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 samples. The bi- otinylated peptide interacts with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes the substrate solution. 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 competi- tive binding of the biotinylated peptide with the standard peptide or samples to the peptide antibody (primary antibody). A standard curve of known concentration can be established accordingly. The unknown concentration in samples can be determined by extrapolation to this standard curve.
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