Development And Specificity Assessment Of Antibodies For Myxobolus Honghuensis(Myxosporea: Bivalvulida)

In order to set up rapid detection method and facilitate the immunological tools to screen target antigens for immunopreventive against Myxosporidiosis, two monoclonal antibodies(MAbs) and polyclonal antibody(PAb) against Myxobolus honghuensis were prepared from mice immunized with soluble protein from sonicated spores. The main results are as following:1. Development of polyclonal antibody(PAb) against M. honghuensisThe ELISA titer of PAb is 1:25600. PAb mainly binds to polar filaments, posterior and the periphery of spores through immunofluorescence analysis. In western blotting assay, PAb reacted with nearly 10 protein bands(180 k Da,130 k Da,78 k Da,75 k Da,62 k Da,52 k Da,42 k Da,36 k Da,34 k Da,28 k Da). We found some cross-reaction using PAb to react with several Myxobolus and Thelohanellus species.2. Development and characterization of monoclonal antibodies(MAbs) against M.honghuensis polar filaments and spore valvesThe ELISA titer of MAb 1C7, MAb 3B7 is 1: 124000, 1:248000. The isotype of MAb 1C7 and MAb 3B7 is Ig M and Ig G1 respectively. Additionally, the immunofluorescence analysis revealed that MAb 1C7 specifically reacted strongly with the polar filaments of spores, MAb 3B7 localized almost wholly on spore valves. From western blotting analysis, MAb 1C7 recognized two prominent protein bands of 130 Kda and 180 Kda, the band of 28 Kda is binding with MAb 3B7. Moreover, we do blastp from Uni Prot database to find BCLF1(Bcl-2-associated transcription factor 1) and ANGI_AOTTR are conformed to the reactive protein with MAb 1C7 and MAb 3B7.