Overview

Phage display is the most commonly used technology for the generation of recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The genotype and phenotype of single domain antibody (sdAb) fragments are linked by fusing the corresponding sdAb gene fragments to the minor coat protein III gene of the filamentous bacteriophage M13, and the result is that sdAbs fused to protein III are expressed on the surface of M13 phage particles. Despite other in vitro methods such as ribosomal display, yeast surface display, or mammalian cell display, phage display is the most widely used method for the generation of sdAbs.

The precondition for successful generation of sdAbs against any target using phage display is the construction of high-quality sdAb libraries used for the selection. Maximum diversity is generated by amplifying sdAb genes from peripheral blood lymphocytes (PBL) or lymphoid tissue isolated from several nonimmunized/immunized donors using polymerase chain reaction (PCR) primers that correspond to all known V_HH/V_NAR gene sequences. This cloning process could be used to generate a diverse sdAb library for subsequent selection by phage display. The phagemids contain a phage morphogenetic signal for the packaging of the recombinant phagemid vector into the assembled phage particles in order to create a display library.

Different Types of sdAb Library

There are several different forms of sdAb libraries that are classified mainly by the cDNA source encoding the sdAb genes, which are mainly divided into either naive, immune, or synthetic libraries. Different types of sdAb libraries can be used depending on the field of sdAb applications. Based on the advanced technology platforms and extensive experience, Creative Biolabs is a long-term expert in the field of sdAb development and provides one-stop solution for sdAb development, including the construction of sdAb library as well as the subsequent development of sdAb.

• Overview of Naïve sdAb Library

The naïve library is unique in which the sdAb gene repertoire used is generally from healthy individuals. In this way, the sdAb libraries would not be exposed to any disease-specific antigens resulting in the true unbiased nature of the sdAb repertoires. Although the large naïve library is somewhat unfocused in its targeting antigen population, it is shown to be highly efficient in the generation of specific sdAbs against different types of antigens.

• Overview of Immune sdAb Library

Immune sdAb library is typically generated from sdAb genes isolated from PBMC of the immunized donors to clone the antigen-specific sdAb in a phage display vector. As sdAb repertories usually undergo in vivo affinity maturation processes during the immunization process, the sdAbs derived from the immune library will generally possess higher affinities if compared to those obtained from the naïve library. The immune repertoires are used to isolate distinct sdAbs against particular antigens, e.g. cell surface antigens of pathogen or tumor markers.

• Overview of Synthetic sdAb Library

The construction of synthetic sdAb library is based on a single domain scaffold with randomly integrated diversity in complementarity-determining regions (CDRs) that can contribute to antigen binding and the primary source of sequence diversity in antibodies. The use of synthetic library provides a vast resource of renewable sdAb reagents and allows control over both library design and selection conditions aimed to fine-tune sdAb characteristics, including affinity, specificity, and manufacturability. The synthetic library facilitates the generation of sdAbs with precisely engineered binding specificities.

Library Construction Workflow

The committed step for generation of sdAb gene libraries strategy for either naïve, immune, or synthetic libraries are following described, including:

• Isolation of the RNA from B-lymphocytes
• Preparation of cDNA by RT-PCR
• Amplification of V_HH/V_NAR sequences from the cDNA pool by PCR
• Restriction enzyme digestion of V_HH/V_NAR and ligation to vector
• Transformation with the purified ligation mixture to generate V_HH/V_NAR library
• Library capacity estimation & QC

Application of sdAb Library

One of the broadest areas of applications for the sdAb library is the generation of sdAbs for large-scale proteome studies. The method allows for the rapid and continuous generation of sdAbs from such libraries with high specificity and high affinity against a wide array of target proteins. The isolated sdAbs from phage display have been applied successfully to evaluate the immunogenic epitope(s) of the antigens and provided potentially valuable sources for basic research, diagnostic, and therapeutic purposes.

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