A NONISOTOPIC IMMUNOASSAY FOR GUANOSINE 3'5'-CYCLIC MONOPHOSPHATE USING A CYCLIC GMP-BIOTIN CONJUGATE AS TRACER

2'-O-monosuccinylguanosine 3':5'-cyclic monophosphate was coupled to N -biotinyl-1,8-diamino-3,6-dioxaoctane after converting succinyl-cGMP i nto its N-hydroxysuccinimide active ester. Isolation and purification of the succinyl-cGMP-biotin conjugate was performed with FPLC(R) using reversed phase chromatography. The synthesis described yielded a conj ugate suitable for use as tracer in immunoassays for the cGMP measurem ent in plasma and urine samples. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for flexible end poin t determination by means of commercially available labeled streptavidi n derivatives. Streptavidin-europium was used in conjunction with the DELFIA(R)-system for time-resolved fluorometric end point measurement (TR-FIA), streptavidin-horseradish peroxidase was used for colorimetri c end point determination (EIA). Both non radioactive immunoassay syst ems showed excellent correlation with the reference radioimmunoassay, good sensitivity and reproducibility. The succinyl-cGMP-biotin conjuga te was shown to be stable for more than two years without any apparent loss of chemical stability or immunological reactivity.