An enzyme-like polyclonal antibody with high catalytic activity was isolated and purified from the antisera of New Zealand rabbits. The hapten of mono-p-nitrophenyl phosphonate ester (4) was synthesized and coupled to bovine serum albumin (BSA) and keyhole-limpet hemocyanin (KLH). Ratios of the hapten/carrier were obtained in the range of 18:1 to 45:1. The conjugate KLH-5 was used as an immunogen to raise polyclonal antibodies in rabbits. Three carboxylates 1, 2, and 3 were used as chromogenic substrates to investigate the catalytic activities of the antibody. The immunoglobulin Gs (IgGs) from the antisera of rabbit-1 and -2 (showing the higher titer of 1:40.000) were purified bg (NH4)(2)SO4 precipitation and affinity chromatography on a protein A-Sepharose CL-4B column and were shown to be homogeneous by SDS-PAGE. This preparation of IgGs could catalyze the hydrolysis of the carboxylate 1 at pH 8.5 and 30 degrees C, the Michaelis constant K-m and the apparent rate constant k(cat) of the catalysis were found to be 13.1 mu m and 22 min(-1), respectively. Further studies of the catalytic antibody revealed the significant rate enhancement (k(cat)/k(uncat) = 5.0 x 10(1) - 5.0 x 10(3)) and the substrate specificity for the hydrolysis of analogous substrates. In the antibody-catalyed hydrolysis of carboxylate 1, the k(cat) exhibited a first-order dependence on [OH-] whereas the K-m was relatively unaffected in the range of pH 7.6 and 9.0.