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

Abstract

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.

Modification of Periodate Oxidation Method to Produce HRP-IgG Conjugate and Test its Stability Overtime

“…According to the protocol of Eivazi [16]. Finally, the conjugates were dialyzed against PBS at +4˚C overnight as per protocol of Nakane and Kawaoi [7].…”

Conjugation of Peroxidase from Brassica oleracea gongylodes for Use as a Label- Prospect of a Novel Enzyme Tag for Immunoassay Systems

“…have found applications in conjugation procedures. (O'Sullivan et al, 1978;Nygren & Hansson, 1981;Jeanson et al, 1988;Ray et al, 2012, Ramesh Kumar et al, 2014. The enzyme salted out at 40-80% ASS was treated with 5, 10 and 15 mM PI and the glycosyl content was estimated.…”

Horseradish peroxidase-labeled rabbit anti-non-structural protein 1 of dengue virus-2 for the diagnosis of dengue virus infections

“…Before labeling with HRP, the anti-NS1 antibody was purified with Sephadex® G-100 (Pharmacia fine chemicals). The anti-NS1 antibody was conjugated to HRP as described by Ramesh Kumar et al 15 First, the HRP was activated by dissolving 2 mg of HRP (Sigma Aldrich, Missouri) in 0.5 ml of distilled water, and 0.2 ml sodium periodate (NaIO4) 0.1 M was added. The solution was gently inverted for 2 hours at room temperature until the color changed from orange to green.…”