Identification and isolation of mycoplasmas by immunobinding

This laboratory has developed an immunobinding assay (IBA) to identify and detect mycoplasmas in a variety of specimens. The specimen is inoculated in volumes of 10 microliters onto nitrocellulose (NC) paper, which is then blocked, fixed, and incubated at room temperature. Specific antimycoplasma polyclonal or monoclonal antibody is first added, followed by peroxidase-labeled antibody directed toward the first immunoglobulin. Alternately, antimycoplasma IgG can be purified and conjugated to horseradish peroxidase for use in a direct assay. Addition of a developing solution results in the formation of purple color when mycoplasmas are present. Titers of rabbit antimycoplasma antisera range from 1:1,000 to 1:30,000. This assay can detect approximately 1 x 10(4) colony-forming units (CFU). This IBA has been used routinely to identify mycoplasmal isolates from 132 infected cell cultures. In addition, the procedure successfully detected Mycoplasma pneumoniae in throat swabs from patients with respiratory illness within 2 h. Perfect correlation was obtained with the IBA and microbiological culture of throat swabs for M. orale, M. salivarium and M. pneumoniae. The procedure was successfully used for other Mollicutes. It detected ureaplasmas in urogenital swabs and corn stunt spiroplasmas in infected corn plants and leafhoppers. A modification of the technique has been developed that identifies mycoplasma colonies on agar. It has also been used to assay for antimycoplasma antibodies in serum.