A monoclonal antibody to the growth hormone receptor of rabbit liver membranes.

A monoclonal antibody which recognizes the [125I]human GH ([125I]hGH)-binding proteins of rabbit liver has been produced using hybridoma technology. A CB6F1/J mouse was immunized over a period of 82 days with a partially purified GH receptor (GHr) preparation. On the 83rd day, spleen cells from the mouse were fused with P3x20 mouse myeloma cells using polyethylene glycol 1540. Hydridomas were produced by selection in hypoxanthine, aminopterin, and thymidine in RPMI/1640 medium and screened for antibody production using a binding inhibition assay. Four antibody-secreting clones were isolated from the same primary well, and one of these was injected ip into mice to generate ascitic fluid. At a concentration of 1:10,000, the ascitic fluid inhibited 50% of the specific binding of [125I]hGH to rabbit liver GHr, and at higher concentrations, the ascitic fluid was capable of inhibiting 95% of the specific binding. The ascitic fluid does not bind [125I]hGH nor does it inhibit [125I]hGH binding to rat liver membranes, rabbit mammary gland, or IM9 lymphocytes. More than 90% of the antibody activity was abolished by goat antimouse immunoglobulin G antiserum. An immunoglobulin fraction from the ascitic fluid, precipitated by ammonium sulfate and coupled to activated CH Sepharose, specifically adsorbed an [125I]hGH binding moiety from Triton X-100-solubilized rabbit liver membranes. After dissociation by brief exposure to 0.1 M glycine (pH 2.0), the moiety retained hGH-binding activity. Preliminary experiments indicate that the antibody will be helpful in purification of the rabbit liver GH receptor.