A new immunochemical method for detecting substance P receptors based on the biotin-streptavidin system
Golovnina, T.A.; Kasheverov, I.E.; Plaksin, D.I.; Iakunina, N.B.; Kovalenko, V.A.; Utkin, I.N.; Tsetlin, V.I.
Bioorganicheskaia Khimiia 18(7): 932-941
1992
ISSN/ISBN: 0132-3423 PMID: 1280142 Document Number: 398645
A novel method for detecting the membrane receptors of Substance P (SP) has been developed. The method does not require radioactive derivatives of SP and is based on quantitation of specifically bound biotinylated SP (Bt-SP), the analysis being performed after destruction of the ligand-receptor complex in acidic conditions and Bt-SP extraction into solution. The acidic extract from the Bt-SP-membrane complex after neutralization is added to immulon microELISA plate coated with affinity purified anti-SP-antibodies. The sensitivity of subsequent detection of Bt-SP enhances by using a new type of the streptavidin conjugate with a polymeric form of horseradish peroxidase. Enzyme immunoassay conditions were optimized using [125I]-labelled SP derivative. The developed method allows the determination of femtomoles of SP in a sample and has a sensitivity comparable to that of radioligand analysis.