The agar solidifies, immobilizing the cells. Antibodies secreted by B cells bind to the SRBC-antigens that are nearby. Complement proteins are then added, which react with the antibody-coated SRBCs, forming membrane attack complexes. The SRBCs that have antibodies bound to them lyse, leaving an area of clearing in the agar. B cells secreting antibodies other than IgM are usually not detected in the direct assay unless the amount of antibody produced is great enough to coat the SRBCs with about 30 nm between the antibodies.
Complement only binds to IgG when IgG can be cross-linked.
Complement proteins are then able to form membrane attack complexes on the SRBC and an area of clearing appears. Th cells help B cells make antibodies. The absence of T helper cells specific for the immunogen prevents a response. This form of B cell tolerance helps prevent B cell activation by self antigens that are abundant in some sites of the body, but not present in bone marrow. High dose immunization with multiple immunogens may be less effective than administration of each immunogen separately.