Library Display-Based sdAb Discovery

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Creative Biolabs is one of the well-recognized experts who are professional in applying advance Hi-Affi™ Phage Display Platform for a broad range of sdAb discovery projects. Our Hi-Affi™ platform can contribute to generating high-quality sdAb libraries from various source with maximized diversity and library capacity of 1011. Subsequently, our scientists are confident in tailoring the best-fit library screening and binder validation procedures to select specific binders with high affinity around 10-8-10-12.

Typical Features of Hi-Affi™ Phage Display Platform

  • Proprietary primers which are designed specifically for VHH/VNAR domain of corresponding species.
  • Comprehensive phagemid vectors with different detection tag (e.g. His, HA, c-myc), suitable for various target screening.
  • Freshly prepared high-quality phagemid vectors, which can achieve advanced-transfection-grade for the most sensitive applications.
  • Optimized ligation strategies to increase the efficiency, ensure the uniform, and keep the diversity of constructed phagemid libraries.
  • Nearly 100% correct insertion rate.
  • Outstanding transfection system which can maximize library capacity to 1011.
  • Comprehensive library screening procedures, specifically designed for the target of interest.
  • Two-step validation strategy, which can contribute to the most reliable data.

The flow diagram for sdAb library screening.

Phage display is one of the most powerful and widely used techniques that can display sdAbs on the surface of phages and then isolate specific sdAbs against the target of interest. Considering the nature difference of various targets, it is crucial to determine the most suitable source for library construction in the very early stage of a sdAb development project. With our professional scientists who have over ten years’ experience in antibody drug discovery, Creative Biolabs has the capacity to tailor the best-fit source and generate high-quality libraries to lay a solid foundation for your project.

Based on the source difference, qualified sdAb libraries are mainly divided into two classes, immune library and non-immune library. Thereinto, the non-immune libraries can also be subdivided into naïve library and synthetic library.

Immune sdAb Library

An immune library is constructed from natural infections or immunized donors to obtain antibodies against a particular target. As V genes collected for immune libraries contain hypermutations and had gone through in vivo affinity maturation, target-specific sdAbs with high affinity can be selected as long as an immune response can be triggered by the target. This is the best approaches that are widely applicable to various immunogenic targets (e.g. soluble protein, peptide, DNA, whole cells).

Creative Biolabs has unparalleled capabilities for the construction of VHH or VNAR based single domain antibody libraries through immunized camel, llama, alpaca or even shark.

Naïve sdAb Library

A naïve library is derived from primary B-cells of non-immunized donors, in which a large repertoire of naturally rearranged V genes is collected for library construction. In general, multiple host animals will be employed in the sdAb repertoire collection and therefore contribute to a universal library that should not contain distinguish bias for specific target. In another word, a qualified naïve library is expected to be able to isolate binders against all types of antigenic targets. In general, the affinity of selected antibodies from the naïve library is proportional to the size of the library. Due to the lack of in vivo affinity maturation against specific antigen, an additional affinity maturation process is sometimes required to further increase the affinity of selected sdAbs.

Creative Biolabs can generate high-quality naïve sdAb library for either research purpose or industry application. In terms of our abundant host animal sources and advanced Hi-Affi™ platform, unique naïve library with 1011 capacity can be generated upon your request.

Synthetic sdAb Library

A synthetic library is based on the in silico design and then generated in a controlled fashion. Differ with immune or naïve libraries, in which the origin of antibody repertoires is amplified from natural source, the diversity of synthetic library relies on properly design. The ratio of natural and designed parts determines the classification of semi- and fully-synthetic libraries. A typical advantage of synthetic library is the composition of CDR regions can be exactly defined and controlled. It is also a novel option to obtain camelized human single domain antibody. Similar to the naïve library, synthetic libraries are also antigen-independent, which can be used for unbiased selection of sdAbs against any targets.

Creative Biolabs has mature designs and reliable solutions for synthetic sdAb library construction. Our scientists provide a series of blueprint for either regular or human sdAb libraries, which can fulfill your project demands.

Premade sdAb Libraries Available in Creative Biolabs

In addition to generating a fully customized sdAb library, Creative Biolabs can also provide a cost-effective option for specific sdAb discovery with a shorter lead time. In the past ten years, our scientists have generated a series of sdAb libraries in-house. These libraries were preselected based on the thermostability, which can now be a great source for your projects. Some of these premade sdAb libraries are even available for licensing, which including:

  • CaVHHL-1: Camel Naïve Single Domain Antibody Library
  • LlaVHHL-1: Llama Naïve Single Domain Antibody Library
  • AlpVHHL-1: Alpaca Naïve Single Domain Antibody Library
  • HuSdL-1: Human Single Domain Antibody Library

The flow diagram for sdAb library screening.

Creative Biolabs has extensive experience in providing highly customized screening strategies for novel sdAb discovery. We have overcome a great deal of challenging targets (e.g. membrane proteins, small molecules, and PTM-modified peptides) and selected specific sdAbs successfully. Focusing on the target property and your specific project purpose, our scientists are pleased to tailor a suitable strategy to achieve your goals, which including but not limited to:

The flow diagram for sdAb library screening.

● Solid-Phase Screening

Solid-phase screening is a valuable method for binding antibodies selection and epitopes identification. We can provide a series of solid-phase for target molecule immobilization, such as PVDF membranes and microtiter plate wells.

● Solution-Sorting Screening

Solution-sorting screening is an optimized method to obtain desired antibody with antigen in solution. Compared with solid-phase screening, it avoids the problem of conformation changes when coating on the solid phase.

● Cell-based Screening

Cell-based screening is a powerful method to select high-affinity and high-specificity antibodies against cell surface antigens. What’s more, it has been applied to screen antibodies for co-crystallization research.

In vivo Screening

In vivo screening is a high-throughput method to isolate and identify the phages bound to specific tissues via the injection of phage libraries into animals. This is also a potential approach to discover sdAbs which can cross the blood-brain barrier.

Ex vivo Screening

Ex vivo screening is a novel phage display library biopanning technology for the discovery of peptide and protein ligands against organ- and tissue-specific macromolecules.

If you are interested in the discovery and development of novel single domain antibodies, please do not hesitate to contact us for more details.

1. Q: How to calculate the capacity of constructed library?

The capacity of constructed library can be evaluated by the number of colonies present on the transformed plates with serial repeated dilutions. Calculate the average of all dilution plates after counting colonies, which indicates the library size (cfu/mL).

2. Q: How to validate the quality of constructed library?

The quality of constructed library can be determined through QC colony PCR. Some colonies are randomly picked from the titration plates and used for colony PCR to analyze the insertion of the correct size.

3. Q: How to evaluate the diversity of constructed library?

The diversity of constructed library can be determined through QC sequencing. Some colonies are randomly picked from the titration plates and used for sequencing and alignment to evaluate the diversity. It is well known that most of the diversity of the antibody molecule is found in the complementarity-determining regions (CDRs).

4. Q: How to choose between the immune library and premade library?

Immune library is the best-fit choice for specific project purposes, such as some specific target antigens, the high-affinity antibody requirement from nanomoles to picomoles, the largest possible number of antibody clones, improved probability to obtain the exact immune response.

Creative Biolabs can provide several excellent premade libraries which have great diversity to be able to derive specific antibodies. They can be used for rapid discovery of high-potency sdAb against any targets and allow to save your time and cost from library construction.

5. Q: Which kind of starting material can be accepted for naïve library construction?

In essence, the naïve library is constructed by the cDNA isolated from non-immune animals. The RNA from several animals can be mixed to generate one qualified naïve library.

Creative Biolabs can accept properly stored tissues and RNA as starting materials for library construction. Please note, at least 50 μg total RNA or 10 μg mRNA is required for one library construction project.

6. Q: How to identify specific binders after the screening process?

After the screening process, Creative Biolabs can offer two general strategies to identify target-specific binders and then perform corresponding functional assays to select desired sdAb candidates.

Strategy 1: Monoclonal Phage-based Binder Identification & Validation
To isolate specific binders against the target of interest, a group of monoclonal phage clones is randomly picked and gone through a series of validation processes to obtain the sequence of identified positive clones. We can also generate the purified sdAbs for further characterization assays, such as affinity measurement.

Strategy 2: Magic™ Therapeutic Antibody Discovery Platform-based Binder Identification & Validation
Creative Biolabs has established the Magic™ Therapeutic Antibody Discovery Platform which is an exclusive high-throughput solution for the identification of all potential novel binders at one-time. Purified sdAbs can be generated from the sequence of identified clones for further characterization assays, such as affinity measurement.

7. Q: What is the lead time for a general sdAb discovery project?

In general, we need around 6-9 months to complete an immunization-based novel sdAb discovery project, thereinto, the immunization process will spend about 3 months to achieve acceptable immune responses. By screening our premade libraries, only 3-4 months is required to identify new sdAb binders.

8. Q: Are there any additional deliverables available for our internal test?

Yes. Generally, we can provide small aliquots of outputs (phages) after library screening as well as supernatants of validated positive clone (phage-free), which may vary due to different project design. Please note, these materials are not included in the default deliverable list because the stability cannot be 100% guaranteed (especially for oversea delivery). Please notify us at the early stage if you want to receive these materials, they may be charged accordingly.

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

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

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

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).

11. Q: Are anti-M13 antibodies available?

Yes. Recombinant anti-M13 Major Coat Protein Antibody (CBMAB-0206MC) or other custom anti-M13 antibodies are available of your choice. Please inquire us for more details.

Deliverables of Library Construction & Screening Stage

1. Project report including the library construction & screening processes.
2. Constructed library (do not apply to premade library screening).
3. Sequence information of the final identified positive binder(s).

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