Monoclonal antibodies to human prolyl 4-hydroxylase

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Abstract
Monoclonal antibodies against human propyl 4-hydroxylase (P-4-H), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human P-4-H and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiterplate well cultures produced antibodies to human P-4-H; 6 of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the 6 lines were further characterized. All of the 6 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 2 pairs of different subunit proteins, .alpha. and .beta.. Only 1 clone which produced antibodies to the .alpha. subunit was obtained, the other 5 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 P-4-H from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas 1 antibody reacted with purified chick and mouse P-4-H when examined by the western blotting technique. This antibody caused a strong inhibition of human P-4-H activity, but the other 5 antibodies had negligible inhibitory effect on the activity of the enzyme.