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Clinical and Diagnostic Laboratory Immunology, September 1998, p. 703-710, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Use of Highly Encapsulated Streptococcus pneumoniae Strains in a Flow-Cytometric Assay for Assessment of the Phagocytic Capacity of Serotype-Specific Antibodies

W. T. M. Jansen,1,* J. Gootjes,1 M. Zelle,1 D. V. Madore,2 J. Verhoef,1 H. Snippe,1 and A. F. M. Verheul1

Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Section Vaccines, Utrecht University Hospital, 3584 CX Utrecht, The Netherlands,1 and Wyeth-Lederle Vaccines and Pediatrics, Rochester, New York 14586-97282

Received 18 December 1997/Returned for modification 9 April 1998/Accepted 9 June 1998

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 µl per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

* Corresponding author. Mailing address: Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Section Vaccines, Utrecht University Hospital, Rm. G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Phone: 31-30-2506533. Fax: 31-30-2541770. E-mail: W.T.M.Jansen{at}lab.azu.nl.

Clinical and Diagnostic Laboratory Immunology, September 1998, p. 703-710, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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