Coyle P V, Wyatt D, McCaughey C, O’Neill H J. identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more useful tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity. is usually a commensal organism of the human lower genital tract. It has been implicated in diseases of the genitourinary tract (30), unfavorable pregnancy outcome (3, 9, 10, 14), and infections of Cortisone acetate premature neonates (31). The high rate of isolation of from the genital tract has made its role in genitourinary tract disease difficult to define. Since only a subpopulation of colonized individuals ultimately develops disease, it has been postulated that only certain subgroups of are associated with disease. comprises 14 serotypes divided into two biovars on the basis of DNA-DNA homology (6), restriction endonuclease DNA digestion (21, 22), polyacrylamide gel electrophoresis of proteins (12, 29), and sensitivity to manganese salts Rabbit polyclonal to LIMD1 (25). The association of particular serotypes with disease is still controversial (11, 15, 17, 26, 27). This controversy could be due in part to the lack of a standardized method for serotyping. Until now, serotyping studies have been performed with polyclonal antibodies (PAbs). However, the use of PAbs for the serotyping of clinical isolates raised problems such as polyreactivity with clinical isolates and lack of reproducibility (15, 28). Of particular interest is usually that PAbs do not exhibit such polyreactivity with reference strains but they show cross-reactivity between serotype 2 and 5 reference strains. This cross-reactivity has been observed by many investigators (1, 2, 8, 18, 19, 20, 24, 27). When clinical isolates were serotyped with a partial set of monoclonal antibodies (MAbs), good reproducibility was obtained and polyreactivity was not seen with clinical isolates (4, 5, 16). However, these promising preliminary results need to be confirmed with a study with a complete set of MAbs directed against the 14 serotypes of serotypes 1 to 10 were supplied by E. A. Freund (Institute of Medical Microbiology, University of Aarhus, Aarhus, Denmark), and those of serotypes 11 to 14 were supplied by J. A. Robertson (Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada). antigens were prepared by growing serotype reference strains in 1 liter of bromothymol blue broth (23). The cells were harvested by centrifugation at 25,000 for 30 min at 4C. The pellet was washed three times with phosphate-buffered saline (PBS) and was resuspended in PBS before storage at ?80C. The aliquoted fractions of the antigenic preparations were used for MAb production as well as for the Western blot analysis. MAb production procedure. The MAbs against serotype 2 and 7 were produced as described previously Cortisone acetate (4). Immunization of Cortisone acetate mice was performed by intraperitoneal injection (4) for serotypes 2 and 7 and by footpad injection (7) for serotypes 5, 8, 10, 11, 12, and 13. Immunization of mice through footpad injection resulted in a shorter immunization period than that obtained by immunization through intraperitoneal injection: 2 weeks instead of 10 weeks. The fusion was performed as described previously (4). Screening of the hybridoma clones was performed by colony immunofluorescence assay (colony-IFA) (4). Positive clones were subcloned twice by limiting dilution. The immunoglobulin isotypes were determined with the Mouse Antibody Isotyping Kit (Life Technologies, Merelbeke, Belgium). Characterization of the MAbs. The serotype specificities of the MAbs were determined by colony-IFA (13). The Western blot assay (WBA) was performed as described.