ELISA is a solid-phase enzyme immunobinding assay, and the most essential of the immune-enzyme assays. In this method, the antigen is attached to an antibody anchored on a solid phase (polystyrene or polyvinylchloride). This helps in retaining the immunological as well as the enzymatic activity.

ELISA can be used for quantitating antibody or antigen using enzyme-linked antibody and a substrate which forms a coloured reaction product; hence ELISA is also referred to as a qualitative or quantitative assay for antibodies.

Principle

The ELISA is performed in 96-well polystyrene plates. The serum is incubated in the wells, each containing a different serum. Positive and negative control serums are included in the 96 samples being tested. Antibodies or antigens in serum are captured by the corresponding antigen or antibody coated on the solid surface. The serum and unbound antibodies or antigens are removed from the plate by rinsing it with wash buffer.

Secondary antibodies attached to peroxidase or alkaline phosphatase enzyme, are added to each well for detecting bound antibodies or antigens. The unbound secondary antibodies are rinsed off after incubating the wells for a defined period. On adding an appropriate substrate, it reacts with the enzyme to develop a colour which is measured as a function or amount of antigens or antibody in the sample. Measurement of the intensity of colour or optical density is done at 450 nm, and the colour intensity indicates the number of antigen or antibody.

This table enlists the different enzymes used for ELISA:

Table : Commonly Used Enzymes and their Substrates in ELISA

Enzymes	Substrates
Horseradish peroxidase	Hydrogen peroxide and o-phenylene diamine
Alkaline phosphatase	p-nitrophenyl phosphate
β-galactosidase	o-nitrophenyl- β-d-galactopyranoside

This table enlists a few auto-antibodies detected by ELISA:

Table : Detection of Auto-Antibodies by ELISA

Antibodies	Target Auto-Antigens	Clinical Relevance
Thyroid microsomal antibody	Thyroid peroxidase	Auto-immune thyroid disease
Mitochondrial antibody (M2)	E2 pyruvate dehydrogenase complex	Primary biliary cirrhosis
Glomerular basement membrane antibody	C terminal end of type IV collagen	Good pasture’s syndrome and anti-glomerular basement membrane nephritis
Double stranded DNA antibody	dsDNA	Systemic lupus erythematosus
Phospholipid antibody	Cardiolipin	Primary phospholipid syndrome and systemic lupus erythematosus.

Procedure

The steps involved in the procedure of ELISA are:

Assay samples or standard solutions are added to the antibody-coated wells, and incubated for defined time period to capture the antigen molecules by the antibody.

After this binding reaction, the reaction mixture is discarded and excessive materials are removed from the wells by washing.

A second antibody that identifies another epitope in antigen is labelled with horseradish peroxidase (HRP) enzyme and then added to the wells.

This enzyme-labelled second antibody binds to the antigen already bound to the first antibody on the bottom of wells. This means that HRP enzyme is also fixed on the bottom of wells. The amount of antigen captured and the fixed enzyme is directly proportional.

The enzyme activity is measured by adding a chromogenic substrate of the enzyme. In case of HRP, tetramethylbenzidine is often used. After incubation for a definite period, the chromogenic substrate is changed to a coloured product. The reaction is stopped by adding a reaction stopper, e.g., diluted sulphuric acid, and absorbance is measured with a plate reader.

A standard or calibration curve is obtained by plotting the concentration of standard solutions.

Types of ELISA

ELISA test is of the following three types:

Indirect ELISA: This method is useful in identification and quantitative determination of antibody. In this method, serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated microtitre well and allowed to react with the antigen attached to the well (figure a). After washing away any free Ab1, the antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody.

After washing away any free Ab2, a substrate for the enzyme is added. The intensity of colour produced is measured by specialised spectrophotometric plate readers, which can measure the absorbance of all the wells of a 96-well plate in seconds. Indirect ELISA is preferred for detecting the presence of serum antibodies against HIV. In this assay, the recombinant envelope and core proteins of HIV are adsorbed on to microtitre wells as solid-phase antigens. The HIV infected individuals will produce serum antibodies to epitopes on these viral proteins which can be detected within 6 weeks of infection by indirect ELISA.

Advantages

1. This method has an elevated level of sensitivity because more than one labelled antibody binds to each primary antibody.
2. Numerous labelled secondary antibodies are commercially available.
3. Since the primary antibody is not labelled, its immunoreactivity is retained to the maximum.
4. This method is versatile as many primary antibodies can be prepared in one species and the same labelled secondary antibody can be used for detection.
5. This method is flexible as a single labelled secondary antibody can be used with many primary detection antibodies.
6. This method is cost efficient as a less number of labelled antibodies are used.
7. With same primary antibody different imaging indicators can be used.

Disadvantages

1. Cross-reactivity with secondary antibody may occur, causing nonspecific signal.
2. This method requires an additional incubation step.

Sandwich ELISA: This method is useful in detection and measurement of antigen (figure 6.2b). In this technique, the antibody is immobilised on a microtitre well. Antigen-containing sample is added and allowed to react with the immobilised antibody. After washing the well, a second enzymelinked antibody having specificity for a different epitope on the antigen is added and allowed to react with the bound antigen. After washing away any free secondary antibody, the substrate is added and the intensity of colour produced is measured.

Advantages

1. This method has high specificity as the antigen is specifically captured and detected due to the use of two antibodies.
2. This method is suitable for complex samples as the antigen is not purified before measurement.
3. This method is flexible and sensitive as direct and indirect detection methods can be used.

Competitive ELISA: This method is useful in estimation of antigen quantity (figure c). In this technique, antibody is incubated in an antigencontaining sample solution. The antigen-antibody mixture is added to an antigen-coated micro titre well.

More the number of antigens present in the sample, less the number of free antibody available for binding to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of primary antibody, determines the amount of primary antibody bound to the well. However, in competitive ELISA, higher the concentration of antigen in the original sample, lower is the absorbance.

Advantages

1. This method is highly specific as two antibodies are used.
2. This method is highly sensitive as both direct and indirect detection methods are used.
3. This method is suitable for complex samples as the antigen is not purified before measurement.

Applications

The technique of ELISA has the following applications:

1. It can be used to detect the presence of antigen or antibody in a sample.
2. It can be used to determine serum antibody concentrations in a virus test.
3. It can be used in food industry to detect potential food allergens.
4. It can be used in disease outbreaks to track the spreading of diseases, e.g.,

HIV, bird flu, common, colds, cholera, STDs, etc.

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