Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAb4D8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 x 10(8) M(-1) and 9.6 x 10(8) M(-1), while their interactions to AK in solution are much weaker, with affinity constants of 7.0 x 10(4) M(-1) and 3.9x10(6) M(-1), respectively. Thus, McAb3D3 and McAb4D8 react preferentially to the immobilized AKs. Since proteins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAb4D8 are directed against non-native AK.