ELISA- A quick review

ELISA– Enzyme-Linked Immunosorbent Assay

The ELISA techniques are widely used for-

1) Quantitative estimation of-

• Hormones
• Amino acids
• Growth factors
• Tumor  markers and
• Other analytes which are present in a very small concentration in biological fluids

2) Qualitative detection of-

• Bacterial or viral antigens
• Antibodies against microbes
• Any antigen, or antibody present in a small concentration  in the biological fluid/tissues

Advantages of ELISA

• The test can be undertaken to detect antigens or antibodies present in very small quantities in tissues or blood.
•  It is a highly economical and sensitive method.
•  An enzyme is used as a label, so it is a non-isotopic immune assay.
•  It is more sensitive than Radio-Immuno Assay and there is no risk of radiation hazards.

Principle

ELISA is based on the Immunochemical principles of the Ag-Ab reaction. The specific interactions between specific antigens and antibodies are used here to detect/ quantitate the specific components of a biological fluid, using the enzyme-labeled antibodies. The substrate of the enzyme is added, coupled with a color reagent; which is colorless in the beginning but during the course of the reaction becomes colored. The intensity of the color formed predicts the concentration of the Ag/Ab present in the given sample.

Steps of ELISA

A) Antigen detection

 Sandwich ELISA (Figure-2)

A specific antibody is fixed to the well of a microtiter plate(Figure-1) The patient’s serum is added in the well and incubated for 30 minutes at 37 degrees Celsius. During this time, if the serum contains the antigen, it is fixed on the antibody. Excess antigens and other unwanted proteins are washed out. Then antibodies tagged with horse-radish peroxidase(HRP) enzyme are added. If the antigen is already fixed, the Ab-HRP conjugate will be fixed in the well. Then a color reagent, containing hydrogen peroxide (H2O2) and diaminobenzidine (DAB) is added. The reaction is as follows-

i) H₂O₂   ————————–>      H2O  +  Nascent oxygen [o]

The reaction is catalyzed by Horseradish peroxidase enzyme

ii) Diaminobenzidine—————————>   Oxidized DAB

   (colorless)                                                            (brown color)

 The reaction takes place in the presence of Nascent oxygen [o]

Thus in the above reaction, an antigen is sandwiched between Ab ( solid phase) and the enzyme-labeled antibody. The development of brown color indicates that the antigen is originally present in the patient’s serum. Colour developed is proportional to the antigen in the serum. Therefore the intensity of the color is measured from which the concentration of the antigen can be calculated. Other chromogens that can be used in ELISA are NBT (Nitro blue tetrazolium-blue color) and NPP (Nitro phenyl phosphate-yellow color).

 

Figure-1- Showing microtitre plate

B) Antibody detection

Indirect ELISA (Figure-2)

This is useful to detect small quantities of antibodies in the blood e.g. to detect HIV antibodies in the patient’s serum, this test can be undertaken by the following method-

Antigen from HIV is coated in the well of a multiwell microtitre plate. The patient’s serum is added and incubated. If it contains the antibodies, it is fixed. The wells are washed. This is done to remove excess unbound antibodies. Then a second antibody (Antibody against human immunoglobulin) conjugated with HRP is added. Then color reagent containing hydrogen peroxide and diaminobenzidine is poured over. The development of brown color indicates the presence of antibodies in the patient’s serum. The color developed is proportional to the antibody concentration. Thus from the intensity of color, the concentration of Ab can be determined(Figure-1).

ELISA is less specific for HIV so the confirmation by other methods is necessary for the final diagnosis of AIDS. 

Figure-2-Showing the steps of Sandwich and Indirect ELISA