This Enzyme Immunoassay kit is designed to detect a specific peptide and its related peptides based on the principle of ÒcompetitiveÓ enzyme immunoassay.
The immunoplate in this kit is pre-coated with secondary antibody and the nonspecific binding sites are blocked. The secondary antibody can bind to the Fc fragment of the primary antibody (peptide antibody) whose Fab fragment will be competitively bound by both biotinylated peptide and peptide standard or targeted peptide in sample. The biotinylated peptide is able to interact with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes the substrate solution composed of 3,3Õ, 5,5Õ-tetramethylbenzidine (TMB) and hydrogen peroxidase to produce a blue colored solution. The enzyme-substrate reaction is stopped by hydrogen chloride (HCI) and the solution turns to yellow. The intensity of the yellow is directly proportional to the amount of biotinylated peptide-SA-HRP complex but inversely proportional to the amount of the peptide in standard solutions or samples. This is due to the competitive binding of the biotinylated peptide and the peptide in standard solutions or samples to the peptide antibody (primary antibody). A standard curve of a peptide with known concentration can be established accordingly. The peptide with unknown concentrations in samples can be determined by extrapolation to this standard curve.