Abstract
Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.
Highlights
Conventional heterotetrameric antibodies are composed of two heavy chains and two light chains
Besides the therapeutic application, which requires complex engineering and optimization steps, nanobodies can be used as research tools
In addition to protein detection and purification, another significant application of nanobodies is the structural characterization of their antigens
Summary
Conventional heterotetrameric antibodies are composed of two heavy chains and two light chains. The heavy chains from heavy-chain antibodies (HCAbs) comprise two constant domains (CH2 and CH3) and one antigen-binding domain that is termed nanobody, or VHH, or single-domain antibody. Nanobodies are functionally equivalent to conventional antibodies’ antigen-binding fragments in recognizing target antigens. Their molecular weights are around 12 to 15 kDa, about one-tenth of conventional antibodies’ size. Nanobodies have longer CDR3, which provides them with more diverse paratopes [3]. The characteristics of small size and high specificity mean that nanobodies are suitable for many more applications than conventional antibodies. Nanobodies are fully qualified to substitute antibodies in assisting research and have more potential usages as their size is much smaller. CDR1+CDR2: partially randomization; CDR3: fully randomization except for cysteine a consensus framework derived from llama genes IGHV1S1-S5
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