ELISA Test

ELISA test stands for EnzymeLinked Immunosorbent Assay. It is a type of serological test and immunoassay technique. In the ELISA test, an enzyme links to the antibodies particularly to detect the presence of proteins like Antigens. The ELISA method was evolved from the RIA technique in the 1960s. Therefore ELISA technique is more or less similar to Radio Immunoassay Antigen (RIA), where the antigen is radiolabelled.

By the increasing concern over environmental pollution, the ELISA technique has been introduced instead of radioisotopes. Therefore, ELISA is a widely used method for the detection or diagnosis of bacterial, viral infections. Nowadays, ELISA kits have been also developed which is most frequently used in many laboratories.

Content: ELISA test

  1. Definition
  2. Principle
  3. Overview
  4. Types
  5. Qualitative measure
  6. Quantitative measure
  7. Advantages
  8. Disadvantages

Definition of ELISA Test

Enzyme-linked immunosorbent assay is the immunological reaction which performs on a solid phase i.e. Microtitre plate that uses enzymes to label the antibodies which indirectly measures the quantity of antigens or antibodies present in the test sample by both quantitatively and quantitatively.

Principle of ELISA Test

ELISA is the immunoassay technique which comes under the category of Labelled immunoassay. Immunoassay depends upon the interaction between an antigen (Ag) and antibody (Ab) which forms Ag-Ab complex refers to “Immunocomplex” that is used to detect the presence and to quantify specific antigens or antibodies.

ELISA being an immunoassay method it is very sensitive and specific in nature where specific antigens or antibodies binds to its homologous antibodies and antigens.

Overview of ELISA

ELISA is performed on a Microtitre plate. A microtitre plate is made up of polystyrene and consist of 96 shallow wells or sometimes more than that.

For example: To detect the presence of antigen

  • First, the protein i.e. Antigen is immobilized on the solid surface of the well.
  • Then the Enzyme-linked antibodies are introduced into the well. Now after introducing these into the well, there will arise two conditions:

Positive ELISA: If the enzyme-linked antibody binds to the specific antigen then this will give a positive result for the ELISA. We can neither see the interaction of antigen and antibody nor the presence of antigen or antibody. Therefore, the colourless substrate is added to the wells where it reacts with the enzyme that is linked to the antigen-antibody complex and release some signals in the form of colour change. Hence, a positive result is indicated by a colour change and higher is the concentration of antigen higher is the signals produced by the substrate.

Negative ELISA: If the enzyme-linked antibody does not bind to the antigen or there is no such interaction of antigen and antibody then it gives the negative result for ELISA. Therefore in negative ELISA, there is neither Ag-Ab binding nor colour change by the addition of a substrate.

Therefore, we can say an ELISA is basically a “Colorimetric assay” which is detected by the colour change in the wells to indicate the presence or absence of antigens.

Types of ELISA Test

ELISA is mainly of four types:

Direct, Sandwich, Indirect and Competitive ELISA

Direct ELISA

It includes the following steps:

DIRECT ELISA

  1. First, add the buffered protein sample into the wells of the microtitre plate.
  2. Then add surface blocking or non-reacting protein like BSA (Bovine Serum Albumin), Casein etc. to block the bottom surface just to cover the extra space and to prevent false binding or false signalling.
  3. After that add Specific primary antibody which is conjugated with the enzyme that directly binds to the protein of interest.
  4. Then add washing buffer to remove the unbound proteins.
  5. At last add substrate, which will further react with the enzyme and produce a significant signal in the form of colour change.

Indirect ELISA

It includes the following steps:

Indirect ELISA

  1. First, add buffer protein sample into the wells of the microtitre plate.
  2. Then add surface blocking or non-reacting protein like BSA (Bovine Serum Albumin), Casein etc. to block the bottom surface just to cover the extra space and to prevent false binding or false signalling.
  3. After that add Specific primary antibody that will bind to the protein of interest.
  4. Then add washing buffer to remove the unbound proteins.
  5. Add secondary antibody which is conjugated with enzyme binds to the antigen-antibody complex.
  6. Then add washing buffer to remove the unbound secondary antibodies.
  7. At last add substrate, which will further react with the enzyme and produce a significant signal in the form of colour change.

Sandwich ELISA

It includes the following steps:

Double antibody sandwich ELISA

  1. In this technique first, add the antiserum to the wells of the microtitre plate.
  2. After that, the antibodies present in the antiserum adhere to the well- surface
  3. Then add the test antigen which couples with the homologous antibodies.
  4. Add enzymes labelled specific antibody which binds with the antigen that is coupled with the antibody adhere to the well surface or to the antigen-antibody complex. This reaction results in the formation of antibody-antigen-antibody complex which resembles a sandwich.
  5. At last, add the enzyme-substrate which reacts with the enzyme which degrades the substrate molecules by the enzyme activity.

Competitive ELISA

It includes the following steps:

  1. First coat the bottom of the well will capture the antibody.
  2. Then add surface blocking proteins with BSA or detergents.
  3. Mix the sample with enzyme-conjugated protein.
  4. After that, add the mixture to the well-containing antibodies adhere to its surface.
  5. Wash the well with washing buffer to remove unbound proteins.
  6. Add substrate to the complex which will react with the enzyme and gives a significant colour that indicates the presence of protein or antigen in the given sample.

Qualitative measure of ELISA

For the qualitative measure or to detect the presence of antigens or antibodies, the Enzymes are used as it is clear from the name itself that is Enzyme-Linked ImmunoSorbent Assay.

In the ELISA method, the most commonly used enzymes are as follows:

  • HRP: It stands for Horseradish peroxidase. HRP uses the following substrate viz. OPD, TMB, ABTS etc.
    • OPD gives Amber colour to the solution.
    • TMB gives Blue colour to the solution.
    • ABTS gives a green colour to the solution.
  • AP: It stands for Alkaline phosphatase. AP makes the use for PNPP substrate which gives a yellow colour to the solution.

Therefore, in ELISA positive and negative results are made on the basis of colour change. If the solution remains colourless even by the addition of substrate then it gives a negative result. If the colour of the solution changes from colourless it will indicate the presence of analyte or protein of interest.

Quantitative measure of ELISA

For the quantitative measure of ELISA, there are some key points that we have to remember like:

  • Sample
  • Standard
  • Positive control
  • Blank samples
  • Standard curve

Sample: The sample is added either in duplicates or triplicates. For example: If we have 40 samples, then the sample is added twice and suppose we have 20 samples then it is added thrice. Therefore the addition of a sample depends upon the number of samples we have. Triplicates of a sample are considered to be better than the duplicates because it reduces the chances of error in the result.

Standard: To prepare standard of the given sample first the sample is diluted in a series of 0.5, 1, 2, 4, 8, 16, and 32.

Positive control: Positive control is the known concentration of the sample.

Blank samples: It contains everything as protein.

Standard-curve: The standard curve is prepared by diluting the concentrated samples to find the unknown concentration of the protein by plotting a graph between adsorption and concentration.

Advantages of ELISA Test

  • ELISA is the qualitative and quantitative assay i.e. it not only detects the presence of the analyte but also detects the concentration or quantity of the analyte.
  • It can screen a large number of samples in a single take.
  • ELISA can be easily automated.

Disadvantages of ELISA Test

  • ELISA does not give any information about the biochemical properties of the analyte such as molecular weight, distribution among living organisms etc.
  • To know the molecular weight and more information about the analyte, technique like Western blotting is employed.
  • ELISA is a very sensitive method to perform hence requires more precautions while performing the experiment.

Therefore, the ELISA is having both advantages along with some disadvantages but inspite of this, it is a very useful technique in medical science and research.

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