Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis

Serotyping of Chlamydia trachomatis strains usually requires three to six serial passages in shell vials to attain sufficient antigen for typing procedures. To circumvent this problem, we developed a rapid low-passage method for serotyping of C. trachomatis clinical isolates. Isolates with an inclusion count of greater than or equal to 500 per well in primary isolation were inoculated directly onto cell culture monolayers in microtiter plates for typing. Primary isolates with a lower initial inclusion count were passed one to two times in shell vials until there were greater than or equal to 20 inclusions per well and were then inoculated onto plates for typing. Inclusions were grown to maturity and reacted with a panel of 17 C. trachomatis-specific monoclonal antibodies in pools. Wells were then reacted with a fluorescein isothiocyanate conjugate and read by FA microscopy, and the reaction patterns were compared with prototype strain reaction patterns to determine the serotype. By the microtiter method, we successfully typed 1,711 consecutive C. trachomatis isolates; 1,215 isolates (71%) were typed with no or with one passage. The first 209 isolates typed by the microtiter method were also typed by the dot-enzyme-linked immunosorbent assay serotyping method; 100% agreement was demonstrated among strains that were typeable by both methods. We conclude that the microtiter method is extremely useful for accurate serotyping of large numbers of isolates and requires greatly reduced technician time.