Application of quantitative immunofluorescence to clinical serology: Antibody levels of Toxoplasma gondii

Abstract

Methods currently used for immunofluorescent reagent standardization require subjective visual comparison of reagents with control materials. Reactivities of reagents in immunofluorescence test kits vary from manufacturer to manufacturer. To solve these problems, a quantitative immunofluorescence (QIF) method which uses a calibrated photometric system and incorporates reducing agents into the mounting medium to reduce fading was developed to replace the visual method of endpoint determination. A uranyl glass slide was used to calibrate the instrument's voltage measurements, permitting daily comparisons and measurement of the instrument reading fluctuations. The QIF method was initially tailored to the determination of serum antibodies to Toxoplasma gondii by measuring the fluorescence intensity of individual tagged organisms. The nonspecific fluorescence intensity resulting from the counterstain was eliminated by of a red-suppressing filter. The dilution-correlated polar fluorescence component was removed by subtraction of the intensity for the matching negative control dilution from each sample dilution intensity. The QIF method showed a 94% correlation with the visual comparison method for 62 clinical specimens.