Description
Assay calibrators (antibody DM1 conjugate) and test serum samples are added directly to wells of a microtiter plate that is coated with specific anti-DM1 antibody. Subsequently, a horseradish peroxidase (HRP) conjugated DM1 is added to each well. During the incubation period, the antibody DM1 conjugate competes with the HRP conjugated DM1 for the limited binding sites of anti-DM1 antibody. An immune complex of well coated “anti-DM1 antibody – HRP conjugated DM1” is formed. The unbound 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, which is terminated with an acidic reagent (i.e. ELISA stop solution). The absorbance is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is inversely proportional to the amount of antibody-DM1 conjugate in the test sample. A calibration curve is generated by plotting the absorbance versus the respective antibody-DM1 conjugate concentration for each calibrator on a 4-parameter or log-logit curve fitting. The concentration of antibody-DM1 conjugate in test samples is determined directly from this calibration curve