A Monoclonal Antibody to the Growth Hormone Receptor of Rabbit Liver Membranes*

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Abstract
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 P3×20 mouse myeloma cells using polyethylene glycbl 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.

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