Preparation And Characterization Of Monoclonal Antibodies Against Tumor-derived Early Pregnancy Factor

Objective: Applying genetic engineering to prepare tumor-derived recombinant EPF, BALB/c mice were immunized by Freund's adjuvant immunization method. The hybridoma cell lines that secreted anti-EPF monoclonal antibodies (McAb) with high affinity were established by hybridoma technique. Preparation of McAb is useful for further study by using the EPF monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) and other methods to diagnosis ultra-early pregnancy and malignant tumors. Methods: The total RNA was extracted from Melanoma. EPF gene fragment was amplified with RT-PCR, cloned into the expression vector pGEX-5X-1. The recombinant plasmid pGEX-5X-1/EPF was constructed in this way and expressed under the induction of IPTG. The expressed EPF protein was purified by GSH affinity chromatography and digested by Xa protein enzyme. The biological activity of EPF was detected by RIT. The tumor-derived recombinant EPF were used as antigen to immunize several 6-8 weeks old female BALB/c mice. Spleen cells from BALB/c mice immunized were fused with murine NS-1 myeloma cells, and positive hybridoma cells were screened by indirect ELISA, cloning to access cells secreting anti-EPF McAb. Ascitic fluid which contain the McAb against EPF were obtained by ventral injection. McAb were purified by Protein- G affinity chromatography, and EPF McAb was identified by SDS-PAGE and Western-blot. Results: Prepared recombinant EPF has high purity and good immunogenicity. Five hybrisoma cell lines secreting anti-EPF antibodies were obtained. The positive fusion cells injected into the BALB/c mice by intraperitoneal injection, ascites were collected,and the McAb (ascitic fluid) was purified by affinity chromatography. SDS-PAGE analysis showed that the purity of antibody was high. Western-blot analysis showed that the matching of antibody and antigen was good. Conclusions: Recombinant EPF has high purity and good immunogenicity. We obtain five hybrisoma cell lines secreting anti-EPF antibodies. EPF monoclonal antibody has high purity, and is well matched with the antigen.