Quantitation of antigen in tissue by immunofluorescence image analysis

A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/micrograms) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.