Preparation And Characterization Of A Specific Monoclonal Antibody Against URG11 Or URG4 Protein

【Background】Urg11 and urg4 are two novel genes which are up-regulated by HBx protein. It is found [1, 2] that urg11 not only could promote cell growth in culture, colony formation in soft agar, and tumorigenecity in nude mice, but also could increase the expression ofβ-catenin to trigge the Wnt signaling pathway in development of hepatocarcinoma (HCC), and that [3, 4] urg4 not only could promote cell growth in culture, colony formation in soft agar, and tumorigenecity in nude mice, but also colud enhance hepatocarcinogenesis and the development of human gastric cancer. The most important finding [5] is that those two genes, combined with another novel genes, could predict the development of HCC three years earlier. Hence, it is much important to further develop the functions of urg11 and urg4, however, monoclonal antibodies against those two moleculars have not generated yet.【Objectives】To develop and identify monoclonal antibody(MAb) against URG11 or URG4 based on synthesized peptide.【Methods】(1) According to the publication [1,3], peptides were synthesized and coupled to keyhole limpet hemocyanin (peptide-KLH) or to bovine serum albumin (peptide-BSA). (2) BALB/c mice were intraperitoneally immunized with peptide-KLH. (3) Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells from the immunized BALB/c mouse whose serum titer of the anti-peptide-BSA greater than 1:10000 determined by ELISA. Supernatant of the growing hybridoma was used to screen for the presence of anti-peptide-BSA antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma. (4) BALB/c mice were boosted with the hybridoma to produce a deal of MAb. (5) A commercially available mouse MAb isotyping kit was used to identify the isotype of this MAb. Immunohistochemistry and Western blotting proved that the new prepared MAb could bind both native and denatured purpose protein.【Results】(1)One hybridoma secreting MAb against URG11 was produced. Immunogolobulin subclass determination identified that this MAb was of the IgG2a/κtype. Western blotting and immunohistochemistry showed that the new prepared MAb against URG11 could specifically recognize URG11 in its denatured and native form. (2) One hybridoma secreting MAb against URG4 was obtained. Immunogolobulin subclass determination indicated that this MAb was of the IgG2a/κtype. Western blotting and immunohistochemistry proved that the new prepared MAb against URG4 could specifically recognize the denatured and native form of URG4.【Conclusions】Those two cell lines(3D2 and 1A8) could stably secret their own purpose MAb. The new prepared MAb could respectively recognize their own purpose protein in experiments of Immunohistochemistry and Western blotting, suggesting that those MAb could bind both native and denatured URG11 or URG4 respectively, which is very important in the biological function study of urg11 or urg4.