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[PubMed] [Google Scholar] 13. be a useful laboratory surveillance tool for studying the epidemiology of pertussis. Based on reactions with specific antisera, can be divided into three types: serotype 2, serotype 3, and serotype 2,3. The major serotyping antigens Hoechst 33258 analog 5 of have been determined to be associated with their fimbriae. Traditionally serotyping is done by the bacterial agglutination test using the slide agglutination method with bacteria mixed with specific serotyping antisera on glass slides. Hybridoma monoclonal antibodies to the serotype 2 and serotype 3 fimbria antigens have also been produced and characterized (7). Attempts to standardize the serotyping method by means of microagglutination have also been made and published (9). Nevertheless, the bacterial agglutination method is still subjective and depends on the ability of the bacteria to form a smooth suspension. Therefore, we have explored the possibility of using an objective method of indirect whole-cell ELISA for the serotyping of isolates. This assay development was evaluated independently at two laboratories with strains of that had been serotyped by the microagglutination method in one laboratory and then tested blindly in a second laboratory by the indirect whole-cell ELISA using Hoechst 33258 analog 5 different batches of the same serotyping monoclonal antibodies. In this communication, we report our findings and compare the two methods for the determination of serotypes of isolates. isolates used in this study were mostly from the culture collection of the Swedish Institute for Infectious Disease Control (SIIDC) and were selected to represent isolates from different periods as well as expressing different serotyping antigens of Fim2, Fim3, and Fim2,3. All isolates were retyped before they were sent blindly to the National Microbiology Laboratory (NML) for testing by ELISA. A few Canadian patient isolates were typed by ELISA at NML and sent Hoechst 33258 analog 5 blindly to SIIDC for testing by the bacterial microagglutination assay. Monoclonal antibodies that recognize serotype 2 and 3 fimbria antigens were made from hybridoma cell lines BPF2 (anti-Fim2) and BPC10 (anti-Fim3), which were originally developed by Brennan, Manclark, and Li (November 1992; U.S. patent 5,162,223) (7). Antibodies from the hybridoma cell lines were produced at the National Institute of Biological Standards and Control and made available to NML and SIIDC. Serotyping of isolates by the bacterial microagglutination method was done as essentially described by Mooi et al. (9). Traditional slide agglutination was carried out according to the method described by Preston (10). Indirect whole-cell Rabbit Polyclonal to TFE3 ELISA was done according to a procedure described for the serotyping of meningococci (1). Briefly, a smooth suspension of a loopful of bacteria grown for 48 h on a Bordet-Gengou agar plate was prepared in pH 7.4 sterile phosphate-buffered saline and heat inactivated at 56C for 1 hour. The inactivated bacterial suspension was cooled and stored at 4C until ready for testing. Such inactivated cell suspensions for the ELISA were found to be stable at 4C for months and can be reused as antigens (e.g., as controls) in multiple assays. Antigen coating was done by adding 100 l per well of the inactivated bacterial antigen, Hoechst 33258 analog 5 diluted in phosphate-buffered saline to give an optical density of about 0.1 at 620 nm, to a Nunc Maxisorp 96-well flat-bottomed Immuno microtiter plate (Nalge Nunc International, Rochester, NY). Detection of binding of the serotyping monoclonal antibodies to the bacterial cells was done by addition of a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G F(ab)2 fragment-specific antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Each assay was done in the presence of both a Fim2-positive control strain and a Fim3-positive control strain. Details of the different serological methods are available from the authors. Optimal dilutions of the anti-Fim2 and anti-Fim3 monoclonal Hoechst 33258 analog 5 antibodies for use in the indirect whole-cell ELISA were determined by titration of each antibody.