Hemoglobin ELISA

This ELISA is used for quantitative determination of hemoglobin in stool. In a first incubation step, the hemoglobin in the sample is bound to anti-hemoglobin antibodies (in excess), which are
immobilized on the surface of the microtiter wells. To remove all unbound substances, a washing step is carried out. In a second incubation step an anti-hemoglobin peroxidase labeled antibody is added. After another washing step, to remove all unbound substances, the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic solution is then added to stop the reaction. The color converts from blue to yellow. The intensity of the yellow color is directly proportional to the concentration of hemoglobin in the sample. A curve of the absorbance unit (optical density, OD) vs. concentration is generated, using the values observed from the standards. The hemoglobin present in the samples is determined directly from this curve.