A flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibodies against Streptococcus pneumoniae.

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Ms Dharmavaram Sravani (1), Dr Geetha N (1), Dr KL Ravikumar (1)
1. Central Research Laboratory,KIMS

Streptococcus pneumoniae, Flow based opsonophagocytic assay, Functional antibodies, Vaccine efficacy.

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Streptococcus pneumoniae remains one of the most significant causes of morbidity and mortality worldwide. The use of vaccines has been promoted as a means of preventing the spread of multidrug-resistant pneumococci. The licenced pneumococcal polysaccharide and conjugate vaccines has been demonstrated to be both secure and, in the vast majority of investigations, effective in lowering the occurrence of invasive illness. Immune responses to pneumococcal vaccines have been evaluated with assays that measure total binding antibody, such as ELISA and functional antibodies such as opsonophagocytic assay (OPA). Current methods for assessing functional antibody immunity are expensive, time consuming and labour intensive. High throughput sensitive and specific automated Flowcytometry assay is an efficient alternate platform that provides large immunogenicity data. Here we describe a rapid, sensitive, and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows the measuring of uptake of fluorescent bacteria labeled bacteria


S.pneumoniae serotype strains 4,6B,9V, 14, 18C,19F, and 23F were labelled with fluorescent dye(FITC) following standard bacterial labelling protocols (Martinz et al 1999 and Jansen et al 1998). Labelled bacteria were used in the Opsonophagocytic assay for control samples, Pre-PPV23 and Post-PPV23 vaccinated serum samples and reference serum. Samples were analyzed using a DxFlex (Beckman Coulter) and 3000 gated cells were analyzed per tube. The opsonophagocytic titter is defined as the reciprocal of the dilution with 50% of the maximal percent uptake for each sample.


Measurement of functional antibody activity was demonstrated by increased fluorescence of HL-60 containing phagocytosed pneumococci. The opsonophagocytosis, i.e., fluorescence, was dependent upon the amount of functional antibody present in each sample and behaved in an antibody concentration-dependent manner. The maximum percent uptake of FITC labelled pneumococci by differentiated HL-60 cells in post vaccination serum was similar for all serotypes tested, with a mean ±1 standard deviation uptake for all serotypes. The reproducibility of the flow opsonophagocytic assay was assessed in triplicates (all serotypes included) of a single quality control serum. The titters for 85% of the assays were within ±1 dilution of the median titer, and the titers for 97% of the assays were within ±1 dilutions of the median titer.  Overall, the results of the flow cytometric assay correlated well (r = 0.92 and P = 0.001) with those of the manual viable assay.


The high-throughput flow cytometric assay developed was successfully optimized for use with Streptococcus pneumoniae. The assay proved to be sensitive and highly reproducible for the measurement of bacterial uptake opsonophagocytosis. The assay can be used in large-scale efficacy studies which assist in the assessment of functional antibodies.


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