Approach to validating an opsonophagocytic assay for Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (pneumococcus) polysaccharide serotype-specific antibodies that have opsonophagocytic activity are considered a primary mechanism of host defense against pneumococcal disease. In vitro opsonophagocytic assays (OPAs) with antibody and complement to mediate opsonophagocytic killing of bacteria have been designed and developed as an adjunct to the standardized serum immunoglobulin G antipneumococcal capsular polysaccharide enzyme immunoassay to assess the effectiveness of pneumococcal vaccines. OPA presents challenges for assay standardization and assay precision due to the multiple biologically active and labile components involved in the assay, including human polymorphonuclear leukocytes or cultured effector cells, bacteria, and complement. Control of these biologically labile components is critical for consistent assay performance. An approach to validating the performance of the assay in accordance with International Conference for Harmonization guidelines, including its specificity, intermediate precision, accuracy, linearity, and robustness, is presented. Furthermore, we established parameters for universal reagents and standardization of the use of these reagents to ensure the interlaboratory reproducibility and validation of new methodologies.

FIG. 1.

The specificity of the pneumococcal serotype 4 OPA was evaluated by determination of the inhibition of opsonophagocytic activity when homologous PnPs (PnPs 4), heterologous PnPs (PnPs 1), unrelated polysaccharide (MnCPs), or no competitor (0 ng/ml) was added to the OPA.

FIG. 2.

The specificity of the pneumococcal OPA was evaluated by determination of the inhibition of opsonophagocytic activity with sera from nonvaccinated adults with 125 μg of homologous, heterologous PnPs (PnPs 1 for the serotype 4, 5, 6B, and 9V OPAs and PnPs 5 for the serotype 1, 14, 18C, 19F, and 23F OPAs), or unrelated MnCPs per ml. Homologous inhibition is shown as solid symbols, while heterologous or unrelated MnCPs inhibition is shown as hollow symbols. Pn, pneumococcal serotype.

FIG. 3.

Linearity of the results for two human serum specimens in the pneumococcal serotype 5 OPA. Each specimen was tested at four different initial concentrations. Linear correlation analysis was performed after log₂ transformation of the data. The resulting slopes and the Pearson correlation coefficients (r) for each line are indicated.

FIG. 4.

Effect of HL60 cell age (expressed in cell passage) on opsonophagocytic activity. Dilutions of a serum specimen from a nonvaccinated adult were tested in the pneumococcal serotype 6B OPA with day 3 HL60 cells (400:1) from postdifferentiated passage 51 (p51; solid line) and 111 (p111; dotted line). Incomplete opsonophagocytic activity (i.e., high baseline numbers of CFU [>20]) was observed when high-passage-number (>80) differentiated HL60 cells were used. However, the OPA titer assigned to the serum specimen remained the same when qualified HL60 cells from a different passage were used.

FIG. 5.

Changes in effector cell/target bacterium ratio on pneumococcal serotype 9V OPA performance. The numbers of CFU obtained in the pneumococcal serotype 9V OPA with one serum specimen are shown by using PMNs or HL60 cells (passage 19) at day 4 postdifferentiation at 400:1 (solid lines) and 50:1 (dotted lines). A single analyst performed the comparison study on the same day using the same bacterial culture to minimize variation.