Monoclonal antibodies to human prolyl 4-hydroxylase

Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.