| Objection: This study is to prepare a kind of bispecific antibody of anti-HIV p24 antigen and anti-human RBC type A antigen, and to build a new way to detect HIV p24 antigen with this bispecific antibody. Now the ways to detect HIV p24 include enzyme linked immunosorbent assay, immuno-chromatography, agglutination test, and so on. The way build in this experiment is an indirect RBC agglutination test, which is simple, fast, sensitive, and no pollution for the enviroment.Methods: The BALB/c mouse is immunized with gene recombinant antigen p24 for four times in 2 months. The spleen cells of immunized mouse is hybridized with Sp2/0 by PEG, and the positive cell clones secreting the antibody to antigen p24 are detected by indirect ELISA. Through three clonings less diversed anti-p24 hybridoma cells are gained. Select a hybridoma cell named 2-E4 with high anti-p24 antibody titer, and anti-human RBC type A hybridoma cell S2, to prepare the hybrid-hybridoma. 2-E4 and S2 is induced respectively by 8-Ag and 5-BrdU with different drug concentration to make them deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and in thymidine kinase(TK) respectively and renamed 2-E4-A and 82-6. Their antibodies' isotypes are tested by goad anti-mouse isotype regent. 2-E4-A and 82-6 are hybridized during their log growing time, and the hybrid-hybridomas are cloned for 3 times and produce 6 hybrid-hybridoma cells. The chromatosome of hybrid-hybridoma 3-Hu and hybridoma 2-E4-A and S2-B are counted, and the antibody of ascites fluid or culture supernatant of 3-Hn is prepared. The positive clones are detected by three methods at the same time: RBC agglutination for monospecific anti-human RBC Type A antibody, indirect ELISA for anti-p24 antibody, and RBC solid-phase adherence for bispecific antibody. The antibody of ascites fluid of hybrid-hybridoma 3-Hn is selected to test for the heat-resisting and freeze-thawing activity. The heat-resisting test is done under 56 for 30 minutes, 37 for 1-7 days,and 4 for 7 days. The freeze-thawing test is: after the ascites fluid is frozened at -20, melt it at room temperature naturely, and repeat this for 6 times. The ascites fluid antibody purified by 33% ammonium sulfate is further purified by chromatography. The p24 affinity chromato-graphic column done to purify the bispecific antibody is in the BrCN activated sepharose 4B. After washing the unspecific proteins, change the washing buffer and the anti-p24/anti-human RBC type A bispecific antibody and the anti-p24 monospecific antibody are gained respectively. This bispecific antibody companied with another anti-p24 monospecificmonoclonal antibody can build a new way of indirect RBC agglutination to detect p24 antigen, and this new way is preliminarily applied to detect serial diluted p24 to determine the sensitivity.Results: Gain 5 hyhridoma cells secreting anti-p24 antibodies, in which 2-E4 is selected for further hybridization and its ascites fliud antibody titer is 1:1600000. The ascites fliud titer of the other hybridoma S2 for further hybridization is 1:16000 by blood agglutination.The isotype of 2-E4 is IgG2a, and S2 is IgGs. 2-E4-A and 82-6 which are induced by 20 u. g/ml 8-Ag and 20 u g/ml 5-BrdU respectively are dead in the HAT culture in a week. 2-E4-A and S2-B are hybridized routinely and after three clonings produce 6 less-diversed hybrid-hybridoma cells. The chromatosome of hybrid-hybridoma 3-Hn are 132+15, that of hybridoma 2-E4 and S2 are 84+9 and 89+9. The bispecific antibody tilers of ascites fluid and culture supernatant of 3-Hn are 1: 16000 and 1:64. At the same time the anti-p24 antibody and the anti-human RBC type A antibody in the ascites fluid of hybrid-hybridoma 3-Hn are tested, and their titer is 1:800 and 1:16000 respectively. Therefore the hybrid-hybridoma can secrete not only bispecific antibody but also anti-human RBC type A monospecific antibody and probably anti-p24 monospecific antibody. The mixed antibodies purified by ammonium sulfate is further purified by p24 affinity chromatography.
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