Quantitative studies of immunofluorescent staining. I. Analyses of mixed immunofluorescence.

Mixed anti-globulin immunofluorescence (or mixed IF) was analysed using the following model system: calf thyroid sections, human anti-nuclear factor (ANF), rabbit antisera to human IgG and fluorescein-labelled human IgG. This system was characterized on the basis of: (1) the immunoelectrophoretic demonstration of antibodies to human IgG in anti-globulins; (2) titration of the antiglobulins and labelled globulins; and (3) determination of molar fluorescein to protein (F:P) ratios. Titration of anti-globulins (rabbit anti-human IgG) by gel precipitation afforded an assay of units' of antibody activity. Similarly, units' of labelled globulin antigen were determined by a gel precipitation titration. Block titrations of these components of the indicator system against ANF serum yielded constant titres or `plateaux' of nuclear staining over a range of units of anti-globulin and of labelled immunoglobulin. Thus, the following predictions can be made. Optimal mixed IF staining may be attained with 4 units or more of an anti-globulin (anti-human IgG) and with 1 unit or more of a fluorescein-labelled human immunoglobulin if the latter has an F:P ratio in the range of about 1:1 to 3:1. The titre of the ANF appears to be proportional to the F:P ratio over this range.