A quantitative approach for immunofluorescence in microscopy: The use of antibody multilayers on nuclei

Quantitative analysis of fluorescent antibody bound to chicken nuclei was carried out by measurement of the fluorescence on nuclei smears in a microscope and after solubilisation in a spectro-fluorometer. Three successive layers of antibody were used: 1. human anti-nuclear factor, 2. fluorescent rabbit γ2 (anti-human-IgG), 3. fluorescent goat γ2 (anti-rabbit-IgG). Titrations gave a straight line in a log versus log plot, the slope depending on the amount of antigen present. Saturation of the antigenic sites was in all cases achieved only at high antigen and antibody concentrations; four molecules of goat anti-rabbit-IgG were bound by one molecule of rabbit-IgG. A quenching of fluorescence in the second layer by antibody in the third layer was not observed. The fluorescence intensity measured was directly proportional to the number of fluorescein molecules (up to three) per molecule of antibody used. The limit for the detection of antibody lies at about 4·104 molecules per μ2 of measuring field. From the data it can be concluded that the antigen concentration in different parts of a tissue or in different tissues is identical if the amount of antibody bound at different antibody concentrations is the same.