The coated well immunoenzymatic assay for the quantitative measurement of analyte utilizes a multiclonal anti‐analyte antibody and an analyte‐HRP conjugate. The assay sample and buffer are incubated together with analyte‐HRP conjugate in pre‐coated plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzyme‐substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the
The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the analyte concentration since analyte from samples and analyte ‐HRP conjugate compete for the anti‐analyte antibody binding site. Since the number of sites is limited, as more sites are occupied by
analyte from the sample, fewer sites are left to bind analyte‐HRP conjugate.
Standards of known analyte 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 analyte. The analyte concentration in each sample is interpolated from this standard curve.
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