A non-isotopic 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 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.
Dressendörfer
R A
RA
Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
Heim
J M
JM
Gerzer
R
R
Strasburger
C J
CJ
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
C094020:N(1)-biotinyl-N(8)-(cGMP-succinyl)-1,8-diamino-3,6-dioxaoctane; D001710:Biotin; D006152:Cyclic GMP