Construction of Naïve Single Domain Antibody Library
Naïve sdAb Library
The preparation of single domain antibody (sdAb) library starting from lymphocytes isolated from immunized animals is a priority consideration because it enables high-affinity binders that underwent in vivo somatic maturation. However, the preparation of an immune library for each new antigen is limited by the immunogenicity, high toxicity, un-availability of antigens as well as more time and higher costs.
Thus, it is urgent to look for an alternative strategy such as naïve sdAb library offered by non-immune animals. The specificity of naïve sdAb library is that it is a one-pot collection, unspecific randomly, and no preferred antigen for any antibody subpopulations. Consequently, it is necessary to create a large library in terms of diversity to increase the chance of identifying sdAbs for any given antigen. Despite the lack of somatic maturation, the use of naïve sdAb library based on phage display allows for the identification of more extensive diversity of sdAbs for any potential antigen and with high affinity in the subnanomolar or picomolar range.
Strategy for Construction of Naïve sdAb Library
The preparation of the naïve library does not significantly differ from the protocol otherwise used for immune sdAb library; one key point is the choice of the raw materials used for the library construction. To construct a suitable naïve sdAb library, peripheral blood lymphocytes collected from the blood of several nonimmunized animals are used for mRNA extraction. Owing to the lack of somatic maturation stimulated in vivo by the immunization process, such a library theoretically should contain a larger number of independent clones (~109) and greatest genetic diversity to allow the retrieval of high specificity and affinity binders to a given antigen.
In practical terms, the theoretical genetic diversity and potential utility of a naïve library increase with an increasing number of lymphocytes initially collected. For practical reasons related to material handling, a large volume of mixing blood samples collected from different individual animals is a prerequisite to ensure diversity. In addition, to avoid material loss, unnecessary diversity reduction during the cloning of the library, each library preparation step should be performed with maximum care without the risk of compromising the accuracy. As a practical alternative, the final library size could also be increased by mixing independently prepared collections to guarantee the highest diversity.
Furthermore, the affinity and specificity of sdAb can be improved by molecular evolution after the selection procedure. Although these binders from naïve library may be of relatively low affinity, second-generation sdAb library using in vitro maturation techniques, such as DNA shuffling, error-prone PCR, and randomized primers can be employed to improve the diversity of library and the affinity of selected sdAb.
Features of Naïve sdAb Library
- No animal immunization and larger library size
- Need a substantial amount of blood samples collected from different individual animals
- Capable of generating sdAbs against any specific antigen
Case Study
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