Quantitative studies of immunofluorescent staining. Relationships of characteristics of unabsorbed antihuman IgG conjugates to their specific and non-specific staining properties in an indirect test for antinuclear factors

Abstract

Reproducible titres of indirect immunofluorescent (IF) staining with antinuclear factor (ANF)-containing sera could be obtained with different antihuman IgG conjugates by quantitative adjustments of their characteristics. Conversely, one ANF yielded a broad range of ANF titre (80-640) upon appropriate adjustments of the conjugate characteristics. The same and related characteristics of the conjugates also afforded a basis for quantitatively defining the conditions under which non-specific staining (NSS) appeared. The salient characteristics of the anti-IgG conjugates include: (1) their strength of antiglobulin (expressed as units/ml of precipitating antibody or mug antibody N/ml); (2) their apparent fluorescein concentration (in mug F/ml); (3) their protein concentration (in mg/ml). Optical and immunologic sensitivity ratios are calculated from these conjugate characteristics. Optical sensitivity (expressed as fluorescein concentration to protein concentration (F/P) ratios), immunological sensitivities (expressed as units/1% protein) and the dilution employed serve to characterize quantitatively anti-IgG conjugates adequately to define their specific and non-specific staining properties.