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Phage Display & Antibody Library Services

Services Platform Cases FAQ Resources

Phage display is one of the most powerful and widely used laboratory technique for the study of protein-protein, protein-peptide and protein-DNA interactions. This technology is mainly based on displaying the interest protein (peptides, antibodies, scaffolds or others) on the surface of employing phage and then be used to interrogate the constructed libraries containing millions or even billions of displayed phages. Theoretically, phage display is an exogenous gene expression method which the gene encoding the interest protein is inserted into bacteriophage coat protein gene then displaying the interest protein on the phage surfaces, resulting in a connection between genotype and phenotype.

Creative Biolabs has long-term devoted to the development and application of phage display technology. With years of experience, our scientists have developed several phage display based platforms and tailored hundreds of particular libraries and thousands of specific antibody products to boost our global customers’ research and project goals. We are pleased to use our extensive experience and advanced platform to offer the best service and the most qualified products to satisfy each demand from our customers.

Phage Display & Antibody Library Solutions

Phage Display Library Construction
Phage Display Library Construction
  • Different types of phage display libraries: scaffold libraries, peptide libraries, antibody libraries, cDNA libraries and others
  • Able to tailor the most suitable phage display system (M13, T4, T7, or lambda) for the library construction
  • Large library capacity from 107 to over 1012 and high affinity from 10-7 to 10-9
Phage Display Library Screening
Phage Display Library Screening
  • Proprietary protocol and tailored biopanning strategies: solid-phase screening, solution-sorting screening, cell-based screening, in vivo and ex vivo screening
  • Successful antibody selection with an extremely high affinity of 10-12 by four rounds library screening
  • In-house premade antibody libraries, peptide libraries, scaffold libraries, etc.
Phage Display Library Screening
PTM Specific Antibody Discovery
  • Specific for post-translational modifications on proteins
Peptidome Discovery
Peptidome Discovery
  • Available for a broad range of peptidome sources
  • Advanced phage display platforms and comprehensive systems for qualitative or quantitative analysis
Anti-idiotype Antibody Discovery
Anti-idiotype Antibody Discovery
  • High specificity and sensitivity Abs against any targets of interest
  • Applicable drugs: monoclonal antibody, polypeptide, protein, compound, cell line, DNA and so on.
  • One-stop solutions from antibody development to preclinical research of therapeutic Abs
Agonistic & Antagonistic Antibody Service
Agonistic & Antagonistic Antibody Service
  • One-stop, customized service packages
  • Wider applications and combination therapy strategies for severe diseases and malignant cancers
  • Extensive experience in antibody discovery, development, and manufacture
  • Dedicated devices, advanced technologies, and powerful platforms

Comprehensive Tech Platform

Phage Display Platform
Phage Display Platform

Phage display has been well-recognized as a pivotal tool in immunological and cellular biological research due to its remarkable contribution to the development of new drugs and vaccines. Furthermore, phage display platform is an equally important tool for the development of vaccines and the generation of diagnostic tests for monitoring disease progression and evaluating treatment efficacy.

Cases

Phage Display Selection

Polyclonal phage ELISA of the five different panning rounds. (Andreas H. Laustsen, 2022) Fig. 1 Polyclonal phage ELISA of the five different panning rounds. (Andreas H. Laustsen, 2022)

The client used our two random linear peptide libraries, TriCo-16 Phage Display Peptide Library and TriCo-20 Phage Display Peptide Library for phage display selection. Fig.1 is polyclonal phage ELISA of the five different panning rounds performed in the phage display selection experiment against myotoxin II.

Antibody Discovery

C5H9v2 mAb is a high-affinity blocker of PD-L1 and can potentiate T-cell responses. (Hikmat H. Assi, 2021) Fig. 2 C5H9v2 mAb is a high-affinity blocker of PD-L1 and can potentiate T-cell responses. (Hikmat H. Assi, 2021)

The client identified PD-L1 blocking antibodies from a fully human single chain variable fragment (scFv) phage display library contracted with Creative Biolabs using recombinant His-tagged human or mouse PD-L1 extracellular domain.

References
  1. Laustsen AH, et al. In Vivo Neutralization of Myotoxin II, a Phospholipase A2 Homologue from Bothrops asper Venom, Using Peptides Discovered via Phage Display Technology. ACS Omega. 2022 Apr 25;7(18):15561-15569. doi: 10.1021/acsomega.2c00280.
  2. Assi HH, et al. Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents. Cancer Immunol Res. 2021 Dec;9(12):1451-1464. doi: 10.1158/2326-6066.CIR-21-0031.

FAQ

Antibody Development by Phage Display Technology - Frequently Asked Questions

• Phage Display - General Questions[↑Top]

  1. Q: What is phage display?

    A: Phage display is a selection technique in which a library of variants of a peptide or protein is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside. This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called "biopanning". Biopanning is carried out by incubating the pool of phage-displayed variants with a target of interest that has been immobilized on a plate or bead, washing away unbound phage, and eluting specifically bound phage. The eluted phage is then amplified in vivo and the process repeated, resulting in stepwise enrichment of the phage pool in favor of the tightest binding sequences. After 3-4 rounds of selection/amplification, individual clones are characterized by DNA sequencing.

  2. Q: What are the advantages of phage display screening over other antibody development method?

    A: In comparison with hybridoma technology, phage display technology offers great advantages. Hybridoma method is only available in mouse, rat, hamster and guinea pig. On the other hand, phage display could be used to generate high-affinity monoclonal antibodies from all popular antibody production species, including but not limited to human, mouse, rat, rabbit, chicken, llama, camel, alpaca, cow, dog, sheep, monkey, and shark. Hybridoma-based monoclonal antibody development can only generate a small number of antibody candidates against a particular immunogen at a time; whereas phage display technology can present the entire antibody repertoire (e.g. 108) of an immunized animal, in which almost 10% of the antibodies are immunogen(s)-specific. With such a huge pool of potential binders, the chance is much better to use phage display technology to discover antibodies of the desired properties. In addition, using hybridoma technology, it is hard to incorporate an enriching step that can selectively isolate antibodies with the desired functionality. In most cases, all the hybridoma clones are produced first and then validated one by one. In contrast, phage display technology allows various enriching strategies: antibodies of desired properties can be enriched thus those without the desired functionality can be excluded from further validation. For example, antibody library screening can be done using the target to capture strong binders while using the controls to block/deplete cross-reactive binders. In summary, this immune antibody library approach allows collecting almost all antibodies in the animals and isolating the strongest binders from the collection. Moreover, counter-selection can be easily incorporated.

  3. Q: What are the difference among M13, T7, and T4 displaying system?

    A: M13 filamentous phage has always been the most popular option and extensively used in various types of research, among many other types of phages. The viral coat consists of five different capsid proteins, including one major capsid pVIII (2,700 copies) and four minor capsids (pIII and pVI at one end while pVII and pIX at the other end). Unlike T4 and T7, M13 is lysogenic phage which is assembled in the periplasm and secreted out of the bacterial membrane without lysing the host.

    The multiple capsid proteins on M13 phage allow the comprehensive display choices for a variety of peptides and proteins with distinctive characteristics. All five capsids have been successfully used for foreign domain display with unique vectors. pIII and pVIII are mostly the preferred choices for M13 phage display.

    Bacteriophage T4 is distinct from M13 in many aspects. T4 has much larger size, tailed structure and double-stranded DNA (dsDNA) genome encoding 50 different proteins. Larger genome DNA allows larger insertions of foreign proteins. Two different domains could be displayed on HOC and SOC, the two non-essential coat proteins. Both N- and C-terminal insertion are available. Without membrane secreting process, host toxicity and confirmation changes could be avoided using T4 phage.

    T7 phages have a shorter lifecycle compared to filamentous phages and lambda phages. The assembly of the progeny phages are in bacterial cytoplasm and released by lysis of cell envelope, therefore there is no size limitation and host toxicity resulting from the secreting process. In addition, T7 phages are very stable at extreme conditions where other phages could not survive.

  4. Q: Can I use M13 phage display system to construct a cDNA library?

    A: Usually, M13 is not suitable for cDNA expression, because the in-frame expression between the leader sequence (required for secretion) and the N-terminus of coat protein pIII or pVIII is required for M13 phage display. In order to fuse the corresponding protein to the coat protein properly, an insert must be in the correct reading frame at both ends and contains no in-frame stop codons. This results in a vanishingly small number of productive clones in M13 cDNA libraries.

  5. Q: What are the differences between pIII and pVIII display?

    A: Minor coat protein pIII and the major coat protein pVIII proved most effective for the display of proteins and peptides.

    pIII could incorporate and present larger inserts compared to pVIII, it is the scaffold of choice in most cases of ORF and protein display. There are 5 copies of pIII protein on the phage virion. Both short peptides and large protein could be fused to pIII. The fusion protein will be present at low valency, 1-5 copies, therefore beneficial in selecting high-affinity binders.

    pVIII (encoded by genes VIII) is the major structural protein of the virus. Approximately 2,800 copies of pVIII are required to coat one full-length wild-type virion. Therefore, pVIII has high copy number and multivalent display. Multivalent display reduces selection for this property, by greatly increasing avidity (effective affinity) to the point where weak and strong ligands cannot be distinguished. On the other hand, multivalency could be a distinct advantage in other applications. Such is the case when it is desirable to accumulate a broad spectrum of peptides as potential leads; the identified peptides can be ordered subsequently in accordance with their affinity and selectivity using monovalent display. In pVIII display systems, there is a sharp limit on the length of the displayed peptide. The pVIII display systems will only accommodate very short peptides, ~100 amino acids, especially in high DNA copy number systems. the pVIII display system is able to display 300-500 copies of the pVIII fusion peptide/protein.

    Creative Biolabs is an expert on both pIII and pVIII display systems, we’ll work closely with you to select the optimal system for your projects.

  6. Q: Do I need an import permit for a phage?

    A: No, phages are considered non-pathogenic to humans, animals or plants.

  7. Q: What media can be used to culture the phage?

    A: TSB/TSA (trypticase soy broth/trypticase soy agar) can be used to grow most of the phages. However, the appropriate media can be various due to different phage display systems.

  8. Q: What is the molecular weight of the coat protein pIII?

    A: For the unprocessed pIII (with a leader sequence but no displayed proteins or peptides), the molecular weight is 44,651 Da; for the mature pIII (without the leader sequence), it is 42,579 Da. However, pIII usually shows an apparent molecular weight of 60-65 kDa by SDS-PAGE, due to the unusual glycine-rich spacer regions between the domains of the protein.

  9. Q: Could I display antibody fragments or proteins at the N-terminus of coat protein pIII with M13KE?

    A: M13KE is not amenable to antibody or protein expression, because in this system each copy of pIII displays the encoded peptide sequence. Any insert that interferes with phage particle assembly or pIII function will not make viable phage. We consider 25-50 amino acids to be the maximum range for this system.

  10. Q: What kinds of E. coli strains can be infected by bacteriophage M13? Does it have the chance to contaminate all cell strains in our laboratory?

    A: The M13 phage is a kind of filamentous bacteriophage, which uses the tip of the bacterial F conjugative pilus as a receptor to facilitate the infection process. Thus, they are only specific for E. coli strains containing the F plasmid (F+). For your strains in your lab, we suggest you check whether they contain the F plasmid. If they don’t, the M13 phage cannot infect them.

  11. Q: Is the phagemid vector used for the construction of custom libraries available?

    A: The vectors used for constructing all phage display libraries are available for sale, for research use only. However, the full sequence of the vector is private, we can only provide the schematic map, the primer, and the restriction enzyme information to you for PCR and DNA sequencing.

  12. Q: Is there any antibiotic resistance for E. coli TG1 host strain?

    A: E. coli TG1 strain does not contain antibiotic resistance, while the phage-infected TG1 strain can be selected by 2YT-AK media.

  13. Q: Could the helper phage be titrated by blue-white screening?

    A: The helper phage provided by Creative Biolabs does not contain lacZα but carry the KanR gene for antibiotic selection, thus cannot be distinguished through blue-white screening.

  14. Q: What is the difference between pfu and cfu?

    A: The pfu, refers to the plaque-forming unit, is a measure of the quantity of individual infectious particles, and is usually used to count bacteriophage.

    The cfu, refers to the colony-forming unit, is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell, and is usually used to count bacteria (e.g. E. coli).

  15. Q: How many clones are provided following binding/screening tests? Are these clones sequenced?

    A: In the clone validation stage, 40-300 clones will usually be tested (more clones could be validated upon request). We can perform phage ELISA, DNA sequencing, and soluble protein ELISA to confirm the activity. Other validation tests can also be performed upon request. Usually > 3-10 unique clones could be discovered. Our customer can be the sole owner of these identified clones.

    At this time, we are proud to offer an advanced high-throughput antibody discovery platform, Magic™ Therapeutic Antibody Discovery Platform (MTADP). After the library screening, MADTP could identify all the unique antibody sequences, with VH-VL pairing information, in the enriched library (~105). While using ELISA, only a very small portion, 40-300 clones, will be analyzed. It's very important to have a large number of antibody candidates if we want to obtain good therapeutic/diagnostic antibodies. Therefore, MTADP is highly recommended as there is a much better chance to identify functional antibodies from a large pool of antibody candidates.

  16. Q: Can we ask supernatant or a small amount of purified antibodies for internal test?

    A: After the screening, a number of random clones will be validated. The supernatant of the positive clones can be provided to the customers as an additional deliverable. We also provide purified scFv/Fab/IgG production services upon request.

  17. Q: Are anti-M13 antibodies available?

    A: Yes, we can offer the anti-M13 antibodies. We can offer the Recombinant Anti-M13 Major Coat Protein Antibody (CBMAB-0206MC) or other custom anti-M13 antibodies of your choice. Please contact our customer service for more details.

  18. Q: Is there any discount for a large number of projects?

    A: Yes, we can offer volume discount for a large number of projects. Please contact our customer service for more details.

• Panning and Screening Related Questions[↑Top]

  1. Q: Which kinds of screening methods can be offered?

    A: There are many different types of screening method can be offered, such as screening using antigens in solution, screening using immobilized antigens, screening on whole cells, and in vivo screening. The success of a project often depends on the method used for screening. We will work with you to determine the best screening method for your project.

  2. Q: What would be the steps of a screening? If everything goes well, when could we expect the antibody?

    A: Usually, for the library screening step, we need to perform 3 to 4 rounds of biopanning to get a good and specific enrichment for the target protein. For naïve libraries, enrichment is usually not observed after the first two or even three rounds of selection; in the fourth round, typically, the enrichment can be found. However, for those based on a pre-existing ligand, enrichment may be observed sooner.

    At the end of each screening, monoclonal phage ELISA and binder phages validation will be performed: a series of individual clones will be validated to select positive binding clones. DNA sequencing will then be performed to the positive clones and identify the final DNA sequences of the unique antibody binders. After that, the selected antibody fragments can be expressed as a phage-free format to further test their binding specificity through the QC soluble ELISA.

    Typically, it will take approx. 3 months to finish the whole screening project, which may vary depending on the complexity of the project. A progress report will be sent to you when a minor achievement is reached or a decision needs to be made by the client, such as the completion of the biopanning, the result for the clone validation.

  3. Q: Which genetic elements that can be used for screening displayed phage against environmental phage?

    A: The display vector contains AmpR. Please use 2YT-G media to culture the phage-infected cells. Also, the M13KO7 helper phage contains KanR, which is necessary for phage packaging. After adding helper phage, the 2YT-AK media can be used to select the desired phage.

  4. Q: Which plate do you recommend for the panning procedure?

    A: For solid-phase screening, almost all the commercial polystyrene plate can carry the task. While if plate-based in-solution screening is preferred, the streptavidin-coated plate is required. Using streptavidin-coated beads is another option for in-solution screening.

  5. Q: How many phages should be used for each round of screening?

    A: The number of phage particles should be around 100× greater than the library size (e.g. 1012 pfu for a library of 1010 clones). Generally, 100 μL library (1012 pfu/mL) per well is used for one panning. This volume can be adjusted for different panning strategies. The titer of the phage library offered by Creative Biolabs is ~1013 pfu/mL. To prepare 1012 pfu/mL, simply dilute 50 μL of the library with 200 μL PBS, and then mix with 250 μL of the 4% PBSM (PBS containing 4% milk).

  6. Q: What is the enriching factor for each round?

    A: Regarding the enriching factor, it represents the ratio of clones eluted from the target coated well to the input. The lower enriching factor the better enrichment. By monitoring the enriching factor, the progress of the selection process can be tracked. For naïve libraries, enrichment is usually not observed during the first two rounds of selection; while after the third or fourth round of screening, about 5-10 folds lower enriching factor can be expected compared to the former rounds of screening to indicate a good enrichment. For immunized libraries or those based on a pre-existing ligand, enrichment may be observed sooner.

  7. Q: After each round of panning, how can I calculate the phage titer correctly? Will the helper phage affect the accuracy of counting? How many helper phages should be added for each panning?

    A: The pfu of input or output phages can be calculated by tittering the phage through infecting TG1 cells with serial dilutions (10-6, 10-8, 10-10, 10-12) of phage stock, plating to 2YT-AG, incubation, and enumeration of the numbers of AmpR colonies that appear. Calculate titer of phage as pfu/mL. The recombinant phages contain the AmpR gene, but helper phages do not and cannot grow on the 2YT-AG plate.

  8. Q: How many helper phages should be added for each panning?

    A: For each panning, add helper phage to a final concentration of 5×109 pfu/mL for the phage packaging.

  9. Q: Do I need to culture TG1 in the M9 medium before each panning step? Is it essential for my experiment?

    A: The TG1 offered in our kit is derived from colonies grown on a plate of M9 minimal agar. It is to ensure the expression of the F pilus, as this is required for phage infection. There is no need to culture them on the M9 minimal plate again before each panning step.

  10. Q: Are there any other details not specified in the protocol of the premade phage library that you feel important for successful panning?

    A: Here are several points that should be noted about the panning protocol.

    1) The stability of Fab and scFv on the surface of the phage is reduced over time. Use freshly prepared phage Abs for panning to maximize the number of phages that bind during panning: this applies to the use of both library phage and phage amplified during panning.

    2) Simple variations in the standard panning protocol, such as changing the constituents of buffers, the washing conditions, and Ag concentration can reduce the levels of nonspecific phage binding and/or the degree of specific phage Ab binding.

    3) The optimal coating concentration for each Ag (coating buffer, concentration, and temperature) should be determined prior to panning, using an appropriate ELISA. The panning protocol described suggests a single optimal coating concentration be used over all rounds of panning (dependent on the Ag being used). Not all approaches to panning maintain a single concentration. Mathematical models of the panning process have demonstrated that the concentration of Ag can influence the selection of Abs. For example, high concentrations of Ag increase the possibility of enriching for low-affinity Abs or Abs that exist at low frequencies in the library. With this in mind, panning strategies using high, low, or varying concentrations of Ag could be used to isolate Abs found at a particular frequency in the library or with a specific affinity.

• Premade Phage Library Related Questions[↑Top]

  1. Q: What should I do when I receive the premade phage library?

    A: Grow the supplied bacteria first. You can keep the phage at 4°C for a few days. For long-term storage, move the kit to -80°C.

  2. Q: Should I store the phage library at -20°C, or at 4°C? How long can it be stored without reducing the complexity?

    A: Phage particles can be stored in an infectious state for one month at 4°C or one year at -80°C. They are less stable at -20°C than at -80°C. Thus, for long-term storage, you can move the kit to -80°C.

  3. Q: What kinds of libraries are offered by your premade phage library kit? What is the helper phage used for?

    A: Two forms of libraries were offered in this kit, one is in phage particle and the other one is in phagemid (RF DNA). The supplied phage particles are the recombinant fully infectious phage particles, which can be used for screening directly. The RF DNA is offered only for the sequencing validation of this library. After each round of screening, the helper phage can be used to amplify the output phages.

    For the offered premade library, antibodies are displayed using plasmid-based "phagemid"-helper phage system. After infecting the host cell, the phagemid only replicates in the host as plasmids and cannot be packaged into a phagemid particle, or recombinant phage, without infection with a helper phage. The helper phage provides the genes for the production of the structural, the packaging and assembly proteins needed for phage morphogenesis. Because helper phage carries mutations in the origin of replication or packaging sequences, the phagemid genome is packaged more efficiently than the helper phage genome during replication. Phagemids transformed E. coli host co-infected with helper phage resulted in the assembly of fully infectious phage particles.

  4. Q: The phage library kit can be used for 10 biopanning projects, shall we aliquot it before panning?

    A: Yes. This kit can be used for 10 independent biopannings. For each panning, we suggest add 1012 pfu phage particles (100 µL of the phage preparation) for the first round of screening. As we offered 1 mL (~1.0×1013 pfu/mL) phage preparation to you, you can aliquot them and stored at -80°C for long-term storage.

  5. Q: Which premade libraries are available for isolating monoclonal antibodies from human antibody libraries?

    A: We have following human antibody libraries, which have a great diversity to be able to derive high-affinity antibodies with affinity ranging from 10 pM to 10 nM.

    HuScL-2: Human Single Chain Antibody Library
    HuFabL-1: Human Fab Antibody Library
    HuScL-3: Human Single Chain Antibody Library
    HuFabL-2: Phage Display Human Naïve & Semi-synthetic Fab Phage Display Library
    HuScL-5: Human Synthetic scFv Library
    HuScL-4: Hunan Naïve scFv Library


  6. Q: In your premade phage display libraries, are the scFvs or Fabs from the same clone expression, just posttranslational digested isoforms?

    A: We have various scFv and Fab libraries, including naive, semi-synthetic and fully-synthetic libraries. They are from different sources.

  7. Q: What's the typical affinity of a custom antibody?

    A: To screen the premade/immune phage display antibody library we can identify antibody with nM to pM range. In addition, we provide high-quality affinity maturation service, we were able to increase antibody affinity ranging from 1.0 nM-20 nM to 10 pM-100 pM.

  8. Q: Can the libraries be used to screen against whole cells?

    A: Yes. Creative Biolabs has extensive experience with cell-based library screening. Cell panning procedure is well established, which allows the selection of phage-displayed antibody library directly on intact cells. For many cell surface antigens, their physiological conformation can only maintain in the form of whole cell and these antigens are very attractive for therapeutics or imaging. Some cell surface proteins could not be expressed and purified in vitro. This strategy overcomes the obstacle of the acquisition of the recombinant antigens. Furthermore, it could be used to select antibodies against novel epitopes which are created by disease-related over-expression or modification.

  9. Q: Can your premade libraries be used for in vivo screening?

    A: Yes. Our premade peptide libraries have been used for selecting organ-specific peptides in vivo successfully.

  10. Q: Is it possible to re-amplify the library in bacteria and obtain additional copy of the bacterial library for the future experiments?

    A: We suggest you not re-amplifying the library, because re-amplification will reduce the diversity of the primal phage library obviously.

  11. Q: Regarding the soluble antibody expression, in your manual, HB2151 is used for periplasmic soluble expression. Do you have any special reasons for using HB2151? Have you ever used other hosts for this purpose? Is it possible to use SS320 instead of HB2151?

    A: There is an amber stop codon TAG between the antibody and the gIII gene. Therefore, for the antibody soluble expression, we need to use non-suppressor bacterial strains, in which the amber stop codon can be recognized and the antibody can be expressed in a soluble pIII-free form and secreted into the periplasm. HB2151 and TOP10 are the most commonly used. Since the SS320 is also a non-suppressor strain, which is constructed by transferring the F’ episome from XLI-Blue (Stratagene) to MC1061 (Bio-Rad), it is able to be used for the antibody periplasmic soluble expression.

  12. Q: Should I grow the phage at 37°C or 30°C for use with your premade library kit?

    A: Usually, the optimal temperature of growing the phage depends on the growth temperature of the host bacterium. For each of our premade phage library, the suggested growth temperature is indicated in the manual.

  13. Q: For the library kit component of "Phage library as plasmid", is it provided for transformation to produce the library? However, there is not any protocol for this purpose. How can we do this?

    A: The library plasmid is offered only for performing the sequencing validation of this library (through sequencing these plasmids, the diversity of the library should be known), but not for transformation to produce the Fab library again. Moreover, the amount of the plasmids included in the kit was not enough for library construction. Thus, we do not provide the protocol for library plasmid transformation in the manual.

  14. Q: We are trying to concentrate the phage library that we obtained. Can we directly infect TG1 cells with the library that we obtained?

    A: If you want to increase the concentration of the phage particle, we suggest you precipitate the phage library directly with phage precipitant (PEG/NaCl: 20% (w/v) polyethylene glycol-8000, 2.5 M NaCl) and re-suspend the pellet with a small volume of PBS. DO NOT amplify the library by infecting the TG1 cells, because the amplification will reduce the diversity of the primal phage library dramatically.

  15. Q: When I am expressing and purifying antibody fragments in pCDisplay 4, can I use anti-Histidine antibody instead of anti-HA antibody for detection of the protein?

    A: Yes. There are two tags in the pCDisplay-4, one is 6x His, and the other is HA. Both of these two tags can be used for the antibody purification and detection.

  16. Q: Could you provide us a detailed vector map (with sequences) of the phagemid used together with the library inserts?

    A: The full sequence of this vector is private, we can only provide the schematic map, the primer, and the restriction enzyme information to you for PCR and DNA sequencing.

  17. Q: Could you provide the size of the amplicon of the pCDisplay-5 vector to us?

    A: The size of the pCDisplay-5 vector is 4.8 kbp. L1 and S6 primer can be used for colony PCR and phagemid DNA sequencing, L1 is the forward primer, and S6 is the reverse primer. The size of the PCR product should be 660 bp.

  18. Q: Since the phagemid is offered in 75% ethanol, do you recommend isolating the plasmid and re-suspending it in water for PCR?

    A: In order to protect the plasmid from degradation, we deliver the plasmid in 75% ethanol. Before the PCR, you can precipitate the plasmid by centrifuging the tube at 20,000g for 5 minutes at 4°C to pellet the DNA, then discard the supernatant, and vacuum the DNA pellet for 5-10 minutes. Because the quantity of the plasmid is only 10 µg, it’s quite normal that you do not see any pellet after the centrifugation. We suggest adding water (10-20 μL) directly and re-suspending the DNA by passing the solution up and down several times through a pipette tip.

  19. Q: Is the phagemid vector of pCDisplay-3M applicable for direct periplasmic expression of the sdAb from selected clone (amber stop codon or similar strategy), and if so, what tag is present?

    A: Yes. The pCDisplay-3M can be used for direct periplasmic expression of the sdAb. There is an amber stop codon TAG between the sdAb and the gIII gene. For the sdAb soluble expression, we need to use non-suppressor bacterial strains, such as HB2151 or TOP10, in which the amber stop codon can be recognized and the sdAb can be expressed in a soluble pIII-free form and secreted into the periplasm. Furthermore, the 6×His tag will be involved to the C-terminal of the sdAb.

• Antigen Related Questions[↑Top]

  1. Q: What do I need to send with my antigen? Do I need to ship my antigen on dry ice? How are you currently storing your protein?

    A: Please include an order form as well as the concentration of the antigen and the buffer component. The paper documents are preferable in a zip-lock bag. You only need dry ice if the storage conditions are -80°C. If the protein is stable, ice packs are fine. Please ship it standard overnight via FedEx or a similar carrier.

  2. Q: Do I have to cleave the tag from my recombinant protein before sending it to you?

    A: Larger tags (GST, MBP, etc.) should be cleaved as they can interfere in specific antibody production. Small tags such as His, Myc, and FLAG do not need to be cleaved prior to antibody production.

  3. Q: What factors will affect an antigen?

    A:
    Antigen type:

    Approach will be selected depending on the target properties (specific epitope, cross-reactivity, how conserved it is, etc.), the end use of the antibody and other specific antigen information. Please contact us to discuss your specific antibody goals so we can suggest the best approach possible.

    Antigen size:

    Haptens and peptides require coupling to carrier proteins to be immunogenic. Antigens greater than 10 kDa are considered immunogenic.

    Antigen purity:

    The immunizing antigen should be the highest purity available. Greater than 90% purity is suggested.


  4. Q: How long should my peptide be?

    A: The length of 12-25 amino acids is recommended as the best length of the peptide. Because the length of 10-12 amino acids is the standard size as based on a B cell epitope. The exception to this length requirement occurs in the case of antibodies to phosphor-specific sites or other modification-specific sites. Phosphorylation of a single residue produces minor conformational changes in an epitope. Peptides of 8-12 amino acids with the modification centrally located are selected to enhance the production of phosphor-specific antibodies.

  5. Q: Which form should my antigen be in, for immunization and screening?

    A: For immunization, if the antigen is soluble we prefer a minimum concentration of 1 mg/mL in a sterile neutral buffer (with no additives) for immunization purposes. Please ensure that your sample does not contain anything harmful/toxic, for example, urea, formalin, sodium azide, glycerol, extreme pH, high salt or high concentrations of imidazole. If you are unsure, please contact us for detailed information. If the antigen is lyophilized, please provide the protocol for reconstitution.

    For using solution-sorting screening strategy, the buffer should avoid containing primary amine (e.g. Tris).

  6. Q: My antigen is a fusion protein. Is this a problem?

    A: Fusion proteins are added for ease of purification or to increase the immunogenicity of the target, but there is a good chance that antibodies will be made against the large tags (GST, MBP, etc.). Please cleave the larger tags. Small tags such as His, Myc, and FLAG do not need to be cleaved prior to antibody production.

  7. Q: What chemistry do you typically use for carrier protein conjugation?

    A: KLH is a straightforward, highly efficient and stable chemistry that will not affect the specificity of the resultant antibody. We recommend adding a cysteine to one of the termini of your peptide (or designing the peptide to use one that is natural) and using the free sulfhydryl group to conjugate to KLH. Several alternative conjugation methods are also available, but only recommended when you have internal or multiple cysteines in your antigen.

  8. Q: I have synthesized my peptide already; can I send it to you for antibodies production?

    A: Yes, Creative Biolabs is one of the well-recognized experts who are professional in antibody production. Please provide a minimum of 10 mg of your lyophilized peptide for KLH conjugation and contact us for a quote.

  9. Q: How many antigens do you need for a custom project?

    A: The quantity of immunogens depends on various factors, such as the type and number of animals used, and the screening strategy. A general guideline would be at least 2.5 mg for small animals and 5 mg for large animals. Of note, if you do not have enough antigen at the moment, you could always send us what you have to initiate the project and send us more later. Please contact us for more detailed instructions according to your specific project.

  10. Q: What's the purity of the antigen we should provide?

    A: In the most general terms, peptide antigens and protein antigens need to be around 90% purity or greater. The immunogens with high purity will induce precise immune response in the host. The impurities have chance to be immunogenic, therefore generating non-specific antibodies. Please note that the requirement of the antigens may vary upon different projects. Please feel free to contact us if you have antigens with a lower purity, we can still get high-quality antibodies with a custom strategy.

  11. Q: What type of antigen should be used for a custom project?

    A: We can use various types of antigens for antibody development, including but not limited to recombinant proteins, antibodies, peptides, whole cells, DNAs, and haptens. We will work together with you, determining the most appropriate form of antigen for your project.

    DNA: For antigens that are hard to be expressed, DNA immunization will be a good option. With the information of the sequence of your target, we'll be able to construct the plasmid encoding the target and use it for immunization.

    Proteins/Peptides: Proteins and peptides are the most commonly used immunogens. We also provide peptide conjugation service if your peptide is not conjugated to the carrier protein.

    Whole cells: The cell line overexpressing the target protein could be used as immunogen. A control cell line without expressing the target protein is also needed. It's specifically suitable for novel antibody discovery against membrane protein (extracellular domain) and the biomarkers on tumor cell lines.

    Haptens: Due to the small size and simple structure, haptens may need to couple with a carrier molecule to increase immunogenicity. We are able to conjugate the hapten with appropriate carrier protein including keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin (BSA), to dramatically elevate immune responses.

  12. Q: What is the smallest size of the antigen that could induce an immune response?

    A: The smallest size of the antigen that could produce immune response depends on the intrinsic property of the protein. We have successfully used a 4 kDa protein for immunization.

  13. Q: Do we provide the antigen/target protein for the screening and if so, how many proteins are required?

    A: If you do not have the target protein, we have antigen expression service which could provide the high-quality antigen. If you have the protein, 1.5 mg would be enough for the regular screening and clone validation experiments.

• Immune Related Questions[↑Top]

  1. Q: How does Creative Biolabs treat their immune?

    A: Creative Biolabs follows Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) published guidelines on antibody production. Creative Biolabs works with OLAW assured institutes for animal experiments. All immunes at Creative Biolabs are treated with respect and enrichment programs are in place to ensure that immune can play and learn. Toys, treats, and nesting materials are provided. When applicable, they also receive human contact and affection on a daily basis.

  2. Q: How do you test the immune effect?

    A: The serum collected before immunization and after the immunization will be tested for reactivity against the immunogen using ELISA. The immunogen is coated onto an ELISA plate, followed by serial dilutions of antiserum that contain the antibody. We can also provide the antiserum for our customers' internal assays.

  3. Q: Will you do test bleeds during the immunization?

    A: Yes, we do test bleeds before the immunization and after the 2nd or 3rd injection. The immune serum will be titrated by indirect ELISA to assess immune response. The serum can also be shipped to clients for evaluation in the client’s specific assays before terminating the animals.

  4. Q: What is the expected titer for a good immunization?

    A: We expect a titer at >10,000. The higher the titer, the better chance of obtaining high-affinity antibodies. We have ever discovered excellent antibody through a titer at 6,000.

  5. Q: What do pre-immune serum contribute to?

    A: Pre-immune serum is a great negative control for a wide variety of experiments.

  6. Q: How long does it take for immunization?

    A: Typically, the immunization takes 2-3 months. The exact schedule depends on the strength of the immune response arose in the host. Additional boosts may prolong the schedule.

  7. Q: Is rabbit monoclonal antibody better than mouse monoclonal antibody?

    A: The typical affinity of rabbit monoclonal antibody is 10-100 times higher than that of mouse antibody. Rabbit monoclonal antibody is highly recommended in the cases as following: 1) Need excellent performance in IHC or WB; 2) Detection of targets with low expression level; 3) Develop ELISA assays with other mouse monoclonal antibodies.

  8. Q: Why do I need two rabbits?

    A: Every animal responds differently to immunization and often immune make different subsets of antibodies to a single antigen. Immunizing two immunes increases the odds of one of the immune providing antibodies with high reactivity and specificity to your protein of interest.

  9. Q: What strain(s) of mice should I use?

    A: Most customers choose to use BALB/c mice. Swiss Webster and A/J mice are also regularly available.

  10. Q: What species are used for making antibodies?

    A: Creative Biolabs can use all popular antibody production species to make monoclonal antibodies, including human, rabbit, chicken, llama, camel, alpaca, cow, dog, mouse, rat, sheep, monkey, and most recently shark.

  11. Q: What species of the antibodies could be humanized?

    A: We have humanized antibodies from many species, such as mouse, rabbit, chicken, canine, and llama.

  12. Q: What species should I choose for my antibody production?

    A:

    Animals Properties
    Mouse Advantages
    1. Lower feeding cost
    2. Ease of maintenance and handling
    3. Strong ability to reproduce
    4. Inducing a good immune response to a great range of antigens
    Disadvantage
    Small amount of serum
    Rat Advantages
    1. Diverse antibody isotypes: IgA, IgD, IgE, IgG and IgM antibody isotypes
    2. Inducing the immune response to mouse antigens
    3. Reduced antigen requirement
    4. No cross-reaction in immune-detection of antigens out of a mouse background
    Disadvantage
    Small amount of serum
    Rabbit Advantages
    1. Can recognize epitopes conserved between rodent and human antigens that are invisible to rodent monoclonal antibodies
    2. Lower feeding cost
    3. Ease of maintenance and handling
    Disadvantage
    Medium amount of serum
    Chicken Advantages
    1. Higher avidity: most mammalian proteins exhibit enhanced immunogenicity in chickens than in mammals due to phylogenetic distance, and thus raise antibodies of higher avidity.
    2. Higher specificity: compared with mammalian IgG, chicken IgY has less cross-reactivity with mammalian proteins other than the immunogen.
    3. Lower background: IgY and IgG are structurally different in the Fc region; IgY does not bind to IgG Fc receptors and causes less false positive staining.
    4. High yield: very low quantities of antigen are required to obtain high and long-lasting IgY titer in the egg yolk; chickens lay eggs regularly, providing a continual source of IgY antibody.
    Disadvantage
    Few antibody isotypes
    Goat Advantages
    1. Distant from human and rodents
    2. Large amount of antiserum, 7-8 times to that of small animal individuals
    3. Suitable for large-scale production
    4. High stability
    Disadvantages
    1. High cost
    2. Long period of immune cycle
    Guinea pig Advantages
    1. More sensitivity
    2. Generating stronger antibody response to some antigens that are high homology with humans, mouse, and rats
    3. Offering novel solutions for some specific targets
    Disadvantages
    1. High cost
    2. Long period of immune cycle
    Camel Advantages
    1. Producing novel antibodies only with heavy chains
    2. Increased functional size of immune libraries
    3. High physicochemical stability
    4. Facile production of multivalent formats
    5. Rapid tissue penetration, fast clearance
    6. Well expressed
    Disadvantages
    1. Relatively longer period of immune cycle
    2. Due to the single domain nature of the antibody, it could be a disadvantage for small antigens, such as hapten

• Phage-derived Antibody Related Questions[↑Top]

  1. Q: Can the antibodies be converted to IgG?

    A: Creative Biolabs can convert Fab/scFv into full-size IgG with no difficulty. In addition, we provide recombinant IgG production in mammalian cells.

  2. Q: How do I store my antiserum/antibody?

    A: Serum could be stored at -20°C for up to one year, or 4°C for less than a month. For longer-term storage, add 0.05% azide and freeze. Purified antibody is most stable when stored at -80°C and multiple freeze-thaws should be avoided. Alternatively, you can add glycerol to a total concentration of 30-50% and store the antibody at -20°C.

  3. Q: How to produce the antibody using the selected phage clone?

    A: The vector (with weak promoter) used for phage display is not suitable for antibody expression directly. The solution is clone the antibody cassette into another expression vector with strong promoter, such as T7.

    Please ensure the removal of the gene III sequence during the amplification of antibody sequence, the gene III is used for phage display.

  4. Q: Can you generate DNA-specific antibodies?

    A: Yes, antibodies can be raised against specific and predetermined DNA sequences. Our advanced screening strategies are designed to differentiate the target nucleotide sequences and the non-specific sequences.

  5. Q: After antibody phage display screening, if we obtain many high-affinity clones, are those clones belong to us?

    A: We can work with a fee-for-service payment model, so all the selected clones belong to our customer.

  6. Q: In your experiences, are human scFvs/Fabs immunogenic to human immune system? Immunogenic could be a problem for us.

    A: There is a possibility of immunogenicity of the human scFv/Fab. However, comparing to antibodies from other species, the fully human antibodies are the ones with the least immunogenicity.

  7. Q: How high the affinity would be a good binder for scFv/Fab?

    A: We have identified antibodies with 10 pM-10 nM affinity previously. There is a great number of reputable references who have used our service and obtained good human antibodies for therapeutic use.

  8. Q: What would be a better expression system for the production of the scFv, for large-scale production?

    A: E. coli is the most common expression system for scFv, due to its high efficiency, low cost, and high yield. Therefore, in most cases, E. coli will be good for large-scale expression.

  9. Q: Can E. coli expressed scFv still remain the antibody function and will not be denatured or misfolded when apply to animal (such as in animal's circulation system)?

    A: One advantage of the mammalian expression system is the low endotoxin level, which is important for in vivo application. As you will use the scFv in the animal, mammalian cell expression may be the best choice when the solubility of the scFv is not a problem. Some scFvs have low solubility, then the E. coli system could express them as the inclusion body and remove the endotoxin for in vivo application.

• Phage Display Antibody Library Construction Related Questions[↑Top]

  1. Q: What are the quantity and the quality requirement of RNA sample for human naïve library construction? To achieve the high titer library, what is the optimal condition to do that?

    A: >500 µg of total RNA with high purity is preferred. In order to construct highly diverse antibody library, it is important to have the starting materials from sufficient numbers of human samples. For example, 2-5 mL blood from 100 people or 5-10 mL blood from 50 people. For a good naïve library, at least a few hundreds of mL or 1-2 L blood are required to ensure the diversity.

  2. Q: How do you know the constructed library is perfect?

    A: The affinity of Abs selected from an immune library is proportional to the size of the library, the larger of the library repertoire, the better of the antibody affinity. Usually, the size of the final library should reach 108. It is large enough for isolating high-affinity antibodies. Furthermore, as a perfect library, it should have a high diversity. The QC results of the final library showed that no common sequence was found from all of the randomly picked clones, which indicated that the diversity of the library was at a really high level.

  3. Q: Regarding the RNA samples for library construction, can you provide protocols for extracting RNA from spleen cells, storage, and shipping?

    A: Regarding the RNA purification, since the RNA is very easy to be degraded, and the quality of the RNA sample is very important for the phage library construction, if you do not have the experience of performing the RNA purification experiment or handling RNA sample, we suggest you prepare the tissue samples with Trizol™ reagent and then directly send them to us. We will provide the professional RNA isolation service for you.

  4. Q: Alternative to Trizol™, would it be possible to send the samples (spleen cells and PBMC) of the immune animals in RNAlater® to you?

    A: As an alternative, you can also use RNAlater® to prepare the tissue or blood sample and send them to us. However, one thing should be noted that for the whole blood sample, we suggest collecting the peripheral blood mononuclear cells (PMBCs) before adding RNAlater®. Do not add RNAlater® to the blood directly.

• Troubleshooting[↑Top]

  1. Q: The bacterial strain supplied in the kit cannot grow.

    A: The bacteria of HB2151 and TG1 are supplied in the form of stab culture. These, never thawed, will last for 2 weeks at 4°C. Upon receipt of the kit, a fresh glycerol culture should be made. If they cannot grow after streaking a plate, a fresh culture will be needed. Additional stab cultures can be ordered for the cost of shipping.

  2. Q: The titer of the amplified phage is low.

    A: In order to amplify M13 phage efficiently, the cultures should be well aerated. We recommend amplification in 100 mL cultures in 500 mL Erlenmeyer flasks, in a shaker set to 300 rpm. Amplification in smaller vessels will result in much lower yields of amplified phage. Meanwhile, that cultures should be infected early in their growth phase. M13KO7 helper phage should be added when A600 reaches 0.8-0.9.

  3. Q: When performing an experiment using random peptide phage libraries, the background binding to the plate of ELISA is too high.

    A: In the panning step, if the target is directly coated on the polystyrene plate, it is possible to select peptides that specifically bind the polystyrene surface (see Adey, N. B. et al. (1995) Gene 156, 27-31). These peptides are typically rich in aromatic residues and will yield high ELISA signals in the absence of target since the ELISA plate is also made of polystyrene. Selection of polystyrene-specific peptides often occurs in the absence of a strong target preference for peptide sequences present in the library: other libraries may yield the desired target specific sequences.

  4. Q: After 5 or more rounds of panning with HuFab library many clones are false clones which only have a partial CH1 domain.

    A: Typically, 2×1011 input phages are added in a round of biopanning, and between 103 and 106 total phages are eluted off following washing. The library should be enriched 104 to 107 folds per round. In theory, since the library diversity is 6×109, the eluted pool of phage should be fully enriched in favor of binding antibodies after 3 or 4 rounds. Once this point is reached, further rounds of amplification and panning will result only in the selection of phage that has a growth advantage over the library phage. For example, vanishingly small levels of false clones will completely overtake the pool if too many rounds of amplification are carried out.

  5. Q: When panning with peptide phage library, the final selected sequences bind BSA, and the protocol does not select against BSA binders.

    A: Most likely the "BSA-binding" sequences are actually binding to the polystyrene surface of the plate rather than the BSA. BSA is a soluble, monomeric globular protein without a defined ligand binding site. The absence of a defined ligand binding site is precisely why BSA is generally used for blocking in phage display applications. This means that the entire surface of the protein has evolved to specifically bind water (the definition of "soluble"). It is unlikely that a peptide could bind specifically to the surface of the BSA during panning. Contrast with the case where the target protein has a defined ligand binding site, such as an antibody. In this case, the surface of the protein has evolved to bind water, but the water in the ligand binding site is bound LESS tightly and can be displaced by the ligand. So when binding a phage library to an antibody, specific ligands in the library are able to displace the water in the ligand binding site of the antibody but do not bind elsewhere on the surface of the antibody. The bottom line is that for small peptide ligands, there is generally not enough potential binding energy to displace water from the non-ligand-binding surface of proteins.

  6. Q: A synthetic peptide corresponding to an ELISA-positive sequence identified from the peptide library does not bind my target.

    A: During the panning, the N-terminus of the selected peptide sequence is free, however, the C-terminus is fused to the phage and does not have a free negatively-charged carboxylate. If a selected sequence only binds the target as intact phage, but not as a synthetic peptide, it is possible that the selected sequence requires additional elements from the adjacent spacer sequence for binding. A simple synthetic peptide with a free carboxy terminus will introduce a negatively charged group at a position and completely abolish binding. Thus, we suggest adding the spacer sequence Gly-Gly-Gly-Ser to the C-terminus, when synthesizing peptides corresponding to selected sequences.

  7. Q: I have done phage display selections and have had issues with analysis of clones. The yield of DNA varies so poor material for sequencing. The yield of phage for ELISA varies dramatically from clone to clone.

    A: The issues are the main weaknesses of phage display method and they are really common. However, these problems can be solved by repeating the experiment several times. Usually, we perform 2-3 times of the phage ELISA test and use the average OD values for result analysis. Meanwhile, a further validation through the soluble antibody ELISA for these positive clones is also needed.

    For DNA sequencing, we suggest you pick up the positive clones first and then culture them in 10-20 mL medium overnight to get enough bacterial cells for plasmids isolation.

  8. Q: Do you have a positive control that I can use for the phage ELISA? I’m also having problems with my positive control not giving a good signal to noise.

    A: There is no positive control which can be used for phage monoclonal ELISA. Usually, our criteria for choosing the positive clones is that: the clones should have an A450 value over 0.5 for the target protein after background (no coating well) subtraction. Besides, the value of the target protein should be around 3 or more folds of the value for the negative control (add helper phage instead of monoclonal phage).

  9. Q: We purchased HuFabL-2 phage display library kit. Regarding the production of soluble Fabs, based on the User Manual the domain 3 of gene III contained in phagemid need to be removed by Mlu1 digestion and re-ligation before transformation.

    A: For the production of soluble Fab via E. coli system, as the vector of pCDisplay-8 which used for phage display is not suitable for antibody expression (the promoter in pCDisplay-8 is lac, which is not a strong promoter), we suggest you should construct the Fab cassette to another expression vector with strong promoters, such as T7, to achieve a better yield. The full sequence of the Fab cassette and corresponding elements (such as VLCK/CL, VHCH1, His tag, and Myc tag) in pCDisplay-8 can be found through the Appendix_2 to 5 of the library manual. You can free feel to design primers to amplify the necessary regions for further tests.

  10. Q: We have a problem in amplifying the helper phage M13KO7. Is there a more detailed description of this helper phage?

    A: M13KO7 is a M13 derivative which carries the mutation Met40Ile in gII, with the origin of replication from P15A and the KanR gene from Tn903 both inserted within the M13 origin of replication. M13KO7 is able to replicate in the absence of phagemid DNA. In the presence of a phagemid bearing a wild-type M13 or f1 origin, single-stranded phagemid is packaged preferentially and secreted into the culture medium.

    For the amplification of M13KO7, several points should be noted:

    1. Check the viability of M13KO7 before the amplification. Infect log-phase TG1 bacterial cells with M13KO7 phage at different dilutions for 30 min at 37°C and plating in top agar onto 2TY plates to check if phage plaque could be formed.

    If not, the helper phage stock would be inactivated. Although M13KO7 Helper Phage is stable at either 4°C or -80°C, too many freeze/thaw cycles may impair the infection ability of the helper phage.

    2. Use fresh phage plaque and fresh TG1 bacterial cells for the amplification.

    3. Make sure that Kan works and is at proper concentration (50 μg/mL). Either decrease or increase the concentration may result in fewer phage particles.

  11. Q: Why do some peptide antibodies fail to bind to native protein?

    A: There are linear and conformational epitopes for a native protein, while peptides most likely have a linear structure. The amino acid sequence of the peptide to be used as immunogen is a crucial step toward the success of such purpose. Creative Biolabs will assist our customers with the selection of the best candidate peptide sequences as immunogens. In addition, more than one peptide from a given protein chain is recommended to increase the success rate of native protein binding.

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