Production of monoclonal antibodies recognising N-ethylmaleimide during attempted generation of monoclonal antibodies to human type I procollagen.
Mice were immunised for the production of monoclonal antibodies to human procollagen (I) using antigen purified from fibroblast conditioned medium. The procedure for procollagen (I) preparation included the addition of proteinase inhibitors N-ethylmaleimide, phenylmethylsulphonyl fluoride and ethylenediaminetetraacetic acid to prevent damage by proteolytic cleavage. Four of the five monoclonal antibodies subsequently produced were found to recognise the thiol proteinase inhibitor N-ethylmaleimide but not type I procollagen prepared in the absence of N-ethylmaleimide. One of these monoclonal antibodies was examined further and shown to recognise beta-galactosidase after it had been reacted with N-ethylmaleimide. As far as we are aware this is the first time that monoclonal antibodies have been produced which recognise N-ethylmaleimide. Our findings indicate an unexpected reaction between procollagen and N-ethylmaleimide and prompt the suggestion that the use of N-ethylmaleimide in the purification of procollagen and other proteins should be reexamined.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D005033:Ethylmaleimide / Q000276:immunology*; D006801:Humans; D011347:Procollagen / Q000276:immunology* / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000276:immunology
D000911:Antibodies, Monoclonal; D011347:Procollagen; D001616:beta-Galactosidase; D005033:Ethylmaleimide