An antibody IgG-coated polystyrene ball was incubated with an antigen and then with affinity-purified Fab'-enzyme conjugate in the presence of normal IgG, F(ab')2, Fab' or Fab'-bovine serum albumin conjugate. After washing by incubation at 30[ddot]C for 10 min with shaking, the enzyme activity bound to the polystyrene ball was assayed. The non-specific binding of the Fab'-enzyme conjugate to the polystyrene ball considerably decreased in the presence of normal IgG and the other related proteins, while the specific binding decreased only slightly. As a result, the detection limit of hCG, human IgE and human α-fetoprotein was improved 3 to 10-fold.