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Journal of Clinical Microbiology, January 2005, p. 156-162, Vol. 43, No. 1
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.1.156-162.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies

Jigui Yu,1 Jisheng Lin,1 William H. Benjamin Jr.,1,2 Ken B. Waites,1,2 Che-hung Lee,3 and Moon H. Nahm1,2*

Departments of Pathology,1 Microbiology, University of Alabama at Birmingham, Birmingham, Alabama,2 U.S. Food and Drug Administration, Bethesda, Maryland3

Received 20 July 2004/ Returned for modification 7 September 2004/ Accepted 14 September 2004

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.

* Corresponding author. Mailing address: 845 19th St. South (BBRB 614), Birmingham, AL 35249-7331. Phone: (205) 934-0163. Fax: (205) 975-2149. E mail: E-mail: Nahm{at}uab.edu.

Journal of Clinical Microbiology, January 2005, p. 156-162, Vol. 43, No. 1
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.1.156-162.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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