Affinity purification of β-endorphin-like material from NG108CC15 hybrid cells by means of the monoclonal β-endorphin antibody 3-E7

Abstract

Neuroblastoma × glioma hybrid cells (NG108CC15) were examined for the presence of β-endorphin-like material. In order to differentiate this β-endorphin-like material from crude cell extract, a procedure for immunoaffinity chromatography was developed. The monoclonal antibody 3-E7 employed possesses the unique property of recognizing the N-terminal sequence of virtually all endogenous opioid peptides, but not their precursors. By means of this immunoaffinity procedure about 90% of exogenous β-endorphin was recovered from 10 ml phosphate buffered saline samples. Affinity chromatography served as first-step purification of crude NG108CC15 cell extract for the separation and concentration of β-endorphin-like material. The eluate of the immunoaffinity gel was subjected either to Sephadex gel filtration or to high pressure liquid chromatography. Under either condition, immunoreactive β-endorphin which eluted with synthetic β-endorphin was detected. The concentration in six different batches varied from 4 to 17 fmol/10 8 cells. This would be 10–200-fold lower than that observed for the enkephalins or dynorphin A/α-neo-endorphin. It is concluded that the utilization of the monoclonal antibody 3-E7 for a first-step purification of cell extracts was an essential pre-requisite for the separation of β-endorphin-like material from the hybrid cells. The presence of enkephalin-like material, of dynorphin A/α-neo-endorphin-like material and of β-endorphin immunoreactive material suggests that NG108CC15 cells are able to generate opioid peptides related to the precursors pre-proenkephalin A, pre-proenkephalin B and pro-opiomelanocortin.