Description
The Eagle Biosciences Human C-Peptide ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human C-peptide in serum and/or EDTA-plasma samples. The C-Peptide ELISA Assay Kit utilizes the “sandwich” technique with selected antibodies that bind to various epitopes of C-peptide. Assay standards, controls and samples are added directly to wells of a microplate that is coated with an anti-human C-peptide specific antibody. Simultaneously, a horseradish peroxidase-conjugated monoclonal C-peptide specific antibody is added to each well. After the first incubation period, the antibody on the wall of the microtiter well captures human C-peptide in the sample. A “sandwich” of “anti-C-peptide antibody --- human C-peptide --- HRP conjugated tracer antibody” is formed. The unbound tracer antibodies and other matrix protein from the test sample 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 human C-peptide on the wall of the microtiter well is directly proportional to the amount of C-peptide in the sample. A standard curve is generated by plotting the absorbance versus the respective human C-peptide concentration for each standard on point-to-point or 4 parameter curve fit. The concentration of human C-peptide in test samples is determined directly from this standard curve. Add 25 µL of Standards, Controls and samples into the designated microwells. Add 100 µL of the above diluted Tracer Antibody working solution to each well. Seal the plate wells securely, cover with foil or similar material to protect from light. Incubate the plate shaking, 450-450 rpm on an ELISA plate shaker at room temperature for 1 hr. ± 5 minutes. Wash each well 5 times by dispensing 350 µL of working wash solution into each well, and then completely aspirating the contents. Alternatively, an automated microplate washer can be used. Add 100 µL of ELISA HRP Substrate into each of the wells. Cover the plate with aluminum foil or similar material to avoid exposure to light. Incubate the plate static, at room temperature for 20 minutes. Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently. Read the absorbance at 450 nm with reference filter at 620 nm or 650 nm