Microfluorometric evaluation of conjugate-specificity with the defined antigen substrate spheres (DASS) system

Six fluorescent antihuman Ig preparations were tested for their Ig class specificity by reacting them with highly purified IgG, IgM, IgA, and OVA coupled covalently to Sepharose beads. OVA was used as a measure for nonimmunologic binding. Bead fluorescence was determined by microfluorometry. The amounts of USS and NSS were expressed quantitatively. These data were compared with the performance of these particular conjugates in a biologic system, namely, monoclonal bone marrow cells. Five of the six conjugates satisfied the requirement of monospecific activity; one did not. At a dilution of 1 : 8, the five monospecific conjugates reacted between five and 50 times stronger with their appropriate antigens than with OVA-coupled beads. Cross reactivity with other Ig classes, after correction for OVA staining was maximally 6%. The conjugate that was nonspecific in the bone marrow system gave very high cross reactivity with the Ig-coupled beads. A good correlation was found between OVA bead staining and nonimmunologic binding of conjugates in bone marrow slides. In this respect, conjugates prepared from antibody preparations isolated by solid immunoadsorbents proved to be superior to globulin or whole IgG fractions. Ig coupled to Sepharose beads seems to represent a very promising substrate for conjugate specificity testing.