125I-labeled protein A as a general tracer in immunoassay: suitability of goat and sheep antibodies.
The immunoassay method in which 125I-labeled staphylococcal Protein A ([125I]PA) serves as a general tracer has been extended to include goat and sheep IgG antibodies. Goat and sheep IgG normally do not react significantly with PA. However, once IgG antibody is bound to immobilized antigen or hapten, binding of [125I]PA is enhanced markedly. Binding efficiencies of [125I]PA to immune complexed goat anti-human IgM, human IgE, methotrexate and sheep anti-IgE were determined and compared quantitatively to rabbit IgG with the corresponding specificity. Immunoassays were developed based on the inhibition of [125I]PA binding as a measure of antibody inhibition by fluid-phase homologous ligand. In terms of sensitivity and specificity, assays using goat and sheep antibodies were comparable to assays developed using rabbit IgG. Goat antibody to the monovalent hapten methotrexate behaved anomalously: for each concentration of IgG tested, there was an optimal amount of methotrexate beads that gave maximum binding of [125I]PA. In the other immune systems, for each antibody concentration maximum binding of tracer was a function only of the amount of immobilized antigen added. In contrast to the results obtained with solid-phase antigen, solutions containing antibody and amounts of antigen ranging from large antigen excess to antibody excess to antibody excess failed to react significantly with PA or [125I]PA.
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000918:Antibody Specificity; D000939:Epitopes; D006041:Goats / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007457:Iodine Radioisotopes; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods; D012756:Sheep / Q000276:immunology; D013205:Staphylococcal Protein A / Q000276:immunology*
D000907:Antibodies, Bacterial; D000939:Epitopes; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D013205:Staphylococcal Protein A