Background: The detection of antibody to hepatitis C virus (HCV) is an important assay for the identification of individuals infected with this virus. However, the confirmation of antibody positivity remains problematic. Currently none of the screening or confirmatory assays provide quantitation of the antibodies present. A microsphere assay was designed to provide improved confirmation.

Methods: Microspheres of 3.6 mum in diameter coated with NeutraAvidin were used to capture biotinylated HCV recombinant proteins. A phycoerythrin goat anti-human immunoglobulin G (IgG) was used to detect specific antibody captured to the microsphere. A human IgG calibrator was designed that was internal to each sample in the microsphere assay.

Results: Detection of HCV-specific antibody using these microspheres was straightforward in most samples, with the lower detection limit set at 0.01 microg equivalents of human IgG per milliliter. In antibody-positive samples, the HCV antibody levels ranged from 0.09 to 55 microg equivalents of IgG per milliliter. Forty-nine of the 54 samples (91%) previously identified as having an indeterminate serologic pattern were negative in the microsphere assay.

Conclusions: The microspheres and biotinylated HCV proteins were stable for longer than 8 months when stored at 2 degrees C to 8 degrees C and coating of the microspheres was reproducible with an interassay coefficient of variation less than 10%. Avidin-coated microspheres provide an easy solid support on which to design an assay provided the capture reagent being used can be biotinylated effectively. Confirmation of antibody to HCV can be performed using this assay format.