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

摘要

A two-site immunometric assay of the undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide. Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy-terminal part of substance P, covalently coupled to the enzyme acetylcholinesterase, was used as tracer antibody. The assay is very sensitive, with a detection limit close to 3 pg/mL. Precision is also very good, with a coefficient of variation of less than 10% in the 10-250 pg/mL, range. More important, the assay is fully specific for SP since cross-reactivity coefficients below 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay was used to measure the SP content of rat brain extracts and was validated by HPLC experiments.