Summary
An enzyme-linked immunosorbent assay (ELISA) was developed in order to serve in detecting and speciating mycoplasmas isolated from cell cultures. Its main features included a biotin-streptavidin amplification step and a solid phase consisting of a microporous membrane. Cell samples in the form of suspensions were applied to nitrocellulose or ion exchange membranes immobilized in commerciallyavailable microtiter, multiwell manifolds. The blocking buffer contained 1% purified α-casein. The primary antibodies were monoclonal and the polyclonal secondary antibody was biotinylated. The enzyme utilized was streptavidin-horseradish peroxidase. The substrate-dye complex consisted of either 4-chloro-1-naphthol and hydrogen peroxide or ortho phenylene diamine (OPD) and hydrogen peroxide. The presence of homologous antiserum in the reaction sequence gave clearly visible, colored reactions on the membrane when 50 ul with approximately 105 or more cfu/ml were present. This new biotin-avidin microporous membrane (BAMM-ELISA) test can be used both to detect mycoplasmas and to speciate them. The BAMM-ELISA is simple, rapid, sensitive, specific and economical. As such, it has potential for aiding in the control of mycoplasma contamination in cell culture, and could prove useful in clinical diagnostic applications as well.
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This study was supported in part by Bionique Laboratories, Inc., and research grants awarded by the National Institutes of Health (SBIR Phase II from NIEHS, R44ES03705) and the New York State Science and Technology Foundation (SSF 84-1). Valuable technical assistance and counsel were provided by Dr. Steven Geary, Angela Alongi and Alexandria Siy. Photography was done through the courtesy of Marina LaDuke of the W. Alton Jones Cell Science Center.
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Gabridge, M.G., Lundin, D.J. & Gladd, M.F. Detection and speciation of common cell culture mycoplasmas by an enzyme-linked immunosorbent assay with biotin-avidin amplification and microporous membrane solid phase. In Vitro Cell Dev Biol 22, 491–498 (1986). https://doi.org/10.1007/BF02623451
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DOI: https://doi.org/10.1007/BF02623451