Journal of Chromatography & Separation Techniques
Open Access

Abstract

Anti-Human IgG-Horseradish Peroxidase Conjugate Preparation and its Use in ELISA and Western Blotting Experiments

Ramesh Kumar K, Sneha Xiavour, Swarna Latha, Vijay Kumar and Sukumaran

Studies of our experiment are related to the binding of enzymes to antibodies. This involves the formation of a stable, covalent linkage between an enzyme & antibody. We used different Enzymes for this studies which are altered in binding action. Enzymes we used-e.g. Horseradish peroxidase (HRPO), urease, or alkaline phosphatase. Studies of Enzyme and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site nor the active site of the enzyme is functionally altered. The enzyme most commonly used in the immunoreagent (the antibody enzyme conjugate) preparation is horseradish peroxides. This enzyme is cheap and can be attached to the immunoreagent by a variety of methods. Moreover many chromogenic substrates for it are also available. Our Experiment studies on conjugation of Horseradish Peroxidase (HRPO) to antibody (anti human IgG) will be carried out by Periodate oxidation method. We have prepared Anti human IgG-HRP conjugate in our laboratory. This will be used for ELISA and western blot experiments and IgG purified from different human serum samples would be used as the antigen in the above experiments. This experiment is carried out in two different steps mentioned as: Preparations of anti-human IgG-Horseradish peroxidase conjugate, Characterization of isolated human IgG.