Abstract
BackgroundNanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems.ResultsWe constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 1011 clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT.ConclusionWe have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0091-7) contains supplementary material, which is available to authorized users.
Highlights
Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs)
Due to their relatively large size of approximately 50 kDa, Fabs yield large oligomeric molecules [7]. scFvs are the smallest intact antigen-binding fragments that can be derived from a conventional immunoglobulin G (IgG) molecule [4]
To construct a large and highly diverse naïve Nb library, as shown in Figure 1, total RNA was isolated from peripheral blood lymphocytes (PBLs) collected from twenty four nonimmunized bactrian camels and a spleen from another non-immunized camel
Summary
Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). With the rapid development of molecular gene engineering techniques, Fabs (50 kDa) and scFvs (27 kDa) have been widely used as alternatives to classic antibodies These antibody fragments can maintain the specificity to antigens. Fabs are the oldest class of monoclonal antibody derived fragments, which are clearly thoroughly explored [6] Due to their relatively large size of approximately 50 kDa, Fabs yield large oligomeric molecules [7]. ScFvs are the smallest intact antigen-binding fragments that can be derived from a conventional IgG molecule [4]. They are recombinant antigen-binding fragments in which the variable regions of light and heavy chains are combined into a single polypeptide. Their susceptibility to temperature and pH [10] make them unsuitable for use in biosensors as diagnostics or as therapeutic candidates
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