Substance P Radioimmunoassay Using Nα-Tyrosyl-substance P and Demonstration of the Presence of Substance P-like Immunoreactivities in Human Blood and Porcine Tissue Extracts

Abstract

A sensitive and specific radioimmunoassay for substance P was developed using synthetic substance P and 125I-N.alpha.-tyrosyl-substance P. Substance P-human .alpha.-globulin conjugate was used for production of anti-substance P antisera in rabbits. Synthetic substance P was used as a standard, and the dextran-coated charcoal method was employed to separate the free peptide from that bound to antibodies. The absence of cross-reactions by physalaemin and eledoisin observed in this system proved its high specificity to substance P. N.alpha.-Tyrosyl-substance P and [Tyr1]-substance P showed the displacement curves to be indistinguishable from that of the standard substance P. Neither substance P5-11 nor substance P6-11 competed with the tracer at the concentration used. The minimum measurable dose of substance P by the assay system was 2.5-5 pg/incubate. Human plasma samples from 42 healthy volunteers of both sexes were contained immunoreactive substance P in amounts that averaged 298 pg/ml in the male and 251 pg/ml in the female. Substance P-like immunoreactivity was also demonstrated in hot-H2O extracts of porcine duodenum, jejunum, ileum, cecum, middle colon, rectum, pancreas, stomach and pituitary. The highest concentration (379 ng/g wet wt of organ) was found in the pituitary, and the ileum (7.9 ng/g wet wt of organ) and jejunum (1.9 ng/g wet wt of organ) were rich in the contents.