A MONOCLONAL-ANTIBODY TO THE LIGAND-BINDING DOMAIN OF THE NEUROKININ-1 RECEPTOR (NK1-R) FOR THE NEUROPEPTIDE SUBSTANCE-P

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclon al antibodies, we selected the mAb12 antibody which specifically cross reacted, through its paratope, with a binding site present on membrane s from rat parotid gland cells, with an affinity close to 2 X 10(-7) M and with membranes from CHO cells expressing human brain NK1 receptor s. Immunocytochemical investigations using mAb12 revealed immunostaini ng whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and imm unocytochemical experiments, the competition occurring between the ant ibody and substance P, or a substance P-protein conjugate, indicates t hat mAb12 recognizes a membrane epitope located at or near the substan ce P binding domain on the NK1 receptor. Immunization of mice with mAb 12 led to the production of specific anti-substance P antibodies, agai n suggesting that mAb12 shares common structural features with the neu ropeptide. This monoclonal antibody can now be used in further biochem ical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for desi gning peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulat ion, inflammation or chronic pain.