MAB5276 Sigma-AldrichAnti-Endorphin β Antibody, clone 3-E7

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. These data indicate that 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.

Six myeloma cell hybrids producing antibodies to human beta-endorphin were isolated from a single mouse spleen. The monoclonal antibodies displayed different binding patterns with the antigen. We report the characterization of one antibody which recognizes the tetrapeptide Tyr-Gly-Gly-Phe representing the message sequence found at the NH2 terminus of all naturally occurring mammalian opioid peptides. Competition experiments in radioimmunoassay and immunohistochemistry show that the antibody fails to bind the beta-endorphin precursor beta-lipotrophin, does not discriminate among opioid peptides that share the same message sequence but have different COOH-terminal extensions, and does not react with pharmacologically inactive derivatives of beta-endorphin. The antibody recognition of the message sequence of natural opioid peptides is sensitive to those molecular changes that affect their receptor binding competence.

Opioid peptide-like (OPL)-immunoreactivity and (the GABA-biosynthetic enzyme) glutamic acid decarboxylase-like (GAD)-immunoreactivity were localized in rat neostriatum and central amygdaloid nucleus (ACE) using a polyclonal sheep antiserum to rat brain GAD and a monoclonal mouse antibody to the N-terminus of beta-endorphin (3-E7) as primary antisera. PAP-immunohistochemistry revealed GAD-immunoreactivity in the majority of neurons in neostriatum and ACE. OPL-immunoreactivity was observed in numerous neurons in ACE, but only in few neostriatal nerve cells. In double immunofluorescence in the same section OPL- and GAD-immunoreactivity colocalized in few medium size cells in the neostriatum, but in numerous neurons in ACE. The existence of opioid peptide containing GABAergic neurons in ACE and neostriatum is demonstrated.

The properties of a mouse monoclonal antibody to beta-endorphin secreted by a clone of hybrid myelomas (3-E7) are described. The antibody displays virtually complete cross-reactivity to met-enkephalin and leu-enkephalin, but no cross-reactivity to beta-lipotropin, alpha-N-acetyl-beta-endorphin and des-Tyr1-beta-endorphin. Substantial cross-reactivity is seen with some other naturally occurring opioid peptides bearing the enkephalin sequence, such as dynorphin, alpha-neo-endorphin and BAM 22, but cross-reactivity is lacking in the case of certain synthetic enkephalin derivatives possessing a D-amino acid in position 2. The data indicate that for the binding of an antigen to the antibody the N-terminal tyrosine moiety is essential. The antibody recognizes, thus, a site which is of functional significance for the interaction of many naturally occurring opioid peptides with the opiate receptor.