Figure 1: Scheme of dot-ELISA affinity test: Protein/peptide is applied onto PVDF membrane using dot blot unit. After incubation with primary antibody, the Ag- Ab complex is created. Stability of such immune complex is tested using chaotropic reagent (NH4SCN), whereby antibodies with lower affinity are washed away where as antibodies with higher affinity resist. Such immunecomplexes are detected by color development using a secondary antibody with HRP (horse radish peroxidase) in the presence of hydrogen peroxide and DAB as chromogen.
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