Binding Characteristics of a Monoclonal β‐Endorphin Antibody Recognizing the N‐Terminus of Opioid Peptides

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Abstract
The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to .beta.-endorphin. An examination of its specificity against a series of human .beta.-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of .beta.-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, .beta.-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and .alpha.-neo-endorphin, whereas .beta.-lipotropin, .alpha.-N-acetyl-.beta.-endorphin, Des-Tyr1-.beta.-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for .beta.-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of .beta.-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. For the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.