The Preparation And Analysis Of Characteristics Of Monoclonal Antibodies Against Five Kinds Of Red Tide Algae

Objective: To obtain several lines of cells that can secrete highly specific, affinitive monoclonal antibodies against five kinds of algal cells which are Chattonella marina, Chattonella ovata, Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens, respectively. The monoclonal antibodies secreted by such hybridomas can specifically bind to algal cells, which implies we can use them to determine the five kinds of algae showed above, and this technology can provide an effective tool for detecting and preventing red tide.Methods: The 5 kinds of algae were mixed well to immunize BABL/c mice and boosted for 3 times. Splenocytes got from mice whose antisera showed high liters were fused with myleoma cells Sp2/0 in some ratio by dipping polyethylene glycol(PEG)4000. The hybridomas were cultivated in selection media HAT, HT, then screened by reverse indirect agglutination test. Furthermore, by indirect ELISA, we coated 96-well microplates with Chattonella marina, Chattonella ovata, Hetewsigma akashiwo and so on to screen clones which could secrete specific monoclonal antibodies. The positive clones were expanded and cryopreserved. The ascites were produced, and their titers were determined, then purified with ammonium sulfale and protein-A.Results: The hybridoma cloning ratio was aboul 37.5 percent Screening by reverse indirecl agglutination lesl, indirecl ELISA, elc, we oblained several cell lines that could secrete specific monoclonal antibodies against algae, of which 9AD2B2 against Chattonella marina and Chattonella ovata, 15CA7 against Hetewsigma akashiwo and Pseudonitzschia pungens, 23BG2 and 23BG9 against Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens. And tthe monoclonal antibodies from clone M18 showed highly significant cross-reactivity among such five kinds of algae, which indicated that they could be used as candidates for antisera against Ihe 5 kinds of algae. In the optimum coating concentration of algae, the tiler of from 9AD2A8 was 1:3200, and the titer of 23BG9E11 was l:1280.And the main ingredients of such two ascites were IgG1(s).Conclusion: It was feasible to obtain hybridoma cell lines which could secretemonoclonal antibodies against the five kinds of algae by hybridoma technology. The monoclonal antibodies secreted by such hybridomas could specifically bind to the five kinds of algae, which implied that they could be used to determine and detect such five kinds of red tide algae.