“Enzyme Linked Immunosorbent Assay”

 

Introduction:

•      ELISA, or Enzyme-linked immunosorbent assay, are quantitative immunological procedures in which the antigen-antibody reaction is monitored by enzyme measurements.

•      The term ELISA was first used by Engvall and Perlma in 1971.

•      The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

•      It is useful and powerful method in estimating ng/mL to pg/mL ordered materials in the solution.

Why known as ELISA?

1.     Antigen of interest is absorbed on to plastic surface (‘sorbent’).

2.     Antigen is recognized by specific antibody (‘immuno’).

3.     This antibody is recognized by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’).

4.     Substrate reacts with enzyme to produce product, usually colored.

Basic Principle of ELISA:

•      Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).

•      The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding.

•      An ELISA can be used to detect either the presence of Antigens or Antibodies in a sample depending how the test is designed.

•     ELISA was developed in 1970 and became rapidly accepted.

    ELISA Qualitative/Quantitative:

Qualitative: determines antigen or antibody is present or absent.

Quantitative: determines the quantity of the antibody. The highest dilution of the specimen usually serum which gives a positive reaction in the test.

    Materials Needed:

Testing sample.

Antibody/Antigen.

Polystyrene microtiter plate.

Blocking buffer.

Washing buffer.

Substrate.

Enzyme.

 

Types of ELISA:

•      Solid phase immunoassay.

•      Enzyme Label.

•      Competitive assay.

•       Non-competitive assay.

Competitive ELISA:

•      Used to determine small molecule antigens. (T3,T4, progesterone etc).

•      Antibody coated micro well.

•      Serum antigen and labelled antigen added together-competition.

•      Antibody-Antigen-enzyme complex bound is inversely related to the concentration of antigen present in the sample.

•      The bound enzyme conjugate reacts with the chromogenic substrate added to produce a color reaction (blue to yellow color).

•      Increased serum antigen results in reduced binding of the antigen-enzyme conjugate with the capture antibody producing less enzyme activity and color (yellow) formation.

•      Substrate product concentration is inversely proportional to the concentration of standard or test antigen added.

                                            

Noncompetitive ELISA:

•      Sandwich Assay.

•      Direct Assay.

•      Indirect Assay.

 

Sandwich Assay:

•      Antigens such as tumor markers, hormones and serum proteins may be determined.

•      Antigen in the sample binds with the capture antibody on the micro well and becomes immobilized.

•      The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody-antigen-antibody/ enzyme bound to the micro well.

•      Enzyme reaction product is directly proportional to concentration of standard or analytical antigen.

Direct Assay:

Antigen and antibody capture ELISA Antigen adsorbed directly detected by labeled enzyme.

Indirect Assay:

•      Antigen directly adsorbed onto the solid phase is first incubated with patient serum, and then with a labeled antibody specific for human immunoglobulin.

•      Detection of infectious agents (HIV, HBV, HCV) and auto antibodies.

•      In indirect ELISA color change is directly proportional to the concentration of specific antibodies in specimen.

Advantages of ELISA:

•      Reagents are relatively cheap and have a long shelf life.

•      ELISA is highly specific and sensitive.

•      No radiation hazards occur during labelling or disposal of waste.

•      Easy to perform and quick procedures.

•      Equipment can be inexpensive and widely available.

•     ELISA can be used to variety of infections.

Disadvantages of ELISA:

•      Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.

•      Enzyme activity may be affected by plasma constituents.

•      Kits are commercially available, but not cheap.

•      Very specific to a particular antigen. Won’t recognize any other antigen.

•      False positives/negatives possible, especially with mutated/altered antigen.

Limitations:

•      Results may not be absolute.

•      Antibody must be available.

•      Concentration may be unclear.

•      False-positive possible.

•      False-negative possible.

Applications of ELISA:

ELISA test can be done on

•      Hormones.

•      Proteins.

•      Infectious Agent (Viral, Bacterial, Parasitic, Fungal).

•      Drug Markers.

•      Tumor Markers.

•      Serum Proteins.

•      Vaccine Quality Control.

•      For GMO.

•      For Rapid Test.

•      IgG, IgM, IgA.

•      In New Born Screening.

•      In Clinical Research.