Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans

Abstract

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 μm in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract, a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10 −6 and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.