Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies

Abstract

Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well (r(2) > 0.95). Although 22 (out of 390; approximately 6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 mug/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.

FIG. 1.

Number of surviving bacteria (y axis) at various dilutions of serum (x axis). Serotype of pneumococci (Pn) was serotype 14 (top panel) or serotype 6B (bottom panel). For the top panel, the serum sample was absorbed with nothing (solid triangle), 5 μg/ml of serotype 14 PS (solid square), or a mixture of heterologous PS (open square). For the bottom panel, the serum sample was absorbed with nothing (solid triangle), 5 μg/ml of serotype 6B PS (solid square), 5 μg/ml of serotype 6A PS (symbol “X”), or a mixture of heterologous PS (open square). The heterologous PS pool had PS (5 μg/ml each) of all the unrelated serotypes, but PS of cross-reactive serotypes was omitted for serotypes 6A, 6B, 19A, and 19F. The cross-reactive PS was tested separately (also at 5 μg/ml). w/o, without.

FIG. 2.

Comparison of opsonization titers of 30 serum samples determined with MOPA4 (y axis) and with single-serotype OPA (SOPA) (x axis). Each panel contains an identity line (dotted line) and two lines indicating twofold deviation from identity (solid lines). The serotypes and the r² values are indicated in each panel. In the bottom row of the panels, two panels show comparisons for serotype 7F. In run 1, six samples (open circles) showed a more-than-twofold deviation, but their results deviated less than twofold in run 2. In most panels, several samples had opsonization titers below the detection limit, which was 4. These samples were assigned an opsonization titer of 2, and the number of such serum samples is indicated in each panel. Pn, pneumococcal serotype.

FIG. 3.

Opsonization titers of one serum sample for 13 serotypes with the E:T ratio of 400:1 (hatched bars), 200:1 (open bars), 100:1 (gray bars), or 50:1 (black bars). Because serotype 3 is an odd member, it was tested in the single-serotype OPA format. All other serotypes were tested in MOPA4 format. Since opsonization titers for serotypes 6A, 9V, and 23F displayed a marked trend of decreasing opsonization titers with decreasing E:T ratios, the experiment was repeated for these three serotypes. This trend was not reproducible (data not shown).