Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors

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Abstract
Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various human cancers by multiple mechanisms, which include direct effects on tumor cells through the splice variants (SV) of the GHRH receptor. Our findings suggest that the tumoral protein encoded by SV 1 (SV1) is a likely functional receptor. The aim of this study was to develop a polyclonal antiserum against a polypeptide analog of segment 1-25 of the putative SV1 receptor protein. Rabbits were immunized with [Ala-23]SV1 (1-25)-Tyr-26-Cys-27-NH2 as a hapten, conjugated to BSA or keyhole limpet hemocyanin. The antisera thus generated were evaluated by RIA for binding to the radiolabeled hapten. The specificity and sensitivity of the antisera were studied on xenografts of RL and HT human non-Hodgkin's lymphomas. The sera raised against keyhole limpet hemocyanin-SV1 hapten, showed binding values of 50-75% at a 1:56,000 dilution. In Western blot analyses, the purified polyclonal antibody recognized a specific signal with a molecular mass of ≈40 kDa in RL and HT lymphomas. This band corresponds to the estimated molecular mass of the GHRH receptor isoform encoded by SV1. RT-PCR and ligand binding studies also revealed the expression of SV1 and the presence of high-affinity binding sites for GHRH on RL and HT tumors. Because the antiserum developed recognizes the tumoral GHRH receptor protein encoded by SV1, it should be of value in various investigations.

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