|
Protocol
|
The coated well immunoenzymatic assay for the quantitative measurement of serum 8-epi-PGF2α utilizes a monoclonal anti-8-epi-PGF2α and a 8-epi-PGF2α-HRP conjugate. The assay asample and buffer are incubated together with anti-8-epi-PGF2α antibody coated plate for sixty and washed. The diluted 8-epi-PGF2α-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping solution is added to stop the reaction, which will then turn the solution yellow.The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the 8-epi-PGF2α concentration since 8-epi-PGF2α from samples and 8-epi-PGF2α -HRP conjugate compete for the anti-8-epi-PGF2α antibody . Since the number of sites is limited, as more sites are occupied by 8-epi-PGF2α from the sample, fewer sites are left to bind 8-epi-PGF2α-HRP conjugate.Standards of known 8-epi-PGF2α concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color (Optical Density) to the concentration of 8-epi-PGF2α.The unknown 8-epi-PGF2αconcentration in each sample is interpolated from this curve.
|
