Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens.

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.