Quantitative studies of peroxidase labeled antibody. I. Indirect staining system analyzed by chessboard titraions

The sensitivity and specificity of immunohistological staining procedures employing horseadish peroxidase labeled antibody were evaluated with the aid of chessboard titrations. Two indirect staining systems, detecting antinuclear antibody and pemphigus intercellular antibody, were examined with reference to indirect immunofluorescence. In both systems, chessboard titration results showed that plateau titers of serum antibodies appeared to be a function of label content. The plateau endpoints reflected the anti-immunoglobulin concentrations of conjgates. With a conjugate of molar enzyme to protein ratio (E/P) of 1.1, the sensitivity of indirect staining appeared to be equivalent to that of immunofluorescent staining performed with a conjugate with molar fluorescein to protein ratio of 4.8. Sensitivity decreased sharply with conjugates of low E/P rations. Three methods of assaying the peroxidase content of conjugates were evaluated for reproducibility and sensitivity in relation to staining properties.