2016). Abstract
    The therapeutic monoclonal antibody omalizumab that is specific for IgE has proven to be an effective addition to the treatment of allergic disease in humans.

    The aims of this study were to demonstrate the safety and immunomodulating effects of a single injection of a monoclonal antibody single chain variable fragments (scFv) specific for canine IgE in normal dogs.

    Three normal dogs were bled for EDTA whole blood samples for 112 days post-injection (dpi). A fourth dog was monitored for 28 days.

    Anti-IgE scFv was pegylated to minimize scFv dimerization. Four normal dogs were injected once subcutaneously with anti-IgE scFv at 1 mg/kg. Flow cytometry was performed on whole blood. Plasma levels of IgE were measured by ELISA.

    None of the four dogs showed signs of anaphylaxis. All dogs demonstrated decreases in IgE(+) cells in lymphocyte-gated events by 14 dpi. Dogs C and D returned to pre-injection levels by 21 days, whereas dogs A and B remained below pre-injection levels until Day 112. Similar differences were seen in IgE-bearing granulocyte-gated cells. Free plasma IgE decreased below pre-injection levels by 47% in Dog A and by 52% in Dog B at 112 days. Dogs C and D did not change by more than 32% from preinjection levels.

    A single injection of monomeric, pegylated scFv with high affinity for dog IgE was demonstrated to be safe. Marked reduction in IgE-bearing lymphocytes and granulocytes accompanied by reduced "free" plasma IgE level in two of four dogs is analogous to omalizumab in humans.

The paper describes that the heavy and light chain variable regions of a mouse monoclonal antibody (mAb 5.91) with high affinity binding to an epitope in the C2 domain of the epsilon chain of canine IgE were sequenced by Creative Biolabs.

  • Frederick C. S. WANG, Mark H. FOGG. “Neutralizing antibody for Epstein-Barr virus associated disease. ” WO 2013130565. Sep 6, 2013 US9376485 B2. Jun 28, 2016 US20150064174 A1. Mar 5, 2015 Abstract
    Described herein are compositions, methods, and uses relating to an EBV-neutralizing antibody.

These patents suggest Creative Biolabs can design and produce fully humanized antibody.

  • Timoshevskaya I. “Single-Molecule Fluorescence Studies of Glycosylated and Aglycosylated Antibodies.” DePaul Discoveries, 2014, 1(1): 11. Abstract
    Antibodies are Y-shaped, flexible proteins whose structures can be studied using Förster Resonance Energy Transfer (FRET) at the single-molecule level. Dye molecules must be attached to these proteins so as to carry out FRET studies of antibodies. In order to label the binding sites of an antibody, dye molecules were attached to a small molecule, or hapten, which the antibody binds to. Evidence for this binding was provided by ultraviolet-visible (UV-Vis) spectroscopy. To label the stem region of a humanized immunoglobulin G (IgG) antibody, the DNA for this antibody was mutated to introduce a cysteine residue to which dyes can be attached. In this research, the DNA was sequenced and checked to provide the desired sequence for protein production.

The paper describes Creative Biolabs found the sequence errors in the variable heavy chain and fixed them, then produced the IgG protein.

  • Kelliher, Michael T., et al. "The effect of sugar removal on the structure of the Fc region of an IgG antibody as observed with single molecule Förster Resonance Energy Transfer." Molecular immunology 60.2 (2014): 103-108. Abstract
    The deglycosylation of immunoglobulin G (IgG) antibodies leads to a diminished immune response. This reduction in immune response is thought to arise from weakened binding of IgG antibodies to effector molecules as a result of a conformational change in the antibody. The nature of this structural alteration is uncertain due to the conflicting results obtained from different experimental methods. We have examined the impact of deglycosylation by the endoglycosidase PNGase F on the structure of the Fc region of a human IgG antibody using single molecule Förster Resonance Energy Transfer (FRET). The FRET efficiency histograms obtained indicate that the structure of the Fc region becomes more flexible upon deglycosylation. This is demonstrated by a change in the width of the energy transfer efficiency peak, which increases from 0.19 ± 0.02 to 0.6 ± 0.1 upon deglycosylation.

The paper describes Creative Biolabs expressed and purified a mutated Humira IgG from HEK 293E cells. We incorporated a cysteine mutation in place of Ser 258 in each heavy chain exactly as the author demanded.

  • Wanczyk H, Barker T, Rood D, et al. “Cloning and characterization of a hybridoma secreting a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-specific monoclonal antibody and recombinant F (ab).” Toxins, 2013, 5(3): 568-589. Abstract
    Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

The paper describes the heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, expressed in HEK 293 cells with a similar binding features to that of the parent hybridoma.

  • Roh, Jooho, et al. "Generation of a chickenized catalytic anti-nucleic acid antibody by complementarity-determining region grafting." Molecular immunology 63.2 (2015): 513-520. Abstract
    In contrast to a number of studies on the humanization of non-human antibodies, the reshaping of a non-human antibody into a chicken antibody has never been attempted. Therefore, nothing is known about the animal species-dependent compatibility of the framework regions (FRs) that sustain the appropriate conformation of the complementarity-determining regions (CDRs). In this study, we attempted the reshaping of the variable domains of the mouse catalytic anti-nucleic acid antibody 3D8 (m3D8) into the FRs of a chicken antibody (“chickenization”) by CDR grafting, which is a common method for the humanization of antibodies. CDRs of the acceptor chicken antibody that showed a high homology to the FRs of m3D8 were replaced with those of m3D8, resulting in the chickenized antibody (ck3D8). ck3D8 retained the biochemical properties (DNA binding, DNA hydrolysis, and cellular internalizing activities) and three-dimensional structure of m3D8 and showed reduced immunogenicity in chickens. Our study demonstrates that CDR grafting can be applied to the chickenization of a mouse antibody, probably due to the interspecies compatibility of the FRs.

The paper mentioned that Creative Biolabs can reshape non-human antibody into other non-human antibodies such as antibody caninization.

  • Chen, Wu, et al. "Synthesis and optimization of wide pore superficially porous particles by a one-step coating process for separation of proteins and monoclonal antibodies." Journal of Chromatography A 1414 (2015): 147-157. Abstract
    Superficially porous particles (SPPs) with pore size ranging from 90Å to 120Å have been a great success for the fast separation of small molecules over totally porous particles in recent years. However, for the separation of large biomolecules such as proteins, particles with large pore size (e.g. ≥ 300Å) are needed to allow unrestricted diffusion inside the pores. One early example is the commercial wide pore (300Å) SPPs in 5μm size introduced in 2001. More recently, wide pore SPPs (200Å and 400Å) in smaller particle sizes (3.5-3.6μm) have been developed to meet the need of increasing interest in doing faster analysis of larger therapeutic molecules by biopharmaceutical companies. Those SSPs in the market are mostly synthesized by the laborious layer-by-layer (LBL) method. A one step coating approach would be highly advantageous, offering potential benefits on process time, easier quality control, materials cost, and process simplicity for facile scale-up. A unique one-step coating process for the synthesis of SPPs called the "coacervation method" was developed by Chen and Wei as an improved and optimized process, and has been successfully applied to synthesis of a commercial product, Poroshell 120 particles, for small molecule separation. In this report, we would like to report on the most recent development of the one step coating coacervation method for the synthesis of a series of wide pore SPPs of different particle size, pore size, and shell thickness. The one step coating coacervation method was proven to be a universal method to synthesize SPPs of any particle size and pore size. The effects of pore size (300Å vs. 450Å), shell thickness (0.25μm vs. 0.50μm), and particle size (2.7μm and 3.5μm) on the separation of large proteins, intact and fragmented monoclonal antibodies (mAbs) were studied. Van Deemter studies using proteins were also conducted to compare the mass transfer properties of these particles. It was found that the larger pore size actually had more impact on the performance of mAbs than particle size and shell thickness. The SPPs with larger 3.5μm particle size and larger 450Å pore size showed the best resolution of mAbs and the lowest back pressure. To the best of our knowledge, this is the largest pore size made on SPPs. These results led to the optimal particle design with a particle size of 3.5μm, a thin shell of 0.25μm and a larger pore size of 450Å.

Creative Biolabs serviced the papain digested humanized recombinant trastuzumab (2 mg/mL) for the author.

  • Timoshevskaya, Irina. "Single-Molecule Fluorescence Studies of Glycosylated and Aglycosylated Antibodies." DePaul Discoveries 1.1 (2014): 11. Abstract
    Antibodies are Y-shaped, flexible proteins whose structures can be studied using Förster Resonance Energy Transfer (FRET) at the single-molecule level. Dye molecules must be attached to these proteins so as to carry out FRET studies of antibodies. In order to label the binding sites of an antibody, dye molecules were attached to a small molecule, or hapten, which the antibody binds to. Evidence for this binding was provided by ultraviolet-visible (UV-Vis) spectroscopy. To label the stem region of a humanized immunoglobulin G (IgG) antibody, the DNA for this antibody was mutated to introduce a cysteine residue to which dyes can be attached. In this research, the DNA was sequenced and checked to provide the desired sequence for protein production.

Creative Biolabs expressed the humanized IgG antibody for the author, and fixed the sequence errors found in the variable heavy chain for the author.

  • Piraino, Mark S., et al. "Single molecule Förster resonance energy transfer studies of the effect of EndoS deglycosylation on the structure of IgG."Immunology letters 167.1 (2015): 29-33. Abstract
    The bacterial enzyme EndoS specifically cleaves glycans bound to immunoglobulin G (IgG) molecules. Because this deglycosylation procedure leads to a diminished immune response, this enzyme has potential applications as a therapeutic for autoimmune disorders. Although the diminished immune response is attributed to a structural change in the Fc region of IgG antibodies, the specific nature of this structural change is not known due to the variety of results obtained by different experimental approaches. In order to better understand how EndoS deglycosylation impacts the structure of the Fc region of IgG antibodies, we have conducted single molecule Förster resonance energy transfer (FRET) studies of dye-labeled, freely diffusing antibodies. A comparison of the FRET efficiency histograms obtained for glycosylated and EndoS deglycosylated antibodies indicates that the Fc region can take on a wider variety of structures upon deglycosylation. This is demonstrated by the presence of additional peaks in the FRET efficiency histogram for the deglycosylated case.

Creative Biolabs expressed the mutated Humira IgG (hIgG), in HEK 293E cells and purified using a HiTrap rProtein A FF column for the authors.

  • Peng, Hong, et al. "Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: K D values, binding pairs, and amino acid sequences." Chemico-biological interactions 240 (2015): 336-345. Abstract
    Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.

Creative Biolabs amplified and sequenced the complete cDNA sequence of Hybridoma cell line 11D8 for the authors, then produced and purified the recombinant antibodies.

  • Mallavia, Beñat, et al. "Peptide Inhibitor of Nuclear Factor-kB Translocation Ameliorates Experimental Atherosclerosis." The American Journal of Pathology 182.5 (2013). Abstract
    Atherosclerosis is a chronic inflammatory disease of the arterial wall. NF-κB is a major regulator of inflammation that controls the expression of many genes involved in atherogenesis. Activated NF-κB was detected in human atherosclerotic plaques, and modulation of NF-κB inflammatory activity limits disease progression in mice. Herein, we investigate the anti-inflammatory and atheroprotective effects of a cell-permeable peptide containing the NF-κB nuclear localization sequence (NLS). In vascular smooth muscle cells and macrophages, NLS peptide specifically blocked the importin α–mediated nuclear import of NF-κB and prevented lipopolysaccharide-induced pro-inflammatory gene expression, cell migration, and oxidative stress. In experimental atherosclerosis (apolipoprotein E–knockout mice fed a high-fat diet), i.p., 0.13 μmol/day NLS peptide administration for 5 weeks attenuated NF-κB activation in atherosclerotic plaques. NLS peptide significantly inhibited lesion development at both early (age 10 weeks) and advanced (age 28 weeks) stages of atherosclerosis in mice, without affecting serum lipid levels. Plaques from NLS-treated mice contained fewer macrophages of pro-inflammatory M1 subtype than those from respective untreated controls. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. NLS peptide also attenuated pro-inflammatory gene expression and oxidative stress in aortic lesions. Our study demonstrates that targeting NF-κB nuclear translocation hampers inflammation and atherosclerosis development and identifies cell-permeable NLS peptide as a potential anti-atherosclerotic agent.

The paper describes a cell-permeable peptide containing the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor (AAVALLPAVLLALLAP)15 and a single NLS from NF-kB (VQRKRQKLMP)16 was synthesized and cyclized (intrachain disulfide bond between cysteines inserted in the NLS motif) by Creative Biolabs.

  • Gernot Stuhler. “Dual antigen-induced bipartite functional complementation.” WO 2013104804. Nov 21, 2013CA2861003A1. Jul 18. 2013EP2802607A2. Nov 19. 2014US20150079093. Mar 19. 2015WO2013104804A2. Jul 18. 2013WO2013104804A3. Nov 21, 2013CN 104159923 A. Nov 19, 2014 Abstract
    The present invention relates to a set of polypeptides and its uses. In particular, the present invention relates to a set of polypeptides whereby this set comprises two polypeptides each of which comprises a targeting moiety "T" binding to an antigen "A" and a fragment of "F" of a functional domain, wherein said two polypeptides are not associated with each other in absence of a substrate that has "A" at (on) its surface and wherein, upon dimerization of "F", the resulting dimer becomes functional. Furthermore, medical and diagnostic uses of said set are described. Moreover, the present invention relates to nucleic acid molecule(s) encoding said set of polypeptides. The present invention also relates to a vector comprising the nucleotide sequence of nucleic acid molecule(s) encoding said set of polypeptides. Furthermore, the present invention relates to pharmaceutical compositions comprising said set of polypeptides. Moreover, the present invention relates to a kit comprising said set of polypeptides.
  • Bao, Xiaofeng, et al. "Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis." Microbiology 157.9 (2011): 2569-2581. Abstract
    Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ⁶⁶-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability.

These papers describe the genome sequencing, including library preparation, Solexa cluster generation and single-end-read sequencing, was performed by Creative Biolabs.

SECTION 4: Single Domain Antibody Services[Top]

Creative Biolabs is specialized in generating single domain antibodies against any targets. We also provide a wide range of services, including construction of immunized single domain antibody libraries using llama and camel, construction of synthetic camelized human single domain antibody libraries using DNA synthesis, biopanning of single domain antibodies libraries and large scale production of recombinant single domain antibodies. Together with Creative Diagnostics, we provide a large list of single domain antibodies.

  • Suratt, Benjamin T. "Suppression of leptin action for treatment of pulmonary infections." U.S. Patent No. 20,150,329,635. 19 Nov. 2015. Abstract
    Provided is a method of assessing risk of developing pulmonary infection in an individual or a population comprising determining circulating leptin levels in the individuals and comparing the levels or normal controls, and if the leptin levels are higher than in the control, identifying the individual to be at risk of developing pulmonary infection. Also provided are methods of reducing the severity of, or preventing pulmonary infections in individuals with elevated leptin levels by administration of agents that that suppress the levels of leptin or interfere with its actions.

Creative Biolabs provides customized services of single domain antibodies.

  • Sunanda SINGH. “Single domain antibodies directed against intracellular antigens.” WO2016065323 A2. Apr 28 2016. US20160115247 A1. Apr 28 2016. WO2016065323 A3. Jun 16 2016. Abstract
    This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.
  • Sunanda SINGH. “Single domain antibodies directed against tnfalpha.” US20160115226 A1. Apr 28 2016. Abstract
    This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.
  • Sunanda SINGH. “Single domain antibodies directed against kras.” US20160115244 A1. Apr 28 2016. Abstract
    This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.
  • Sunanda SINGH. “Single domain antibodies directed against stat3.” US20160115248 A1. Apr 28 2016. Abstract
    This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.

The cells were grown to 50% to 70% confluence, then disrupted in freshly prepared ice-cold lysis buffer as described above for 45 minutes on ice. Lysates were then centrifuged, the supernatant collected, and protein concentration was determined as described above. Total protein (1 mg) was incubated with 1.5 mg of Dynabeads (Invitrogen) containing the bacterial anti-STAT3 VHH13 (SEQ ID NO:3) or negative control (KRAS, Creative Biolabs, Shirley, NY) for 1 hour at 4°C.

SECTION 5: Yeast Two-Hybrid[Top]

Creative Biolabs provides the services on a strict fee-for-service basis. We are professional in using Gal4 and LexA based Y2H systems. We are able to perform yeast two-hybrid screening against integral membrane proteins and membrane-associated/proximal proteins using a modified split-ubiquitin membrane yeast two-hybrid (MYTH) system.

  • Horgan C P, Hanscom S R, Kelly E E, et al. “Tumor susceptibility gene 101 (TSG101) is a novel binding-partner for the class II Rab11-FIPs.” PloS one, 2012, 7(2): e32030. Abstract
    The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.

The paper describes Creative Biolabs performed a yeast two-hybrid screen and assay against an adult human brain cDNA library.

SECTION 6: Antibody Development in Various Animal Species[Top]

Creative Biolabs provides antibody production service using a comprehensive list of host species.

  • Roy, Urmi, et al. "Structural investigation of tumor differentiation factor." Biotechnology and applied biochemistry 59.6 (2012): 445-450. Abstract
    Tumor differentiation factor (TDF) is a 17 kDa protein produced by the pituitary and secreted into the bloodstream, with no definitive function and incomplete characterization. TDF has the following four cysteine (Cys) residues: Cys17, Cys70, Cys97, and Cys98. To understand the function of TDF, we (1) overexpressed and characterized recombinant TDF (rTDF); (2) investigated native, secreted TDF; and (3) assessed potential disulfide connectivities using molecular modeling. Our results from Western blotting (WB) experiments suggest that rTDF is mostly expressed as insoluble, monomeric, and dimeric forms. Mass spectrometry analysis of the overexpressed rTDF identified a peptide that is a part of TDF protein. WB of the native, secreted TDF detected it as a 50 kDa band. In addition, investigation of TDF by molecular modeling suggests that the Cys residues may form disulfide bridges between Cys17-Cys98 and Cys70-Cys17.

The paper describes anti-TDF antibodies were custom-made by Creative Biolabs using TDF peptide P1 (TDF-P1) as an immunogen. Creative Biolabs’ scientists synthesized TDF-P1 peptide and coupled them to KLH, then used for immunization of rabbits. The correct sequence of TDF-P1 was confirmed by MS and the specificity of the antibody was confirmed by pre-incubation of anti-TDF antibodies with its peptide antigen in an inhibition assay.

  • Ekmekcioglu, Suhendan, et al. "Zyflamend mediates therapeutic induction of autophagy to apoptosis in melanoma cells." Nutrition and cancer 63.6 (2011): 940-949. Abstract
    Melanoma is the most aggressive form of skin cancer. The rising incidence of melanoma and its poor prognosis in advanced stages are compelling reasons to identify novel therapeutic agents. Though isolated dietary components such as lycopene, resveratrol, and isothiocyanate compounds have been shown to provide limited protection against cancer development, the use of whole herbs and herbal extracts for the treatment of cancer remains of great interest. As suggested by earlier studies, the antiinflammatory activity of many plants available as intact products or as extracts has long been considered for supplemental therapeutics for cancer. Zyflamend, a unique multiherbal extract preparation, is a promising antiinflammatory agent that has also been suggested to regulate multiple pathways in cancer progression. As Zyflamend contains ingredients that can suppress tumor cell proliferation, invasion, angiogenesis, and metastasis through regulation of inflammatory pathway products, we hypothesized that this preparation might inhibit melanoma proliferation. To test this hypothesis, we studied the effect of Zyflamend on melanoma proliferation. Here, we present that Zyflamend inhibits melanoma growth by regulating the autophagy-apoptosis switch. Based on the responsible molecular mechanisms of Zyflamend, our study highlights the importance of the use of herbal preparations for the prevention and treatment of cancer.

The paper describes the inducible nitric oxide synthase (iNOS) antibody used in IHC was purchased from Creative Biolabs.

  • Doran, Todd M., and Thomas Kodadek. "A Liquid Array Platform for the Multiplexed Analysis of Synthetic Molecule–Protein Interactions." ACS chemical biology 9.2 (2013): 339-346. Abstract
    Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative composed of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships, and certain types of serological analyses.
  • Thomas Kodadek, Todd DORAN. “Liquid array platform for multiplexed analysis of molecule-protein interactions.” WO2014138028 A1. Sep 12 2014. Abstract
    The invention provides a method for high-throughput analytical detection of ligand-protein interactions. Polyethyleneglycol-coated amino-functionalized polystyrene microspheres are treated to block surface amino groups and separated into subpopulations, each of which is treated with two or more aminoreactive fluorescent dyes in a defined ratio, which serves to encode each subpopulation for identification in a fluorescence-activated cell sorter (FACS). Each subpopulation is then bonded via surface amino groups to a respective ligand. The protein under evaluation is associated with another fluorescent dye. Each fluorescent dye has a unique emission wavelength the intensity of which can be quantified by the FACS. The ratio of light emission of the two or more encoding dyes serves to identify each subpopulation of microspheres and the protein-associated dye indicate the binding of the respective ligand to the protein analyte.

These papers describe the author used a stock solution containing 14 μM IgY from a chicken immunized with ADP3 from Creative Biolabs.

  • Tenkerian, Clara, et al. "mTORC2 Balances AKT Activation and eIF2α Serine 51 Phosphorylation to Promote Survival under Stress." Molecular Cancer Research 13.10 (2015): 1377-1388. Abstract
    The mTOR nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism, and cancer. Phosphorylation of the α-subunit of translation initiation factor eIF2 at serine 51 (eIF2αS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response, but its connection to eIF2αS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2αS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2αS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2αS51P by impaired mTORC2 depends on the inactivation of AKT, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biologic function of eIF2αS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and AKT activity. TSC-mutant cells exhibit increased PERK activity, which is downregulated by the reconstitution of the cells with an activated form of AKT1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by AKT1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacologic inhibition of PERK or genetic inactivation of eIF2αS51P. Thus, the PERK/eIF2αS51P arm is an important compensatory prosurvival mechanism, which substitutes for the loss of AKT under ER stress.

Creative Biolabs produced rabbit antiserum for mouse PERK phosphorylated at T779 by immunizing animals against a chemically synthesized phosphopeptide of mouse PERK conjugated with keyhole limpet hemocyanin (KLH).

  • Morimoto, Jumpei, et al. "Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands." Bioconjugate chemistry 25.8 (2014): 1479-1491. Abstract
    Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors.

Creative Biolabs produced anti-ADP3 chicken IgY by immunizing a chicken with ADP3.

  • Li, Yanchun, et al. "F (ab’) 2 fragments of anti-oxidized LDL IgG attenuate vascular inflammation and atherogenesis in diabetic LDL receptor-deficient mice." Clinical Immunology (2016). Abstract
    Considerable evidence is available supporting the atherogenic role of immune complexes (IC) formed by modified forms of LDL and their corresponding antibodies in humans and other species. In this study, we assessed the effect of IgG F(ab')2 fragments of murine anti-mouse oxLDL, which binds oxLDL forming IC that cannot interact with Fcγ receptors, on the development of atherosclerosis in diabetic LDL receptor-deficient (LDLR-/-) mice. Immunohistochemical study showed that treatment with the F(ab')2 fragments for 8weeks significantly reduced the content of macrophages and interleukin 6 expression in atherosclerotic lesions. Furthermore, histological study showed that treatment with the same F(ab')2 fragments significantly reduced atherosclerotic lesions in diabetic LDLR-/- mice. Taken together, this study demonstrated for the first time that F(ab')2 fragments of anti-oxLDL IgG inhibited vascular inflammation and atherogenesis in diabetic LDLR-/- mice and uncovered a possible new avenue for therapy in patients at high risk to progress to cardiovascular complications.

The mice were fed an atherogenic diet containing 1.25% cholesterol and 21% milkfat for 5 months to increase their LDL level and were used to isolate LDL which was afterwards oxidized and adequately characterized before being sent to Creative Biolabs where it was used to immunize Balb/c mice.

  • Woods, Alisa G., et al. "Identification of tumor differentiation factor (TDF) in select CNS neurons." Brain Structure and Function 219.4 (2014): 1333-1342. Abstract
    Identification of central nervous system (CNS) molecules elucidates normal and pathological brain function. Tumor differentiation factor (TDF) is a recently-found protein secreted by the pituitary into the blood. TDF mRNA was detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. However, TDF has an unclear function. It is not known whether TDF is expressed only by pituitary or by other brain regions. It is also not known precisely where TDF is expressed in the brain or which cells produce TDF. Database searching revealed that this molecule shares no homology with any known protein. Therefore, we investigated the distribution of TDF in the rat brain using immunohistochemistry (IHC) and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions. Double-staining for TDF and glial fibrillary acidic protein (GFAP), an astrocyte marker, showed no co-localization. Double-staining for TDF with NeuN, a neuronal marker, showed co-localization. Not all NeuN positive cells were positive for TDF. Western blotting (WB) using NG108 neuroblastoma and GS9L astrocytoma cell lysate revealed TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized in neurons, not in astrocytes. This is the first report of any cellular localization of TDF. TDF may have specific roles as a pituitary-derived hormone and in the CNS, and appears to be produced by distinct CNS neurons, not astroglia.

Creative Biolabs generated two rabbit polyclonal anti-TDF antibodies: anti-TDF-P1 antibody (anti-TDF-P1-Ab) and anti-TDF-P1P2P3 antibody (anti-TDF-P1-Ab) for the author.

  • Sokolowska I, Woods A G, Gawinowicz M A, et al. “Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells.” Journal of Biological Chemistry, 2012, 287(3): 1719-1733. Abstract
    Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

The paper describes TDF-P1 peptide, with the amino acid sequence H2-RESQGTRVGQALSFLCKGTA-COOH, was synthesized, and rabbit poyclonal TDF generated against TDF-P1 by Creative Biolabs.

SECTION 7: Fluorescent In Situ Hybridization (FISH)[Top]

Creative Biolabs offers a full array of custom fluorescence in situ hybridization (FISH) service from probe design, chromosome/cell preparation to expert result interpretation.

  • Du, Zhi-Qiang, et al. "Novel microRNA families expanded in the human genome." BMC genomics 14.1 (2013): 98. Abstract
    Most studies on the origin and evolution of microRNA in the human genome have been focused on its relationship with repetitive elements and segmental duplications. However, duplication events at a smaller scale (<1 kb) could also contribute to microRNA expansion, as demonstrated in this study.

    Using comparative genome analysis and bioinformatics methods, we found nine novel expanded microRNA families enriched in short duplicated sequences in the human genome. Furthermore, novel genomic regions were found to contain microRNA paralogs for microRNA families previously analyzed to be related to segmental duplications. We found that for microRNA families expanded in the human genome, 14 families are specific to the primate lineage, and nine are non-specific, respectively. Two microRNA families (hsa-mir-1233 and hsa-mir-622) appear to be further expanded in the human genome, and were confirmed by fluorescence in situ hybridization. These novel microRNA families expanded in the human genome were mostly embedded in or close to proteins with conserved functions. Furthermore, besides the Alu element, L1 elements could also contribute to the origination of microRNA paralog families.

    Together, we found that small duplication events could also contribute to microRNA expansion, which could provide us novel insights on the evolution of human genome structure and function.

The paper describes Creative Biolabs performed FISH on a normal primary neonatal dermal fibroblast cell line of European origin (PCS-201-010 from ATCC® Primary Cell Solutions™) following standard procedures with short probe length (~1 kb).

  • Genetic Engineering & Biotechnology News (2009): GEN., Volume 29, Issues 1-8 (pp48, pp56). NY, USA: GEN Pub. Inc. Abstract
    Genetic Engineering & Biotechnology News(GEN) is the most widely-read publication covering tools, techniques, and technologies in the biotechnology industry; a site-wide license is important for academic, corporate, and government institutions to foster global collaborations among these sectors and to keep them abreast of the urgent issues that will affect the field.
    Covering bench to bedside for over 35 years, GEN's unique news and technology focus includes the entire bioproduct life cycle from early-stage R&D, to applied research including OMICS, biomarkers, as well as diagnostics, to bioprocessing and commercialization.
  • Marzioni, Marco, et al. "PDX-1 mRNA expression in endoscopic ultrasound-guided fine needle cytoaspirate: Perspectives in the diagnosis of pancreatic cancer." Digestive and Liver Disease 47.2 (2015): 138-143. Abstract
    Endoscopic ultrasound-guided fine needle aspiration is routinely used in the diagnostic work up of pancreatic cancer but has a low sensitivity. Studies showed that Pancreatic Duodenal Homeobox-1 (PDX-1) is expressed in pancreatic cancer, which is associated with a worse prognosis. We aimed to verify whether the assessment of PDX-1 in endoscopic ultrasound-guided fine needle aspiration samples may be helpful for the diagnosis of pancreatic cancer.

    mRNA of 54 pancreatic cancer and 25 cystic lesions was extracted. PDX-1 expression was assessed by Real-Time PCR.

    In all but two patients with pancreatic cancer, PDX-1 was expressed and was found positive in 7 patients with pancreatic cancer in which cytology was negative. The positivity was associated with a probability of 0.98 (95% CI 0.90-1.00) of having cancer and the negativity with one of 0.08 (95% CI 0.01-0.27). The probability of cancer rose to 1.00 (95% CI 0.97-1.00) for patients positive to both PDX-1 and cytology and fell to 0.0 (95% CI 0.00-0.15) in patients negative for both.

    PDX-1mRNA is detectable in samples of pancreatic cancer. Its quantification may be helpful to improve the diagnosis of pancreatic cancer.

This author mentioned that Fluorescent in Situ Hybridization (FISH) was performed according to the instructions of Creative Biolabs.

SECTION 8: In Situ Hybridization (ISH)[Top]

Creative Biolabs offers a full array of custom in situ hybridization (ISH) service from probe design, tissue acquisition to expert interpretation of gene expression results.

  • Gregg, Jennifer L., et al. "Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture micro dissection."BMC cancer 10.1 (2010): 165. Abstract
    The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM).

    LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry.

    The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues.

    The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

The paper describes the commercially available prostate tissue microarrays (TMAs) used in this study were purchased from Creative Biolabs.

  • Chaerkady R, Harsha H C, Nalli A, et al. “A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma.” Journal of proteome research, 2008, 7(10): 4289-4298. Abstract
    Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. In this study, our objective was to identify differentially regulated proteins in HCC through a quantitative proteomic approach using iTRAQ. More than 600 proteins were quantitated of which 59 proteins were overexpressed and 92 proteins were underexpressed in HCC as compared to adjacent normal tissue. Several differentially expressed proteins were not implicated previously in HCC. A subset of these proteins (six each from upregulated and downregulated groups) was further validated using immunoblotting and immunohistochemical labeling. Some of the overexpressed proteins with no previous description in the context of HCC include fibroleukin, interferon induced 56 kDa protein, milk fat globule-EGF factor 8, and myeloid-associated differentiation marker. Interestingly, all the enzymes of urea metabolic pathway were dramatically downregulated. Immunohistochemical labeling confirmed differential expression of fibroleukin, myeloid associated differentiation marker and ornithine carbamoyl transferase in majority of HCC samples analyzed. Our results demonstrate quantitative proteomics as a robust discovery tool for the identification of differentially regulated proteins in cancers.

The paper describes IHC for myeloid-associated differentiation marker was carried out using tissue microarrays from Creative Biolabs.

  • Chaerkady, Raghothama, et al. "18O Labeling for a quantitative proteomic analysis of glycoproteins in hepatocellular carcinoma." Clinical proteomics 4.3-4 (2008): 137-155. Abstract
    Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

    In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent (18)O/(16)O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to (18)O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

    This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.

The paper describes the study used formalin fixed, paraffin embedded tissue, liver tissue sections and liver cancer tissue microarrays were obtained from Creative Biolabs.

  • Harris, Molly A., et al. "Cancer stem cells are enriched in the side population cells in a mouse model of glioma." Cancer research 68.24 (2008): 10051-10059. Abstract
    The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal, multipotentiality, and tumor initiation upon transplantation. By testing for these defining characteristics, we provide evidence for the existence of CSCs in a transgenic mouse model of glioma, S100beta-verbB;Trp53. In this glioma model, CSCs are enriched in the side population (SP) cells. These SP cells have enhanced tumor-initiating capacity, self-renewal, and multipotentiality compared with non-SP cells from the same tumors. Furthermore, gene expression analysis comparing fluorescence-activated cell sorting-sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human glioblastoma multiforme cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion, this study shows that CSCs exist in a mouse glioma model, suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the CSC hypothesis.

The paper describes the human glioma tissue arrays were purchased from Creative Biolabs.

  • Gass, Justin T., and M. Foster Olive. "Transcriptional profiling of the rat frontal cortex following administration of the mGlu5 receptor antagonists MPEP and MTEP." European journal of pharmacology 584.2 (2008): 253-262. Abstract
    The development of selective type 5 metabotropic glutamate receptor (mGlu5) antagonists, such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP), has revealed an important role for these receptors in various disorders of the nervous system including depression, anxiety, epilepsy, Parkinson's disease, drug addiction, and alcoholism. In this study, we used microarray technology to examine changes in gene expression induced by repeated administration of the mGlu5 antagonists MPEP and MTEP. Male Wistar rats (n=5 per treatment group) were administered MPEP (10 mg/kg), MTEP (10 mg/kg) or vehicle intraperitoneally twice daily for 5 days. Approximately 30 min following the final drug administration, rats were sacrificed and frontal cortices were then dissected and examined for changes in gene expression by cDNA microarray analysis. Changes in gene expression with p-values less than 0.01 were considered to be statistically significant. The expression of 63 genes was changed by both MPEP and MTEP, with 58 genes down-regulated and 5 genes up-regulated. Quantitative PCR verified the magnitude and direction of change in expression of 9 of these genes (r2=0.556, p=0.017). Pathway analysis revealed that many of the biological processes altered by repeated MPEP and MTEP treatment were related to ATP synthesis, hydrolase activity, and signaling pathways associated with mitogen-activated protein kinase (MAPK). Our results demonstrate diverse effects of MPEP and MTEP gene expression in the frontal cortex, and these results may help elucidate the mechanisms by which these compounds produce beneficial effects in animal models of various disorders of the central nervous system.

The paper describes Creative Biolabs can not only offer microarray product but also help the client in analyzing of microarray data.

  • Davis, Cristina E., et al. "Effects of phospholemman expression on swelling-activated ion currents and volume regulation in embryonic kidney cells."Neurochemical research 29.1 (2004): 177-187. Abstract
    Phospholemman (PLM) is a 72-amino-acid phosphoprotein that is a major substrate for cAMP-dependent protein kinase, protein kinase C, and NIMA kinase. In lipid bilayers, PLM forms ion channels selective for Cl-, K+, and taurine. Effluxes of these abundant intracellular osmolytes play an important role in the control of dynamic cell volume changes in many cell types. We measured swelling-activated ion currents and regulatory volume decrease (RVD) in human embryonic kidney cells stably overexpressing canine cardiac PLM. In response to swelling, two clonal cell lines overexpressing PLM had increased swelling-activated ion current densities and faster and more extensive RVD. A third clonal cell line overexpressing mutant PLM showed reduced ion current densities and a diminished RVD response. These results suggest a role for PLM in the regulation of cell volume, perhaps as a modulator of an endogenous swelling-activated signal transduction pathway or possibly by participating directly in swelling-induced osmolyte efflux.

The paper describes the study used the fibronectin-coated coverslips from Creative Biolabs.

  • Sang, Nianli, et al. “Repression of endometrial tumor growth by targeting SREBP1 and lipogenesis.” Cell Cycle 11.12 (2012): 2348-2358. Abstract
    The aberrantly increased lipogenesis is a universal metabolic feature of proliferating tumor cells. Although most normal cells acquire the bulk of their fatty acids from circulation, tumor cells synthesize more than 90% of required lipids de novo. The sterol regulatory element-binding protein 1 (SREBP1), encoded by SREBF1 gene, is a master regulator of lipogenic gene expression. SREBP1 and its target genes are overexpressed in a variety of cancers; however, the role of SREBP1 in endometrial cancer is largely unknown. We have screened a cohort of endometrial cancer (EC) specimen for their lipogenic gene expression and observed a significant increase of SREBP1 target gene expression in cancer cells compared with normal endometrium. By using immunohistochemical staining, we confirmed SREBP1 protein overexpression and demonstrated increased nuclear distribution of SREBP1 in EC. In addition, we found that knockdown of SREBP1 expression in EC cells suppressed cell growth, reduced colonigenic capacity and slowed tumor growth in vivo. Furthermore, we observed that knockdown of SREBP1 induced significant cell death in cultured EC cells. Taken together, our results show that SREBP1 is essential for EC cell growth both in vitro and in vivo, suggesting that SREBP1 activity may be a novel therapeutic target for endometrial cancers.

The paper describes the formalin-fixed and paraffin-embedded tumor specimens used in this study were from Creative Biolabs.

  • Postovit, Lynne-Marie, et al. "Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells."Proceedings of the National Academy of Sciences 105.11 (2008): 4329-4334. Abstract
    Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

The paper describes a breast carcinoma progression tissue microarray used for immunohistochemical staining for Nodal was supplied by Creative Biolabs.

  • American Association for Cancer Research, William H. Donner Foundation (2008). Cancer Research (pp142). Baltimore, USA: Waverly Press.  

The paper describes the normal human lung and 67 different lung tumors were purchased from Creative Biolabs.

  • Pei, Zhengtong, et al. "Very Long-Chain Acyl-CoA Synthetase 3: Overexpression and Growth Dependence in Lung Cancer." PloS one 8.7 (2013): e69392. Abstract
    Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65-76%, and the ability of tumor cells to form colonies in soft agar suspension by 65-80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer.

The paper describes the normal human lung and 67 different lung tumors were purchased from Creative Biolabs.

SECTION 9: Immunology Experimental Technique[Top]
  • Hendrix, Mary Jessica, et al. "Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells." U.S. Patents No. 8,669,239. Mar 11, 2014. EP 2412383. Feb 1, 2012 U.S. Patents No. 8,106,004. Jan 31, 2012. U.S. US8975037 B2. Mar 10, 2015 U.S. Patents No. 12,375,443. Apr 29, 2010 U.S. Patents No. 11,829,070. Feb 23, 2010 EP 2074209. Jul 1, 2009 CA 2658786. Jan 31, 2008 WO2008014426. Jan 31, 2008. Abstract
    The invention provides compositions comprising one or more isolated factors from a microenvironment of human embryonic stem cells (hESCs), including, but not limited to, Lefty and inhibitors of Nodal. The invention also provides methods of utilizing factors derived from human embryonic stem cells (hESC) and their microenvironment to treat and prevent tumor formation and progression and to inhibit tumor cell aggressiveness. The invention further provides methods of inhibiting tumor cell growth and/or treating aggressive tumors in a mammal comprising administering to the mammal, having at least one tumor cell present in its body, an effective amount of an inhibitor of Nodal activity.

These patents describe Creative Biolabs’ scientists performed the immunohistochemical staining for Nodal in a breast carcinoma progression TMA for the client.

  • Chenguang Wang, Jie Zhou. “Compositions and methods for treating cancer with aberrant lipogenic signaling.” EP2864339 A4. Apr 20, 2016

The patent describes Creative Biolabs served Formalin-fixed and paraffin-embedded tumor specimens for them used in this study.

SECTION 10: Recombinant Protein Products[Top]

Creative Biolabs offers extensive expertise across four expression systems for producing your recombinant proteins, monoclonal antibodies, vaccine antigens or disease biomarkers. Whether for structural studies, functional assays, target validation or high throughput screens, Creative Biolabs can give a hand. We can supply proteins expressed by the following systems: Bacterial expression systems (E. coli / Bacillus); Yeast expression systems (P. pastoris / S. cerevisiae); Baculovirus-insect cell expression systemsMammalian expression systems (CHO / 293); membrane protein production (New).

  • Hetényi, Anasztázia, et al. "Competitive inhibition of TRPV1–calmodulin interaction by vanilloids." FEBS letters (2016). Abstract
    There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.

Uniformly 13C- and 15N-labelled CaM was purchased from Creative Biolabs.

  • Turkson, James. "Drug composition cytotoxic for pancreatic cancer cells." U.S. Patents No. 8.685.941. 1 Apr. 2014. EP 2370082. May 30, 2012 WO 2010065444. Jun 10, 2010 CA 2745265. Jun 10, 2010 Abstract
    Disclosed herein are compositions comprising a drug combination that comprises ZD and S3I-201, Das and S3I-201, ZD and AG490, or Das and AG490. The disclosed drug combinations target two or more functional elements such as EGFR or Src and Stat3 or Jaks in pancreatic cancer cells. Also disclosed herein are methods of using the disclosed compositions to cytotoxically affect pancreatic cancer cells and methods of making the disclosed compositions.

These patents study used recombinant human EGF (hEGF) from Creative Biolabs.

  • Thomas Kodadek, Todd DORAN. “Liquid array platform for multiplexed analysis of molcule-protein interactions.” WO 2014138028. Sep 12, 2014. Abstract
    The invention provides a method for high-throughput analytical detection of ligand-protein interactions. Polyethyleneglycol-coated amino-functionalized polystyrene microspheres are treated to block surface amino groups and separated into subpopulations, each of which is treated with two or more aminoreactive fluorescent dyes in a defined ratio, which serves to encode each subpopulation for identification in a fluorescence-activated cell sorter (FACS). Each subpopulation is then bonded via surface amino groups to a respective ligand. The protein under evaluation is associated with another fluorescent dye. Each fluorescent dye has a unique emission wavelength the intensity of which can be quantified by the FACS. The ratio of light emission of the two or more encoding dyes serves to identify each subpopulation of microspheres and the protein-associated dye indicate the binding of the respective ligand to the protein analyte.

The patent study used ADP3 from Creative Biolabs.

  • Chenguang Wang, Jie Zhou. “Compositions and methods for treating cancer with aberrant lipogenic signaling.” WO 2014004054. Jan 3, 2014 CN104662028 A. May 27, 2015 EP2864339 A1. Apr 29, 2015 Abstract
    The technology described herein relates to pinacolyl boronate substituted stilbenes for the treatment of cancers, e.g. cancers expressing abnormally high levels of SREBP1.
  • Kim, Mi‐Sun, et al. "Comparative study of various growth factors and cytokines on type I collagen and hyaluronan production in human dermal fibroblasts." Journal of cosmetic dermatology 13.1 (2014): 44-51. Abstract
    Dermal fibroblast is a primary cell type responsible for synthesis and remodeling of extracellular matrix in human skin. Type I collagen and hyaluronan are main components that have roles in skin fibrosis, wound healing, tissue remodeling as well as skin aging. Several studies have reported cytokine-dependent changes in collagen expression or hyaluronan production; however, the cytokines' effect was controversial in human dermal fibroblasts.

    To clarify the role of various growth factors, cytokines or chemokines on the production of interstitial type I collagen and hyaluronan in dermal fibroblasts.

    We confirmed the presence of various corresponding receptors and assessed the effects of 33 human recombinants on the production of type I collagen and hyaluronan using the assay system in dermal fibroblasts.

    Platelet-derived growth factor (PDGF)-AA, PDGF-BB, epidermal growth factor (EGF), transforming growth factor (TGF)-β1, MCP-1, IP-10, interleukin (IL)-1α, IL-1β, and IL-15 were effective on both type I collagen and hyaluronan production, as compared with no stimulated control. On the other hand, IL-10 and IFN- α caused a significant decrease in type I collagen production, and IL-8 and GM-CSF caused a decrease in hyaluronan production compared with no cytokine-treated control. Interestingly, some chemokines, such as MCP-1 (CCL2), RANTES (CCL5), eotaxin-2 (CCL24), IP-10 (CXCL10), or fractalkine (CX3CL1) significantly induced the type I collagen or hyaluronan production.

    Various growth factors and cytokines on the regulation of type I collagen and hyaluronan in human dermal skin probably function as key factors in skin remodeling and skin aging. Our profile may help to apply to cosmeceutical area maintaining as young skin through the increase in extracellular matrix.

These papers describes nearly all human recombinant cytokines they used in the study are purchased from Creative Biolabs.

  • Lopez-Rivera, Esther, et al. "Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2." Cancer research 74.4 (2014): 1067-1078. Abstract
    Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.

The paper describes iNOS was supplied by Creative Biolabs.

  • Cheng, Yong, Niamh X. Cawley, and Y. Peng Loh. "Carboxypeptidase E/NFα1: A new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways. " PloS one 8.8 (2013): e71578. Abstract
    Mice lacking Carboxypeptidase E (CPE) exhibit degeneration of hippocampal neurons caused by stress at weaning while over-expression of CPE in hippocampal neurons protect them against hydrogen peroxide-induced cell death. Here we demonstrate that CPE acts as an extracellular trophic factor to protect neurons. Rat hippocampal neurons pretreated with purified CPE protected the cells against hydrogen peroxide-, staurosporine- and glutamate-induced cell death. This protection was observed even when hippocampal neurons were treated with an enzymatically inactive mutant CPE or with CPE in the presence of its inhibitor, GEMSA. Purified CPE added to the culture medium rescued CPE knock-out hippocampal neurons from cell death. Both ERK and AKT were phosphorylated within 15 min after CPE treatment of hippocampal neurons and, using specific inhibitors, both signaling pathways were shown to be required for the neuroprotective effect. The expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), was up-regulated after hippocampal neurons were treated with CPE. Furthermore, hydrogen peroxide induced down-regulation of BCL-2 protein and subsequent activation of caspase-3 were inhibited by CPE treatment. Thus, this study has identified CPE as a new neurotrophic factor that can protect neurons against degeneration through the activation of ERK and AKT signaling pathways to up-regulate expression of BCL-2.

The paper describes the purified recombinant WT mouse CPE was custom generated by Creative Biolabs.

  • Lee, Cheol, et al. "The molecular profiles of neural stem cell niche in the adult subventricular zone." PloS one 7.11 (2012): e50501. Abstract
    Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.

The paper describes mouse CPE the author used in the study was expressed and purified by Creative Biolabs.

  • Telikicherla, Deepthi, et al. "Overexpression of ribosome binding protein 1 (RRBP1) in breast cancer." Clinical proteomics 9.1 (2012): 7. Abstract
    The molecular events that lead to malignant transformation and subsequent metastasis of breast carcinoma include alterations in the cells at genome, transcriptome and proteome levels. In this study, we used publicly available gene expression databases to identify those candidate genes which are upregulated at the mRNA level in breast cancers but have not been systematically validated at the protein level. Based on an extensive literature search, we identified ribosome binding protein 1 (RRBP1) as a candidate that is upregulated at the mRNA level in five different studies but its protein expression had not been investigated. Immunohistochemical labeling of breast cancer tissue microarrays was carried out to determine the expression of RRBP1 in a large panel of breast cancers. We found that RRBP1 was overexpressed in 84% (177/219) of breast carcinoma cases tested. The subcellular localization of RRBP1 was mainly observed to be in the cytoplasm with intense staining in the perinuclear region. Our findings suggest that RRBP1 is an interesting molecule that can be further studied for its potential to serve as a breast cancer biomarker. This study also demonstrates how the integration of biological data from available resources in conjunction with systematic evaluation approaches can be successfully applied to clinical proteomics.

The paper describes the author used two types of TMAs including one breast cancer progression TMA that was purchased from Creative Biolabs.

  • Jaganathan, Soumya, Peibin Yue, and James Turkson. "Enhanced sensitivity of pancreatic cancer cells to concurrent inhibition of aberrant signal transducer and activator of transcription 3 and epidermal growth factor receptor or Src."Journal of Pharmacology and Experimental Therapeutics 333.2 (2010): 373-381. Abstract
    Many molecular aberrations occur in pancreatic cancer. Although aberrant epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) are implicated in pancreatic cancer, therapies that target only one of these entities are undermined by signaling cross-talk. In the human pancreatic cancer lines, Panc-1 and Colo-357, pY845EGFR, pY1068EGFR, pY1086EGFR, and pY1173EGFR levels and pY416c-Src are concurrently elevated with aberrantly active Stat3 in a complex signaling cross-talk. Thus, understanding the signaling integration would facilitate the design of effective multiple-targeted therapeutic modalities. In Panc-1 and Colo-357 lines, pY845EGFR, pY1068EGFR, and pY1086EGFR levels are responsive to c-Src inhibition in contrast to pY1173EGFR, which is EGFR kinase-dependent. Constitutively active Stat3 is sensitive to both EGFR and Src inhibition, but the early suppression of aberrantly active Stat3 in response to the inhibition of EGFR and Src is countered by a Janus kinase (Jaks)-dependent reactivation, suggesting that Jaks activity is a compensatory mechanism for Stat3 induction. The inhibition of EGFR, Src, or Stat3 alone induced weak biological responses. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis and decreased the migration/invasion of pancreatic cancer cells in vitro. Significantly, the concurrent inhibition, compared with monotargeting modality, induced stronger human pancreatic tumor growth inhibition in xenografts. We infer that the tumor growth inhibition in vivo is caused by the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer.

The paper describes the recombinant human EGF used in this study is from Creative Biolabs.

  • Murthy S R K, Dupart E, Al-Sweel N, et al. “Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion.” Cancer letters, 2013, 341(2): 204-213. Abstract
    Carboxypeptidase E (CPE), a prohormone processing enzyme is highly expressed and secreted from (neuro)endocrine tumors and gliomas, and has been implicated in cancer progression by promoting tumor growth. Our study demonstrates that secreted or exogenously applied CPE promotes survival of pheochromocytoma (PC12) and hepatocellular carcinoma (MHCC97H) cells under nutrient starvation and hypoxic conditions, but had no effect on their proliferation. CPE also reduced migration and invasion of fibrosarcoma (HT1080) cells. We show that CPE treatment mediates survival of MHCC97H cells during metabolic stress by up-regulating the expression of anti-apoptotic protein BCL-2, and other pro-survival genes, via activation of the ERK1/2 pathway.

The paper describes recombinant mouse full length CPE custom expressed in HEK293 cells and purified by Creative Biolabs.

  • Bah, Germanus S., Vincent N. Tanya, and Benjamin L. Makepeace. "Immunotherapy with mutated onchocystatin fails to enhance the efficacy of a sub-lethal oxytetracycline regimen against Onchocerca ochengi." Veterinary parasitology 212.1 (2015): 25-34. Abstract
    Human onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, has been successfully controlled by a single drug, ivermectin, for over 25 years. Ivermectin prevents the disease symptoms of severe itching and visual impairment by killing the microfilarial stage, but does not eliminate the adult parasites, necessitating repeated annual treatments. Mass drug administration with ivermectin does not always break transmission in forest zones and is contraindicated in individuals heavily co-infected with Loa loa, while reports of reduced drug efficacy in Ghana and Cameroon may signal the development of resistance. An alternative treatment for onchocerciasis involves targeting the essential Wolbachia symbiont with tetracycline or its derivatives, which are adulticidal. However, implementation of antibiotic therapy has not occurred on a wide scale due to the prolonged treatment regimen required (several weeks). In the bovine Onchocerca ochengi system, it has been shown previously that prolonged oxytetracycline therapy increases eosinophil counts in intradermal nodules, which kill the adult worms by degranulating on their surface. Here, in an "immunochemotherapeutic" approach, we sought to enhance the efficacy of a short, sub-lethal antibiotic regimen against O. ochengi by prior immunotherapy targeting onchocystatin, an immunomodulatory protein located in the adult female worm cuticle. A key asparagine residue in onchocystatin was mutated to ablate immunomodulatory activity, which has been demonstrated previously to markedly improve the protective efficacy of this vaccine candidate when used as an immunoprophylactic. The immunochemotherapeutic regimen was compared with sub-lethal oxytetracycline therapy alone; onchocystatin immunotherapy alone; a gold-standard prolonged, intermittent oxytetracycline regimen; and no treatment (negative control) in naturally infected Cameroonian cattle. Readouts were collected over one year and comprised adult worm viability, dermal microfilarial density, anti-onchocystatin IgG in sera, and eosinophil counts in nodules. Only the gold-standard antibiotic regimen achieved significant killing of adult worms, a profound reduction in microfilarial load, and a sustained increase in local tissue eosinophilia. A small but statistically significant elevation in anti-onchocystatin IgG was observed for several weeks after immunisation in the immunotherapy-only group, but the antibody response in the immunochemotherapy group was more variable. At 12 weeks post-treatment, only a transient and non-significant increase in eosinophil counts was apparent in the immunochemotherapy group. We conclude that the addition of onchocystatin immunotherapy to a sub-lethal antibiotic regimen is insufficient to induce adulticidal activity, although with booster immunisations or the targeting of additional filarial immunomodulatory proteins, the efficacy of this strategy could be strengthened.

Creative Biolabs produced the recombinant mutated onchocystatin in Escherichia coli.

  • Ka-Yiu San, Hui Wu. “Intergrated biodiesel process.” US20140335578 A1. Nov 13 2014. Abstract
    Methods of using crude glycerol to make fatty acids are provided, as well as integrated methods of converting glycerol waste from biodiesel production into more biodiesel. Bacteria and other microbes engineered to produce free fatty acids from glycerol are also provided.

The paper describes YeastXceed™ Technology is available from Creative Biolabs.

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