Analysis of Enzyme-Linked Assays - Essay Example

The substrate specificity of HRP in forming complex I am very high; and the only reagents that can combine with HRP to form an active substrate complex are hydrogen peroxide (H2O2), methyl hydrogen peroxide (MeHOO), and ethyl hydrogen peroxide (EtHOO). The specificity of the enzyme-substrate complexes for hydrogen donors in equations 2 and 3 is quite low, as a result, AH2 can be a variety of compounds, and many different bindings or linkings are possible. In this way, antibodies are conjugated with enzymes to amplify the signal via the catalytic properties of the enzymes. HRP conjugates are ideally used for ELISA or enzyme-linked immunosorbent assays. HRP has a fast catalytic rate and can generate more products in a shorter incubation time, allowing for greater sensitivity. HRP-labeled antihuman IgG can be used to bind to target antigens in an assay system to localize even trace amounts of biomolecules.

These biomolecules produce tyrosin bonds by reaction with HRP to produce (Adopted from Rob, P.M. et al. 2002). When used for an assay this will give rise to a reaction like Different types of linked assays and their properties: There are mainly two types of ELISA assays. 1. Sandwich.2. Competitive.Sandwich: This is also known as a two-antibody sandwich ELISA. This is used for the determination of antigen concentration in unknown samples. With a purified antigen standard, it can determine the absolute amount of antigen in a sample.

This requires two antibodies, capture, and detection, that bind to epitopes that do not overlap. Two monoclonal antibodies that recognize discrete sites on the antigen or two polyclonal affinity purified antibodies are used. The capture antibody is purified and bound to the solid phase. The antigen is added, and antigen-antibody binding occurs. This creates a sandwich. Quantification of the assay is then made by measuring the amount of labeled second antibody bound to the matrix by the use of a colorimetric substrate (Please see the picture).