Description
For Samples: Cell culture fluid & body fluid & tissue homogenate serum or blood plasma
Intended Uses: This HIF1a ELISA kit is intended for laboratory research use only and not for use in diagnostic or therapeutic procedures. The stop solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of HIF1a in the sample, this HIF1a ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus HIF1a concentration. The concentration of in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Principle of the Assay: The coated well immunoenzymatic assay for the quantitative measurement of HIF1a utilizes a polyclonal anti-HIF1a antibody and an HIF1a-HRP conjugate. The assay sample and buffer are incubated together with HIF1a-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 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 HIF1a concentration since HIF1a from samples and HIF1a-HRP conjugate compete for the anti-HIF1a antibody binding site. Since the number of sites is limited, as more sites are occupied by HIF1a from the sample, fewer sites are left to bind HIF1a-HRP conjugate. Standards of known HIF1a concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color (O.D.) to the concentration of HIF1a. The HIF1a concentration in each sample is interpolated from this standard curve