An enzyme-linked immunosorbent assay for the rapid quantification of intracellular and extracellular guanosine 3',5'-cyclic monophosphate in cultured glial cells.
A sensitive enzyme-linked, indirect immunosorbent assay (ELISA) for the determination of guanosine 3',5'-cyclic monophosphate (cGMP) in glial cells is described. The assay uses an antiserum produced against succinylated cGMP and is based on the competition between endogenous cGMP and a fixed amount of immobilized antigen. The antibody exhibited a high degree of specificity with negligible cross reactivity with other nucleotides or related compounds. The standard curve, linearized using a logit-log transformation, had an operating range of 1 fmol/50 microl to 5 pmol/50 microl. The sensitivity of the assay was significantly increased by acetylation of standards and samples. Recoveries of cGMP from samples of cultured cells ranged from 85% to 105% with a mean recovery of 97% +/- 7%. Levels of cGMP measured with the ELISA were in agreement with the corresponding values obtained using radioimmunoassay. The present method provides for a cheap, sensitive and simple alternative to the commercially available cGMP assays.
Wellard
John
J
Physiologisch-chemisches Institut der Universität, D-72076 Tübingen, Germany.
Blind
Brigitte
B
Hamprech
Bernd
B
D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry; D002138:Calibration; D002478:Cells, Cultured; D003429:Cross Reactions; D006152:Cyclic GMP / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007202:Indicators and Reagents; D009457:Neuroglia / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay; D012015:Reference Standards; D015203:Reproducibility of Results
D000941:Antigens; D007202:Indicators and Reagents; D000469:Alkaline Phosphatase; D006152:Cyclic GMP