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Principle
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The DetectX® Direct Cyclic GMP (cGMP) Immunoassay kit is designed to quantitatively measurecGMP present in lysed cells, EDTA plasma, urine, saliva and tissue culture media samples. Pleaseread the complete kit insert before performing this assay.For samples where the levels of cGMP are expected to be relatively high, the regular format forthe assay can be used. For samples with expected low levels of cGMP, an optional acetylationprotocol can be used.The kit is unique in that all samples and standards are diluted into an acidic Sample Diluent,which contains special additives and stabilizers, for cGMP measurement. This allows plasma, urineand saliva samples to be read in an identical manner to lysed cells. Acidified samples of cGMPare stable and endogenous phosphodiesterases are inactivated in the Sample Diluent. A cGMPstandard is provided to generate a standard curve for the assay and all samples should be readoff the standard curve. A clear microtiter plate coated with an antibody to capture mouse IgGis provided and a neutralizing Plate Primer solution is added to all the used wells. Standards ordiluted samples, either with or without acetylation, are pipetted into the primed wells. A cGMPperoxidaseconjugate is added to the standards and samples in the wells. The binding reaction isinitiated by the addition of a mouse monoclonal antibody to cGMP to each well. After a 2 hour incubation,the plate is washed and substrate is added. The substrate reacts with the bound cGMPperoxidaseconjugate. After a short incubation, the reaction is stopped and the intensity of thegenerated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength.The concentration of the cGMP in the sample is calculated, after making suitable correction for thedilution of the sample, using software available with most plate readers.
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