Method Description

The indirect immunofluorescent antibody (IFA) assay is a 2-stage "sandwich" procedure. In the first stage, the patient serum is diluted in Pretreatment Diluent for IgM and phosphate buffered saline (PBS) for IgG, added to appropriate slide wells in contact with the substrate, and incubated. Following incubation, the slide is washed in PBS, which removes unbound serum antibodies. In the second stage, each antigen well is overlaid with fluorescein-labeled antibody to IgM and IgG. The slide is incubated allowing antigen-antibody complexes to react with the fluorescein-labeled anti-IgM and anti-IgG. After the slide is washed, dried, and mounted, it is examined using fluorescence microscopy. Positive reactions appear as cells exhibiting bright apple-green cytoplasmic fluorescence against a background of red negative control cells. Semi-quantitative endpoint titers are obtained by testing serial dilutions of positive specimens.(Package inserts: Arbovirus IFA IgM and Arbovirus IFA IgG Instructions for Use; Focus Diagnostics; Rev. 03, 02/17/2023)