ELISA test Microbiology Notes: Procedure, Principle, Types, and Application.

What is ELISA test ?

ELISA test is a widely used serological technique. The full form of ELISA is “enzyme-linked immunosorbent assay“. ELISA is used for the detection of antibody, antigen, proteins, and glycoproteins in blood.

ELISA test was first discovered by two Swedish scientists, Eva Engvall and Peter Perlman In 1971, which revolutionized medicine.

In this technique an enzyme-linked antibody used for the assay of desire antigen or antibody from the test sample, that is why it called enzyme-linked immunosorbent assay.

ELISA Used for the detection of different types of diseases such as AIDS, Lyme disease, pernicious anemia, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, varicella-zoster virus, which causes chickenpox and shingles, Zika virus.

Principle of ELISA test

It is a wet lab technique. In ELISA Test an enzyme-labeled secondary antibody mixed with antibody-antigen Complex, as a result, the enzyme will be combined with the test antigen or FC portion of the test antibody.  

After That, a colorless substrate is added to this mixture. If any antigen or antibody present in the mixture then the enzyme will catalyze the substrate and it will form a colored, fluorescent, or chemiluminescent product.

The intensity of the formed color is proportional to the number of antibody or antigen present in the tested sample. The intensity of the color is measured by the ELISA reader.

In ELISA test different types of enzymes and substrates are used such as; 

1. Horseradish Peroxidase enzyme for substrate o-phenylenediamine dihydrochloride.
2. Alkaline phosphatase enzyme for substrate P Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase. 
3. The beta-galactosidase enzyme also used for the ELISA test.

Types of ELISA

There are presently four types of ELISA test such as;

• Direct ELISA Test (For Antigen Detection)
• Indirect ELISA Test (For Antigen Detection)
• Sandwich ELISA (For Antibody Detection)
• Combined ELISA (For Antibody Detection)

1. Direct ELISA Test

Direct ELISA used for the detection of antibodies. This ELISA test is much faster and easier as compared to others. This process is conducted in the following steps.

Procedure

1. At first, the test antigen (the antigen you have chosen for the ELISA test) mixed with a buffer solution.
2. Then this antigen containing buffer solution added to a microtiter plate (a specific plate containing multiple “wells” used as small test tubes), and allowed them to get immobilized or adhere to the bottom.
3. Then a solution containing non reacting proteins (bovine serum albumin or casein) added to each microtiter well.
4. Then a primary antigen or an enzyme-linked antibody added to this microtiter well which will bind with the tested antigen.
5. After that specific substrate for this enzyme is added to each microtitre, often the enzyme catalyzes the substrate which forms a color compound.
6. The intensity of the formed color is measure by using a spectrophotometer. The intensity of the color depends on the number of primary antibodies present in the microtiter well.

There are few disadvantage and advantage of this test;

Advantage

• Faster and easier processes as compare to other ELISA test.
• Required less precursor.

Disadvantages

• less flexibility in terms of the primary antibody.
• immobilization of the antigen is not specific.

2. Indirect ELISA

Indirect ELISA detects antibodies from the test sample. Used for the detection of serum antibodies against human immune deficiency virus (HIV), HIV. This technique will detect HIV within 5 weeks.

Procedure

1. First of all, antigen immobilized on the surface of a microtiter well.
2. Testing sample (serum) containing primary antibody is added to an antigen-coated microtiter well and allowed them to react with the antigen immobilized to the well’s surface.
3. After that, the remaining free primary antibodies are removed by using ELISA washer.
4. After that, an enzyme-linked secondary antibody added to this microtiter well to detect the antigen-antibody reaction. This secondary antibody will react with the primary antibody.
5. Then remaining free secondary antibody (secondary antibodies which not reacted with the antigens) washed away using ELISA washer.
6. Specific substrate for enzyme added to each microtiter well.
7. The intensity of the formed color is measured by the spectrophotometer and compared with the amount of product generated when the same set of reactions is performed using a standard curve of known Ab1 concentrations.

Advantages

• It’s a cheaper process.
• Required a few labeled ab.
• Offers high sensitivity and flexibility

Disadvantages

• Time-consuming.
• Required extra labor

3. Sandwich ELISA

Sandwich ELISA used for the detection of antigen in the test sample. In this method the antigen sandwich between two antibody that is why it’s called sandwich ELISA. This ELISA used for the detection of Hepatitis B Antigen.

Procedure

1. First of all, antibody immobilized on the surface of a microtiter well.
2. Then a sample containing an unknown amount of antigen added to this microtiter well and allowed them to react with the immobilized antibody.
3. After that, microtiter well is washed away using ELISA washer to remove the unbounded antigens.
4. Then an enzyme Linked secondary antibody added to this microtiter well which is specific for a different epitope on the antigen, allowed the secondary antibody to react with the bounded antigens.
5. Then again remaining free secondary antibodies are removed from the microtiter well using ELISA washer.
6. The selected substrate for the used enzyme is added to this microtiter well and observe for the color change.
7. The intensity of the formed color is measured by the spectrophotometer and compared with the amount of product generated when the same set of reactions is performed using a standard curve of known Ab1 concentrations.

Advantages   

• High sensitivity as compared to other ELISA.
• Shows a highly specific reaction due to the presence of two antibodies for one antigen.

Disadvantages

• Only monoclonal antibodies can be used as matched pairs, for the detection of a specific epitope.
• It is a tedious process to procure monoclonal antibodies 

Aslo Read: Virulence Factors or Factors Responsible for Pathogenicity of a Pathogens

4. Competition/Inhibition ELISA

The competitive ELISA used for the detection of antigen in the test sample. Competitive or competition ELISA completed by these following steps;

1. In this technique, at first, the antibody (primary antibody) is incubated with a sample solution containing antigens.
2. After that this antigen-antibody complex is added to an antigen-coated microtiter well.
3. The more antigen present in the initial solution-phase sample, the less free antibody will be available to bind to the antigen-coated well.
4. After that unbonded antibodies are removed from the microtiter well by using ELISA washer.
5. Then an enzyme-linked secondary antibody added to this microtiter well which is specific for the isotope of primary antibody. It will help to determine the amount of primary antibody bound to the well.

In the competitive assay, the higher the concentration of antigen in the original sample, the lower the final signal.

Advantages

• reproducibility and flexibility.
• Negligible sample processing is required.

Disadvantages

1. Limitation of basic ELISA applied.

Application of ELISA test

There are sevral important application of ELISA such as;

1. Use for the detection of HIV.
2. In the food industry, it used for the detection of potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs.
3. In toxicology ELISA used for rapid presumptive screening for certain classes of drugs.
4. ELISA used for the detection of different diseases, such as dengue, malaria, Chagas disease, Johne’s disease, etc.
5. It is also being used as in-vitro diagnostics in medical laboratories.
6. ELISA also detects Mycobacterium antibodies, rotavirus in feces, hepatitis B markers in serum, hepatitis C markers in serum, enterotoxin of E. coli in feces.

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Reference

• Kuby Immunology by Thomas J. Kindt (Author), Barbara A. Osborne (Author), Richard Goldsby (Author).
• Healthline, “What is an ELISA test?“
• Wkipedia,”ELISA test“
• MyBiosource, “What is ELISA? (Enzyme-linked Immunosorbent Assay)“