Construction of Immune Single Domain Antibody Library
Introduction
The immune library is the priority consideration strategy for obtaining target-specific single domain antibodies with high affinity. Preparation of immune libraries first needs the immunization of animals against targets of interest through which antigen-specific HCAbs undergo in vivo affinity maturation. Affinity maturation in camelids relies to a larger extent on somatic hypermutation; this process precisely tunes the CDRs to recognize any given antigens. Subsequently, immune libraries are readily obtained by cloning the V-gene repertoire from peripheral blood lymphocytes of immunized animals. In general, the construction of small libraries (~106-9 individual transformants) already represents the immune sdAbs repertoire of lymphocytes presenting in the bloodstream of the immunized animals. The unique specificity and high affinity of sdAbs retrieved from these immune libraries can be ensured (low nM or pM levels) because of somatic maturation of immunized camelids/sharks.
Advantages of Immune Library
- Highly enriched target-specific sdAbs
- The most common and standard technique (2-4 months for one specific antigen)
- High affinity of obtained binders from nM to pM
Brief Workflow for Immune Library Construction
The workflow is a step-by-step description to immunize a camelid/shark, to clone the VHH/VNAR repertoire from the immunized animal, which can be used to enrich and select the antigen-specific sdAbs within this immune phage display library. The classical strategy to generate immune single domain antibody library requires five steps:
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Immunization of camelid/shark
To obtain the affinity-matured immune response, the first step is to immunize camelids/sharks with antigens at regular intervals according to standard immunization protocols. The immunization strategy determines what antibody repertoire is raised in the animal and, together with the selection protocol, determine the antigen specificity, affinity, and epitope recognition of the selected antibodies.
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Recovering peripheral blood lymphocytes to extract total RNA
After the last immunization, samples of blood can be collected. The blood sample should contain sufficient HCAbs expressing B cells to retrieve the affinity-matured sdAbs, and the peripheral blood lymphocytes of the immunized animal are used to extract the total RNA.
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Amplifying the VHH/VNAR gene fragments by PCR from the lymphocyte cDNA
The first-strand cDNA is prepared by reverse transcription as a template. Efficient PCR amplification of the VHH/VNAR gene fragments encoding the antigen-binding domain is then conducted by properly designed primers.
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Restriction enzyme digestion and ligation of VHHs/VNAR to phagemid vector
The ends of the VHHs/VNAR amplicons are introduced to restriction enzyme sites for ligating into the phage-display phagemid vector.
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Transforming the ligated material in a suitable bacterial host to construct the library
The ligated DNA material representing the immune sdAb repertoire of B cells is subsequently transformed into E. coli through multiple rounds to generate sdAb library consisting of at least millions of independent transformants.
Case Study
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