Application of an ectopic expression system for the selection of protein-isoform-specific antibodies. The monoclonal antibody K1C3 is specific for the RCK1 potassium channel

Abstract

Monoclonal antibodies were raised against a fusion protein consisting of a fragment of 141 amino acids of the C-terminal region of the rat brain voltage-gated K(+)-channel protein (RCK1) and the lambda N protein (fusion protein I). Selection of K(+)-channel-specific hybridoma cell lines was performed by means of an ELISA employing a fusion protein consisting of the K(+)-channel-specific peptide sequence and glutathione S-transferase (fusion protein II). For final selection of RCK1 isoform-specific antibodies, a panel of Xenopus oocytes was employed, each injected with cRNA coding for a specific RCK isoform (RCK 1, 2, 4 or 5). Several days after injection, cryosections of embedded oocytes were obtained and were employed in immunohistochemical analysis of antibody binding. Of five hybridoma supernatants from stable growing hybridoma cell lines, selected by the fusion-protein ELISA, one monoclonal antibody (denoted K1C3) recognized exclusively the RCK1-protein isoform, with the other four exhibiting different levels of cross-reactivity with other K(+)-channel isoforms, or with unknown protein(s) of non-injected oocytes. The expression of the RCK1 protein in the postnatal brain was studied using, as far as we are aware, the first example of the application of such isoform-specific antibodies.