USE OF HIGHLY ENCAPSULATED STREPTOCOCCUS-PNEUMONIAE STRAINS IN A FLOW-CYTOMETRIC ASSAY FOR ASSESSMENT OF THE PHAGOCYTIC CAPACITY OF SEROTYPE-SPECIFIC ANTIBODIES

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