2-SITE IMMUNOMETRIC ASSAY FOR SUBSTANCE-P WITH INCREASED SENSITIVITY AND SPECIFICITY

A two-site immunometric assay of the undecapeptide substance P (SP) ha s been developed. This assay is based on the use of two different anti bodies specifically directed against the N- and C-terminal parts of th e peptide. Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antib odies. A monoclonal antibody directed against the carboxy-terminal par t of substance P, covalently coupled to the enzyme acetylcholinesteras e, was used as tracer antibody. The assay is very sensitive, with a de tection limit close to 3 pg/mL. Precision is also very good, with a co efficient of variation of less than 10% in the 10-250 pg/mL, range. Mo re important, the assay is fully specific for SP since cross-reactivit y coefficients below 0.01% were observed with other tachykinins, SP de rivatives, and SP fragments. The assay was used to measure the SP cont ent of rat brain extracts and was validated by HPLC experiments.