Characterization of four distinct monoclonal antibodies specific to BmK AS-1, a novel scorpion bioactive polypeptide

Four monoclonal antibodies designed as 2#, 3#, 4# and 5# have been raised against a novel bioactive polypeptide BmK AS-1 purified from the Chinese scorpion Buthus martensi Karsch. All of these antibodies exhibited specific affinity with antigen by ELISA and Biosensor assay. Western blot analysis showed that 3# and 4# were able to recognize the denatured antigen, but not 2# and 5#. These antibodies could cross-react with BmK AS, but not with other types of BmK neurotoxins such as BmK I (an alpha-like toxin) and BmK IT (an excitatory insect-selective toxin), and in which only 5# can partially react with BmK IT2 (a depressant insect-selective toxin). Immunocytochemical staining demonstrated that 3#, 4# and 5# antibodies can visualize the antigen bound to the membrane of SK-N-SH neuroblast cells, with the exception of 2#. This suggests that either conformation alteration of receptor binding might be prone to nonvisualization or the epitope recognized by antibody 2# might be overlapped with receptor binding sites of antigen. The antibodies developed in the study should provide powerful new tools for investigating the structure/function relationship and pharmacological mechanism of scorpion neurotoxins.