Objective To establish a multiplexed opsonophagocytic killing assay (MOPA) for detection antibodies against Streptococcus pneumoniae and to evaluate the immune effect of pneumococcal vaccines.
Methods Assay stocks containing seven vaccine-related serotypes of Streptococcus pneumoniae and HL-60 cell bank were produced according to the MOPA method published by University of Alabama at Birmingham, USA. The antibiotic sensitivity, resistance and optimal dilution of assay stocks were determined. The cell surface markers and cell viability of HL-60 cells were monitored after differentiated for 5 – 6 days by using flow cytometer. Meanwhile, the opsonization indices (OI) of standard reference serum 007SP and reference serum 09CS against seven serotypes were measured with MOPA method. Then MOPA method was employed to detect functional antibody of 6 pairs of sera samples obtained before and after vaccination.
Results The dilution factors of all assay stocks were > 300. The expressions of cell surface markers CD11b, CD35 and CD71 were > 80%, > 70% and < 15%, respectively after differentiated for 5 – 6 days. The proportions of live cells and apoptotic cells were > 70% and < 21%. The nonspecific killing rates of complement were < 30%. The OI of standard reference serum 007SP and reference serum 09CS were stable and were within the quality control ranges. After vaccine immunization, high antibody levels were detected in sera samples and the killing curves were of general sigmoid shape.
Conclusion MOPA method was successfully established in this study, which provide the basis for evaluating the efficiency of pneumococcal vaccines.
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