Methods: 96-well plates were coated with capsular polysaccharides from S. pneumoniae (serotypes 4, 14, and 23F) or C3a antibody. Plates were then washed, blocked and washed again. The polysaccharide coated-plate was incubated with serum from SAD patients and controls supplemented with human complement. Serum was then transferred to the C3a-coated plate. The plate was washed and then incubated with biotinylated C3a antibody. After washing, the plate was incubated with streptavidin-HRP. The plate was washed again and then developed with TMB substrate; absorbance was measured at an OD of 450 nm.
Results: In this preliminary study, our complement fixation assay distinguished controls (healthy control, CVID patient treated with IVIg and IgG deficient patient with good response to pneumococcal polysaccharide vaccine) (n=3) from SAD patients (n=3). P values were <0.05 for each serotype. Discrepancy between a specific serotype titer and the result of complement fixation assay were noted both in controls and SAD patients.
Conclusions: Complement fixation assay is a new functional approach to diagnose SAD in adult patients. We will now apply this method to a larger number of patients in order to evaluate if our assay can correlate better than serotype measurement with the severity of the infectious phenotype.