To Perform Enzyme-Linked Immunosorbent
Assay^38
Enzyme-linked immunosorbent assay (ELISA) is an immunoassay that combines the
specificity of antibody and antigen usually detected in presence of enzyme conju-
gated with antibody. The enzyme linked with the antibody reacts with specific
substrate to produce colored product which is related to antigen or antibody
concentration.
38.1 Principle........................................
Enzyme-linked immunosorbent assay is based on immunochemical principles of
antigen-antibody reaction. The antibody to be determined is coated on an inert
surface. The sample containing protein is applied on antibody-coated surface. The
protein-antibody complex is then reacted with second antibody specific to protein.
This second antibody is enzyme labelled (alkaline phosphatase, horseradish peroxi-
dase, orβ-galactosidase). The unbound enzyme-linked second antibody is washed,
and enzyme activity is determined by adding suitable substrate specific to enzyme
bound with second antibody (i.e., diaminobenzidine for horseradish peroxidase).
This gives a colored product that is directly proportional to the protein being
estimated (Fig.38.1).
The above mentioned method is called indirect ELISA. Other variants of ELISA
are also in use.
38.2 Sandwich ELISA
Sandwich ELISA is named so because the analyte to be measured is bound between
two primary antibodies–the capture antibody and the detection antibody. In this
process, the antibody is immobilized on microwell plate, and the sample containing
antigen is added to this well that reacts with immobilized antibody. The well is
washed to remove unbound antigen, and second antibody that has been conjugated
#Springer Nature Singapore Pte Ltd. 2018
V. Kumar, K. D. Gill,Basic Concepts in Clinical Biochemistry: A Practical Guide,
https://doi.org/10.1007/978-981-10-8186-6_38
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