A Flow Cytometric Opsonophagocytic Assay for Measurement of Functional Antibodies Elicited after Vaccination with the 23-Valent Pneumococcal Polysaccharide Vaccine

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5,6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with ≥97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P ≤ 0.01) was found between the two assays for the seven serotypes tested. The flow cytometric assay is rapid (∼4 h) with high throughput (∼50 serum samples per day per technician) and provides a reproducible measurement of serotype-specific functional antibodies, making it a highly suitable assay for the evaluation of the immune responses elicited by pneumococcal vaccines.