Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

Abstract

A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. ß-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound ß-D-galactosidase activity considerably decreased with less decrease in specifically bound ß-D-galactosidase activity. As a result, the detection limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol) and human growth hormone (10 fg, 0.5 amol) by the present enzyme immunoassay were 30-fold lower than those by the conventional enzyme immunoassay, in which antigens were incubated with monoclonal mouse antibody IgG₁-coated polystyrene balls and rabbit antibody Fab′-ß-D-galactosidase conjugates.