CHARACTERLZATION OF THE MONOCLONAL ANTI-SUBSTANCE P ANTIMODY DERIVED FROM NC1/34

The rat-mouse heterohybridoma NC1/34 secretes a specific anti-substance P antibody (α-SP MAb). The relative affinities of this α -SP MAb for SP, SP fragments and selective neurokinin receptor (NKR) antagonists were determined by inhibition. N-terminal fragments of SP, C-terminal mono- and di-peptide fragments of SP and NKR antagonists did not prevent α -SP MAb binding to SP-BSA conjugate bound to the plates. The relative affinities of a-SP MAb for SP and its C-terminal fragments were: Sp6-11> Sp7-11> SP=Sp5-11, Sp4-11, Sp3-11 and Sp2-11> Sp8-11 and SP9-11. Alanine substitution studies showed that none of these peptides bound to monoclonal a- SP MAb, but all bound to polyclonal anti-SP Ab (serum). We conclude that the α -SP MAb binds to epitopic sites on SP's C-terminal. Amino acids 6 and 7, and, to a lesser extent, 8 and 9, determine affinity. The C-terminal carboxyl group has to be accessible for binding to occur.