Description
Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to calprotectin. After a short incubation period, the plate is washed and horseradish peroxidase (HRP) conjugated human calprotectin specific monoclonal antibody is added to each well. After the second incubation period, a “sandwich” of solid-phase antibody - human calprotectin – HRP conjugated monoclonal antibody” is formed. The unbound monoclonal antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human calprotectin in the test sample. A standard curve is generated by plotting the absorbance versus the respective human calprotectin concentration for each standard on a point-to-point or 4-parameter curve fitting. The concentration of fecal human calprotectin in test samples is determined directly from this standard curve of the Calprotectin ELISA Assay