We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to Neisseria meningitidis serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean r ؍ 0.96; P < 0.001). OPA could be used in meningococcal vaccine evaluation.Meningococcal disease continues to be a significant cause of morbidity and mortality in the United States and worldwide (3). The primary mechanism of protection from disease is through antibody-and complement-dependent bactericidal activity (9). Antibodies to capsular polysaccharides of groups A and C of Neisseria meningitidis have been shown to confer protection from meningococcal disease (1,7).N. meningitidis is efficiently phagocytosed by polymorphonuclear (PMN) cells (5,8,10), and antibodies specific for group A and C polysaccharides are bactericidal in the presence of peripheral blood polymorphonuclear leukocytes and complement (19). Monocytes can also phagocytose Neisseria species (12,13,16,23). The relative importance of opsonophagocytic killing as a defense mechanism against N. meningitidis has been previously demonstrated (2,4,18,20,22). Opsonophagocytic assays (OPA) offer several advantages over the standard serum bactericidal assay (SBA). OPA for other pathogens such as Streptococcus pneumoniae have been developed which are semi-automated, use noninfectious targets, provide useful laboratory correlates of protection, and can be multiplexed (14).This report describes a patent-pending multiplexed flow cytometric OPA for measurement of antibody-and complementdependent cell-mediated antimeningococcal activity against serogroups A, C, Y, and W135. Postvaccination sera were collected 1 month after immunization with a quadrivalent meningococcal vaccine (Menomune; Connaught Laboratories, Philadelphia, Pa). Sera from nonimmunized adults (n ϭ 15) and postvaccination adults (n ϭ 12) were obtained through the Emory University Donor Services, Atlanta, Ga., and used as quality controls (15). Sera were stored at Ϫ70°C and heat inactivated at 56°C for 30 min just before being tested, to inactivate endogenous complement activity. We compare this flow OPA technique to a previously standardized SBA (15).(Part of this study was presented at the 11th International Pathogenic Neisseria Conference, Nice, France, 1998.) Homologous capsular polysaccharide from meningococcal serogroups A, C, Y, and W135 (Connaught Laboratories) at a concentration of 1 mg/ml was oxidized by the addition of 218 l of a 0.046 M sodium periodate solution for 30 min, protected from light, at room temperature. The reaction was stopped by the addition of 0.1 ml of glycerol per ml of reaction solution. The polysaccharide solutions were then dialyzed overnight against 0.1 M sodium bicarbonate buffer (pH 8.0).Aliquots (100 l) from the commercial preparation of 1-m-diameter carboxylate-modified FluroSpheres (Molecular Probes, Eugene, Oreg.) were coupled with each oxidized meningoc...
