[Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection].

OBJECTIVE

To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection.

METHODS

The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA (HTS-ELISA). Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established.

RESULTS

The titers of mouse anti-sera reached 1:10(5);. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity (over 10(9); L/mol). Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg "d, y, r, w" epitopes, respectively. The sandwich ELISA was established through using #3-11 (Anti-HBsAg "a" epitope) as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions.

CONCLUSION

The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.