IMMUNOCHEMICAL CHARACTERIZATION OF MICRURUS-NIGROCINCTUS-NIGROCINCTUSVENOM WITH MONOCLONAL AND POLYCLONAL ANTIBODIES

Eleven murine monocloncal antibodies (MAbs) against Micrurus nigrocinc tus nigrocinctus venom were produced and partially characterized. When M. n. nigrocinctus venom proteins were separated by SDS-PAGE under no n-reducing conditions four sharp and three diffuse bands were observed . The sharp bands had migration rates comparable to reduced standards of 10, 12, 50 and 72 kDa. The diffuse bands migrate in the range of re duced standards from 14.5 to 32 kDa. When venom proteins were separate d under reducing conditions the same sharp bands and an additional pro minent 14.5 kDa band were observed. Three antibodies (MAbs 4, 21 and 2 8) recognized the diffuse bands in western blots of non-reducing SDS-P AGE, whereas MAbs 7G, 22 and 26 reacted with only the 72 kDa protein. MAbs 21 and 28 reacted with the 14.5 kDa band whereas MAb 7G recognize d the 72 kDa band in blots of reducing SDS-PAGE. Two M. nigrocinctus a ntivenoms cross-reacted by ELISA against nine neurotoxic snake venoms, as well as with gamma-toxin from Naja nigricollis and notexin. One an tibody (MAb 9A) was used to affinity purify a fraction (called nigroxi n) from M. n. nigrocinctus venom. Nigroxin showed phospholipase and my otoxic activities and appeared as a single 15 kDa band in SDS-PAGE und er reducing conditions. However, three bands with slight differences i n charge were resolved by urea-PAGE, representing isoforms named nigro xin a, b, and c. Nigroxin induced a dose-dependent release of peroxida se trapped in negatively charged liposomes. Nigroxin induced myonecros is and increased the plasma creatine kinase levels in mice, when injec ted intramuscularly. The plasma membrane of cultured L6 myoblasts was permeabilized by nigroxin, as evidenced by the release of H-3-uridine nucleotides from prelabelled cells. This effect was completely abolish ed after preincubation with MAb 9A, although this antibody failed to n eutralize the enzymatic activity of nigroxin. Nigroxin was also recogn ized by MAbs 4, 7H, 21, 27 and 28. Additionally, the epitope recognize d by MAb 27 is also present in notexin and beta-bungarotoxin.