A Non-Isotopic Immunoassay for Guanosine 3′:5′-Cyclic Monophosphate Using a Cyclic GMP-Biotin Conjugate as Tracer

Abstract

2′-O-monosuccinylguanosine 3′:5′-cyclic monophosphate was coupled to N-biotinyl-1,8-diamino-3,6-dioxaoctane after converting succinyl-cGMP into its N-hydroxysuccinimide active ester. Isolation and purification of the succinyl-cGMP-biotin conjugate was performed with FPLC® using reversed phase chromatography. The synthesis described yielded a conjugate suitable for use as tracer in immunoassays for the cGMP measurement in plasma and urine samples. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for flexible end point determination by means of commercially available labeled streptavidin derivatives. Streptavidin-europium was used in conjunction with the DELFIA®-system for time-resolved fluorometric end point measurement (TR-FIA), streptavidin-horseradish peroxidase was used for colorimetric end point determination (EIA). Both non radioactive immunoassay systems showed excellent correlation with the reference radioimmunoassay, good sensitivity and reproducibility. The succinyl-cGMP-biotin conjugate was shown to be stable for more than two years without any apparent loss of chemical stability or immunological reactivity.