Antibody Labeling with HRP

Objectives
To learn coupling of antibody to enzyme Horse Radish Peroxidase (HRP)
To desalt the labeled antibody

Introduction
Based on the principle that an antibody specifically reacts with its corresponding antigen, several procedures have been developed in
which Antibodies or antigens labeled with appropriate indicators are used for the localization, detection or quantitation of substances of
biological interest. The earliest indicator molecules (labels) developed were the fluorochromes,introduced by A Coons and collaborators
in 1941,followed by the use of enzymes that produce colored reactions, the first one being Horse radish peroxidase .The first labeling
of antibodies was achieved by John Marrack in 1934. The most common used labels (tracers) are Radio elements, fluorochromes, heavy
metals and enzymes.
Enzymes as indicators were introduced by PK Nakane and GB Pierce (in 1966) in the USA and S Avrameas and J Uriel in France. Because
of the catalytic nature, Enzymes offer the possibility of high amplification associated with either simple or sophisticated systems of
detection and quantitation. Depending on the substrates available, these enzymes can be used for the localization or the measurement
of antigen or antibodies or both. Antibodies or antigens are labeled with enzymes either by covalent or by biospecific non-covalent
linkages. A number of cross-linking agents have been used to obtain covalent antibody-enzyme conjugates, but currently used agents
are only glutaraldehyde,m-periodate and maleimide derivatives.
Horseradish peroxidase (HRP) and Calf intestine alkaline phosphatase are the enzymes mostly used in immunoenzymatic techniques.
HRP is a glycoprotein which catalyzes the transfer of two electrons from a substrate to hydrogen peroxide and produces an oxidized
substrate along with water. In the case of protein detection, HRP substrates are designed to generate a chromogenic,
chemiluminescent or fluorescent signal upon oxidation and are relatively small having a molecular weight of 40kDa. This small size
allows greater penetration into sample tissues and cells and reduces the likelihood of interfering with the conjugated protein function.
HRP also has four lysines that are available for conjugation, which improves the efficiency of cross linking to a protein of interest.

Principle
Glycoproteins such as horseradish peroxidase and glucose oxidase and most antibody molecules can be activated for conjugation by
treatment with periodate. Polysaccharide residues in the HRP oxidized with sodium periodate to produce reactive aldehyde groups that
can conjugate with amino groups of IgG molecule and produce Schiff bases. This reaction should be performed in the dark to prevent
periodate breakdown and for a limited period of time (15-30 minutes) to avoid loss of enzymatic activity (figure 1). The relatively labile
Schiff bases can be stabilized by reduction to a secondary amine linkage with sodium borohydride (Reductive animation). The
borohydride is removed (Desalting) by gel filtration column. The efficiency of the enzyme conjugated antibody is tested by direct DotELISA and is expressed as the titre value of the conjugate.

Developed under a Research grant from NMEICT, MHRD

by
Amrita CREATE (Center for Research in Advanced Technologies for Education),
VALUE (Virtual Amrita Laboratories Universalizing Education)
Amrita University, India 2009 - 2015
http://www.amrita.edu/create
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