Development of a Sandwich ELISA Method for Detecting Mycoplasma Contamination in Cell Culture

Abstract

The aim of this study was to develop sandwich ELISA method for the detection of common mycoplasma contaminants in cell culture. We produced the mouse monoclonal antibodies (MAbs) and rabbit antibodies against Mycoplasma arginini, M. hyorhinis, M. orale and Acholeplasma laidlawii. Binding specificities of MAbs and rabbit antibodies to the immunized antigens were confirmed by indirect ELISA and Western blot analysis. Among the established MAbs and rabbit anti-mycoplasmas, MAb MH03 against M. hyorhinis, and rabbit anti-A. laidlawii showed high cross reactivities to the four Mycoplasma species. We constructed a sandwich ELISA using MH03 as capture, horse radish peroxidase labeled rabbit anti-A. laidlawii as detector, and could detect M.arginini with the detection limit of 0.5 ㎍/ml and M. hyorhinis, M. orale and A. laidlawii with the detection limit of 0.1 ㎍/ml. These results indicate that the sandwich ELISA developed in this study could be applied for the detection of the four major Mycoplasma contaminants in cell culture.