title: 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.
authors.affiliation: Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
authors.forename: R A
authors.initials: RA
authors.lastname: Dressendörfer
authors.forename: J M
authors.initials: JM
authors.lastname: Heim
authors.forename: R
authors.initials: R
authors.lastname: Gerzer
authors.forename: C J
authors.initials: CJ
authors.lastname: Strasburger
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D002853:Chromatography, Liquid; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D007118:Immunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C094020:N(1)-biotinyl-N(8)-(cGMP-succinyl)-1,8-diamino-3,6-dioxaoctane; D001710:Biotin; D006152:Cyclic GMP