Microfluorometry of antigen-antibody interactions in immunofluorescence using the defined antigen substrate spheres (DASS) system. Sensitivity, specificity and variables of the method

Abstract

Antigens covalently bound to spherical agarose beads were used as an artificial substrate for quantitative immunofluorescence as described previously. The defined antigen substrate spheres system (DASS system) has been further investigated in regard to important variables of the method. The influence of the density of the beads suspension, of the antigen concentration per bead volume and of the incubation time on the resulting fluorescence intensity was investigated. An immunofluorescence staining procedure standardized according to these investigations resulted in a reproducible assay. The sensitivity of the system was tested using conjugates and antisera with defined antibody content. Anti-IgG antibodies could be detected with the direct immunofluorescence technique in a concentration of 100 ng/ml and anti-ovalbumin antibodies with the indirect immunofluorescence technique in a concentration as low as 10 ng/ml. The specificity of the DASS system has also been investigated with classical methods for testing the specificity of immunofluorescence reactions, such as the neutralisation and, the blocking test. It was found that specific reactions could be completely inhibited by absorption with the appropriate antigen and significantly reduced but not completely abolished by blocking with unlabelled antiserum. The possibilities of the DASS system in fundamental and applied immunofluorescence studies are discussed.