ELISA Technique in Immunochemistry


Immunochemistry, as the name suggests, is a branch of chemistry that deals with chemical aspects of the immune system. It mainly involves studying and interpreting antigen-antibody interactions. Immunochemistry offers simple, rapid, robust yet sensitive, and easily automated methods for routine analyses in clinical laboratories. Immunoassays are based on highly specific binding between an antigen and an antibody. An epitope (immunodeterminant region) on the antigen surface is recognized by the antibody’s binding site. The type of antibody and its affinity and avidity for the antigen determines assay’s sensitivity and specificity. Depending on the assay format, immunoassays can be qualitative or quantitative. They can be used for the detection of antibodies or antigens specific for bacterial, viral, and parasitic diseases as well as for the diagnosis of autoimmune diseases. Immunoassays can measure low levels of disease biomarkers and therapeutic or illicit drugs in patient’s blood, serum, plasma, urine, or saliva.

Immunoassays are bioanalytical methods in which the identification and quantitation of the analyte (protein, lipid, nucleic etc) is done by using the specific relationship of antigen and an antibody. The Immunoassay may involve primary and secondary labelled antibodies. The primary antibodies are those that binds to antigen and the secondary antibodies binds to primary antibodies and partially to the antigen. Analysis is achieved by measuring the labelled activity (e.g., radiation, fluorescence, or enzyme) in either of the bound or free fraction.

ELISA (which stands for enzyme-linked immunosorbent assay) is a technique to detect the presence of antigens in biological samples using an conjugated enzyme whereas in Radioimmuno Assay (RIA), we use radioactive molecules.

Principle –

In ELISA, an antibody conjugated with an enzyme reacts with the colorless chromogenic substrate like Tetramethylbenzidine (TMB) and produces colorful product. Such substrates are called chormogenic because of their ability to form colored product. Enzymes like galactosidase, alkaline phosphatase ad horseradish peroxidase are employed for ELISA. The ELISA is preferred more over RIA because of its sensetivity, safety and it is less costly. The antibodies used in ELISA are obtained from Monoclonal or Hybridoma Technology.


Based on the different procedure, various types of ELISA’s have been developed to conduct quantitative and qualitative studies using antigen and antibody specific relationship. The unknown concentration can be find out from the standard curve of known concentration of antibody.

Direct ELISA –

In Direct ELISA, the antigens are coated in the wells of the microtiter plate. The antibodies conjugated with enzyme are added. The excess or free antibodies are washed. The chromogenic substrate is added. The chemical reaction between conjugated enzyme and chromogenic substrate produces colored produced. This is measured using spectrophotometer. It is widely used for qualitative and quantitative studies of antigen.

Indirect ELISA –

In this type, antigens are coated in wells of microtitre plate. The primary antibodies (Ab1) (from serum/sample) are added in antigen-coated wells. They are incubated and allowed to react with each other. The free or excess antibodies (which are not bound to antigen) are washed. Then Secondary antibodies (Ab2) called anti-iso-type antibodies are added and they bind primary antibodies (Ab1) (which are already associated with coated antigens). These secondary antibodies are conjugated with enzymes. The free or excess secondary antibodies are washed. Now, the substrate is added which is specific for the conjugated enzyme. The enzyme and substrate reaction results into color formation of the product, which could be, measured by spectrophotometric plate readers. The Indirect Elisa is widely used for HIV detection, where viral antigens are coated on microtiter plate and over which patient’s serum is added (primary antibodies) followed by secondary antibodies. If patient is HIV positive, he/she will produce antibodies against HIV virus and hence it will bind to the coated antigens and test would be positive. Whereas if the patient is not infected with HIV virus, then there won’t be any antibodies in the patient serum and no antigen antibodies reactions would occur hence no color formation. Therefore, the test would be negative.

Sandwich ELISA –

In this type, the primary antibodies (Ab1) are coated and immobilized in the wells of microtitre plate in place of antigen. The sample-containing antigen is added and incubated. It allows occurring the antigen and antibodies reactions. The wells are washed in order to remove excess of antigens. The enzyme conjugated secondary antibodies (Ab2) are added. The secondary antibodies also bind to the sample antigen but at different epitope. The excess or free secondary antibodies are removed by washing. Then substrate specific to conjugated enzyme is added and then colored product is measured.

 Competitive ELISA –

The third type is competitive ELISA. In this, the antibodies are incubated with sample containing antigen. The reacted antigen-antibodies are added in the microtitre wells, which are already coated with the second antigen. The motive of competitive ELISA is to detect the quantity of antigen present in the sample. If more number of antigens is present in the sample then less number of antibodies will be available to bind second antigen (coated on wells). If less antigens were present in the sample than more number of antibodies would be available to bind the second antigen. The secondary antibodies conjugated with enzymes are added. They bind to the epitope of the primary antibodies. The excess secondary antibodies are washed. Substrate is added. The conjugated enzyme and substrate react forming the colored product, which can be measured. And hence it is used to measure the availability of primary antibodies. Less Primary antibodies means more antigen in the sample and vice versa. Therefore, lower the absorbance higher is the concentration of antigen in the sample.

Types of ELISA – Owen, J. A., Punt, J., Stranford, S. A., Jones, P. P., & Kuby, J. (2013). Kuby immunology. New York: W.H. Freeman.

Chemiluminescence –

Chemiluminescence is the production of light (electromagnetic radiation) caused by chemical reaction. In this ELISA technique, in place of chromogenic substrate, luxogenic (light producing) substrate is used. The most commonly used luxogenic compound is luminol. The Horseradish Peroxidase enzyme (HRP) react with luminol in presence of H2O2 and produces light generating product. This technique is more sensitive than chromogenic ones.

ELISA Chemiluminescence


  • The procedure is simple.
  • High specificity and sensitivity, because of an antigen–antibody reaction.
  • High efficiency, as simultaneous analyses can be performed without complicated sample pre-treatment.
  • Generally safe and eco-friendly, because radioactive substances and large amounts of organic solvents are not required.
  • Cost-effective assay, as low-cost reagents are used. 


  • Identification of cancer biomarkers for the early detection of cancer
  • Diagnosis of infections like HIV
  • Detection of antibodies in diseases (for example, mycobacterium antibody in tuberculosis)
  • Detection of West Nile Virus by MAC-ELISA
  • Measurement of cytokines or soluble receptors in cell serum or detection of platelets in serum
  • Pregnancy Tests








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