ELISA technique

Enzyme linked immunosorbent assay ELISA is an assay technique of biotechnology for detection of various enzymes, proteins, antibodies and hormones. The detection is based on antigen-antibody interactions. Antigen molecules are immobilized on surface of 96 wells polystyrene plate. Adding antibody solution on plate results in antigen-antibody complexes. Assessing the activity of conjugate enzyme (attached with antibody molecule) via incubation with substrate molecule provides basis for detection of specific antibody molecules.

This technique has significant role in detection of certain chemicals and therapeutic products such as antibodies when prepared in labs.

Background and History

Earlier in lab researchers, the researchers used radioactive-labeled antigens or antibody molecule. The radioactivity of these analytes was a major limitation and a health risk for researchers as well. Therefore, a better detection assay was required.

In 1960, two different teams developed the enzyme linking technique supervised by researchers S.Avrameas and G.B.Pierce. During the same period, Wide and Jerker Porath published the immunosorbent technique. Later in year 1971, two more papers were published by researchers at Stockholm University in Sweden. All of these researches led to the development of ELISA technique.

The conventional ELISA technique involves chromogenic substrates. Development of particular coloration upon substrate degradation helps in detection of analyte. Now days, more advanced ELISA technique is used. This involves electrochemical luminescence and fluorogenic signals for detection. These have more specificity and efficiency to detect the minute amount of analyte in reaction mixture.

Basic principle

The underlying principle of ELISA technique is antigen-antibody interactions. Coating the ELISA plate with antibody mixture provides the opportunity to antigen molecule to bind with antibodies. The binding of antigens and antibodies molecule serves the detection and selection purpose. If the antibody molecules are conjugated with the enzymes, then adding a substrate to the reaction mixture produce colored product on substrate degradation by enzyme molecules. Therefore, it aids in detection of specific analytes in research labs.

Major steps involved in ELISA technique

1. Plate coating

ELISA technique uses specific polystyrene plate of 96 wells. The first step for detection assay is to coat this plate with specific antibody molecules. This is in fact the immobilization of antibodies on surface of ELISA plates. A coating buffer is used in this step.  The next step involves washing to rinse off the extra buffer.

2. Blocking

Blocking the site is the next step. A buffer containing unrelated proteins mixture is added to ELISA plates. These proteins block the free spaces on plate which the antibody molecules left in first step.

3. Detection

The further step is detection. The analyte sample is added to coated plates. For instance, an enzyme conjugated antigen molecule is poured on plates. The conjugate molecules are usually strepavidin conjugate with alkaline phosphate or horseradish peroxidase. The antigens bind to specific antibody molecules. This step requires incubation at particular temperature too facilitate the antigen-antibody interaction and binding.  The extra or unbound analyte is rinsed off by washing.

ELISA assay steps

4. Results interpretation

Last step is addition of substrates molecules. As soon as the enzyme hydrolyzes the substrate molecule, visible coloration develops. Absorbance is measured by spectrophotometer. The results are analyzed for final description of results.

Types of ELISA

There are four types of ELISA based on how many antibody molecules are used and which one has conjugate enzyme molecules. The general steps of coating the plates, blocking are similar. These four types are:

  1. Direct ELISA

In this technique, only one primary enzyme-linked antibody molecule is used. This primary antibody binds to the antigen molecules which are coated on the surface of ELISA plate. There is no need of secondary antibody molecule. The enzyme linked with the primary antibody reacts with the substrate and the visible coloration occurs. Taking absorbance of these signals helps in detection of analyte.

Direct ELISA


  • Rapid and easy with few steps
  • Less reagents are used which mean less error occurs


  • Antigen immobilization sometimes pose an issue mainly because of the proteins nature of enzymes, that the target protein may also bind to plate
  • No signal amplifications, therefore less sensitivity

2. Indirect ELISA

The indirect ELISA technique involves two types of antibodies labeled as primary and secondary antibodies respectively. Moreover, it’s the secondary antibody molecules with conjugate enzyme.

Firstly, the primary antibody molecules serve the purpose of binding to antigens coated on plate. Then, the secondary antibodies attach with the primary antibodies. Adding the substrate develops coloration as soon as the enzyme hydrolyzes the substrate molecules.

Indirect ELISA


  • High sensitivity as more than one type of antibody molecules are used
  • Different primary antibodies are applicable to bind with the secondary antibody, therefore this is economical as well.


  • Takes more time and expertise than direct ELISA
  • Possibility of cross reaction of secondary antibodies

3. Sandwich ELISA

The first two type’s direct and indirect ELISA technique involves coating of antigen molecule on the surface of plates. On the other hand, sandwich ELISA involves coating of plates with antibody molecules. These are termed as captured antibody molecules. Moreover, this technique involves two detection antibodies. The first is un-labeled primary and the second is enzyme-conjugate secondary antibody.

Firstly the antigen binds with the antibody molecule coated on specific plate. Then, the primary detection antibody attaches with the antigen followed by attachment of secondary antibody with primary one. Adding the substrate develops coloration upon hydrolysis by the enzyme. Finally, absorbance helps the detection of analyte.

Sandwich ELISA assay


  • The sensitivity is 2-5 time better than direct and indirect ELISA
  • Two type of detection antibodies make it highly specific and sensitive


  • Antibodies may cross link which complicates the detection of analyte. Sometimes, requires specific standardized kits.

4. Competitive ELISA

The technique for competitive ELISA is comparatively complex. This is also known as inhibition ELISA technique as it involves an antigen inhibitor. Both the actual antigen and the inhibitor antigen compete with each other to bind with the primary antibody molecules.

In this type,  the incubation of primary antibodies mixture with analyte (which also contains desired antigens) allows formation of antigen-antibody complexes. Some of the antibody molecules are left unbound as these are greater in number than antigen molecules. Further, this sample with antigen-antibody complexes is added to ELISA plates. The plates are already coated with inhibitor antigen. The unbound antibody molecules in sample bind to the inhibitor antigen. On the other hand, the antigen-antibody complexes are unable to bind. Therefore, these complexes get washed off. Lastly, adding substrate and measuring the absorbance of coloration detects the analyte. This is competitive ELISA technique when plates are coated with inhibitor antigen. Similarly, we can also coat the plates with labeled antibody molecules.

Four types of ELISA


  • No need of sample purification
  • Robust
  • Highly flexible as all direct, indirect and sandwich ELISA can adapt to competitive form


  • Relatively complex
  • Need expertise
  • Few limitations as all other ELISA techniques can adapt to this competitive form

Elispot assay

This refers to enzyme linked immunospot assay. This is quiet similar to sandwich ELISA technique. This serves the purpose of detection and measurement of proteins-secreting cells at single cell level.

In-cell ELISA

This technique measures the levels of target protein by cells. The cells are coated on surface of polystyrene plate. Moreover, the remaining steps of technique are quiet similar to sandwich ELISA.

Qualitative and quantitative ELISA assay

On the basis of how and what the ELISA technique detects, it can provide both qualitative and quantitative results.

The qualitative type just detects the presence or absence of desired antigen or antibody molecule in analyte. On the other hand, the quantitative form measures the amount and levels of desired antigen or antibody in analyte mixture.


  • This assay technique detects the analytes both qualitatively and quantitatively. This makes it vital research tool.
  • This serves in diagnostic and research work.
  • It can detect allergens and pathogens such as in food items.
  • Can help in diagnosis of allergic diseases. Also, viral infection such as HIV, HBV, HCV
  • Detection of monoclonal antibodies
  • Detection of hormones such as in pregnancy and other disease
  • This assay technique is no doubt a valuable analytic and research tool. It can efficiently detect food allergens and pathogens, diagnose diseases including HBV, HCV and HIV, yet its cost is a limitation to make it a common diagnostic tool. Only few diseases are diagnosed by ELISA assay.




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