A phage-displayed single chain variable fragment that interacts with hepatitis B core antigen: Library construction, selection and diagnosis

Abstract 

Background

Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen.

Objectives

To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen.

Study design

Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (V_H) and light (V_L) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples.

Results

A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10ng of HBcAg can be detected by the assay.

Conclusion

The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.

Abbreviations: HBcAg, hepatitis B core antigen, HBsAg, hepatitis B surface antigen, HBeAg, hepatitis B e antigen, HBxAg, hepatitis B X antigen, ScFv, single chain variable fragment, ABTS, 2,2′-azino-di-3-ethyl-benzthiazoline-sulfonate, PNPP, p-nitrophenyl phosphate, SDS, sodium dodecyl sulfate, CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfate, mAb, monoclonal antibody, AP, alkaline phosphatase, pfu, plaque forming unit

Keywords: Phage display, Combinatorial antibody libraries, Hepatitis B core antigen, Diagnosis, Phage-ELISA