Radioimmunoassay of substance P in lung perfusate

A radioimmunoassay of the undecapeptide substance P (SP) and its application in rabbit lung superfusate has been developed. The assay was based on the use of a radioactive tracer, 125I-SP-Tyr8, which showed, with an excess of antibodies, a specific binding greater than 90% and a nonspecific binding lower than 2%. This tracer was stable for 4 weeks at -20 degrees C. Its specific radioactivity was 384 Ci/mmol. The assay's lower limit of detection was 10 pg/ml (0.7 fmol). The determination of SP in rabbit lung perfusate required extraction and concentration using octadecylsilane cartridges. Under these conditions, the recovery of SP from experiments on lung perfusates (n=4) was 75.0+/-4.5%, and the intraassay and interassay coefficients of variation were 7.3+/-1.9% and 10.7+/-1.3%, respectively. Amounts of SP released in rabbit lung perfusates were determined following stimulation of pulmonary C fibers with capsaicin (10(-4) M). During the stimulation period, the values of SP from lung perfusates (n=6) increased significantly when compared with baseline values.