Microfluorometric evaluation of the specificity of fluorescent antisera against mouse immunoglobulins with the defined antigen substrate spheres (DASS) system.

Abstract Highly purified MOPC-21 IgG 1 , MOPC-173 IgG 2a , MOPC-195 IgG 2b , MOPC-104 E IgM, and MOPC-315 IgA paraproteins, heterogeneous mouse IgG, Fab and Fc fragments of heterogeneous IgG were prepared and coupled to Sepharose beads. These beads were then used as artificial substrates to test the specificity of fluorescent antisera against mouse immunoglobulins by microfluorometry. By comparing the visual evaluation of stained plasma cells and measurements on beads, the highest permissible percentage impurity in a conjugate was determined. It was 14% for a fluorescein iso thiocyanate (FITC) and 6% for a tetramethyl rhodamine iso thiocyanate (TRITC) conjugate. By application of these criteria, 1 out of 7 tested commercial antisera and 6 out of 8 conjugates prepared in this laboratory proved to be satisfactory. The most common impurities were anti-light chain antibodies, as revealed by their reaction with Fab. With the bead system, a good impression of the specificity of an antiserum can be obtained. It gives, however, only approximate information on whether the conjugate will cause a high background staining in the biological specimen.