Introduction of ELISA
Table of Contents
ELISA stands for Enzyme-Linked Immunosorbent Assay. It comes under antigen and antibody reaction tests and is useful for the identification of antigens or antibodies of the following specimens serum, urine, CSF, sputum, semen, supernatant of culture, stool, etc. It is also applicable for qualitative as well as quantitative determination of antigens or antibodies. In qualitative test determines antigen or antibody is present or absent whereas in quantitative determines the quantity of the antibody in titer and the titer is the highest dilution of the specimen usually serum which gives a positive reaction in the test.
Requirements for Test
Solid-phase support:– 96-well microtiter well or polystyrene beads or tubes. Microtitre is well-round, flat, or C-shaped and it is coated with antigen or antibody. Antibodies or antigens are absorbed onto plastic surfaces in an alkaline buffer(carbonate or bicarbonate, PH -9.0) at 37 ⁰C for 1 to 2 hours at room temperature overnight. Wash off unbound reagent. Add blocking solution (1% sodium casein or gelatin or BSA) for 30-60 minutes at 37⁰C. Rewash and then sugar solution (1% glucose, sucrose, mannose, or maltose) for 30 minutes at room temperature. Plates are then dried rapidly using steam of nitrogen or a vacuum and finally store at 4⁰C.
Washing solution:- Phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/T). Diluent buffer:-Phosphate buffer saline (PBS) containing 0.05% Tween 20. Enzyme–substrate system:- It consists of an enzyme linked to a specific antibody or antigen and a specific substrate containing chromogens. Stop solution, Enzyme -substrate Initially the substrate should be colorless. After degradation by the enzyme, it should be strongly colored. Enzymes and their respective substrate, chromogen, and stop solution are as follows for Alkaline Phosphatase (enzyme), para Nitrophenylphosphate (pNPP) (substrate), para-Nitrophenylphosphate + diethandamine+ magnesium chloride (MgCl2)( chromogen), 1 M NaOH (stop solution), Horseradish Peroxidase (H2O2 ), Tetramethylbenzidine + Phosphate – Citrate buffer, 1 M and H₂SO₄, Horseradish Peroxidase, H2O2, O – Phenylenediamine + HCl, 1 M HCl.
It is of the following types-
- Direct ELISA (sandwich ELISA -For Ag detection),
- Indirect ELISA (For Ab detection),
- Competitive ELISA, and
- Capture ELISA.
Principle of Direct ELISA
In this technique, the antigen is sandwiched between the two Abs. The microtitre wells are coated with an Abs that is specific to the antigen. It is then reacted with antigen first (sample) and then an antibody, specific for different epitopes of the antigen, is added (which is already tagged with enzyme) and it leads to the color product after the addition of a specific substrate. After that stop solution is added and it is then measured by an ELISA reader (spectrophotometer). The intensity of the color is directly proportional to the concentration of antigen present in the specimen. For more clarity, observe the above figure carefully.
Principle of Indirect ELISA
In this technique, the microtitre wells are coated with an antigen. It is then reacted with an antibody first( specimen) and then a secondary antibody (anti-human globulins) is added, which is already tagged with enzyme and leads to the color product after the addition of a specific substrate. After a few minutes ( time according to manufacturers) stop solution is added and it is then measured by an ELISA reader. The intensity of the color is directly proportional to the concentration of antibodies present in the specimen. For more clarity, observe the above figure carefully.
Principle of Competitive ELISA
Two specific antibodies are employed in this method. Serum antibody (excess) and enzyme-labeled Abs for the antigen. The antibodies compete for the binding site on the same antigen, hence called competitive. For more clarity, observe the above figure carefully.
Cut off Value
Cut-off value: provided in the kits by the manufacturer. The cut-off value defines a range in which 90% of the normal population is negative below the cut-off value and 10% of the normal population is positive above the cut-off value. ELISA is a semi-quantitative method. The calculation is done as follows. The units of ELISA are optical density (OD) ratio: Sample value= sample OD/cut-off OD
ELISA Ab capture method for Immunoglobulin Mu (IgM)
Immunoglobulin Mu is present early in the course of illness. When immunoglobulin gamma (IgG) and IgM are present in the same specimen, IgG competes for the binding site on the antigen fixed to a solid phase and consequently, there is no binding of anti-IgM antibody conjugate to the antigen. The IgM detection method is an antibody capture immunoassay. In this method, IgG directed against IgM (i.e., antihuman IgM) is attached to well. Patient serum containing IgM is added. Any IgM present is captured by anti-human IgM present well (i.e. IgG). The viral antigen is added and incubated. The enzyme-conjugated antibody is added and incubated. Substrate color is measured, which is directly proportional to IgM present in serum.
ELISA Ag Capture Method
For detection of Chlamydial antigen in an endocervical swab. Well coated with Ab against the antigen. Washing of swabs is added. Antichlamydial antibody linked with enzyme is added. Substrate color is then measured by an ELISA reader. The intensity of the color is directly proportional to the concentration of antigen present in the specimen.
Procedure of ELISA
Follow fig. Enzyme-Linked Immunosorbent Assay (ELISA) Clear Concept.
|Yellow color||Qualitatively Reactive|
|Equal and greater than the cut of value (COV) in the ELISA Reader||Reactive|
|Lower than COV||Non-reactive|
|Control (negative and positive)||NC and PC should be non-reactive and reactive respectively otherwise test invalid|
Interfering Factors of ELISA Test Results
The following factors which affect the ELISA result are as follow as-
- Plate Assay: The shape and quality of the wells, the material of the plate, potential pre-activation, even or uneven coating
- Buffer: pH, contamination
- Capture and detection antibody: incubation time, temperature, specificity, titer, affinity
- Blocking Buffer: cross-reactivity, concentration, contamination
- Target antigen: conformation, stability, epitopes
- Enzyme conjugate: type, concentration, function, cross-reactivity
- Washes: contamination, frequency, volume, duration, composition
- Substrate: quality/manufacturer
- Detection: instrument-dependent factors
- The reader or human error
Advantages of ELISA
- It is a specific and sensitive assay with wide application.
- Equipment is cheap and easily available.
- Reagents are cheap with long shelf life.
- Assays may be rapid.
- Simultaneous assay, variety of labels
- It is the potential for automation.
- The test is lacking radiation hazards.
Disadvantages of ELISA
- There is a chance of contamination.
- Expertise required to label and purify conjugates
- Susceptible to interference from non-specific factors
Keynotes on ELISA
- Direct ELISA is for the detection of antigens.
- Indirect ELISA is for the detection of antibodies.
- Sandwich or Competitive ELISA is for the detection of antigens.
- The invalid test should be repeated.
- Textbook of Medical Laboratory Technology by Praful B. Godkar, Darshan P. Godkar
- Textbook of Medical Laboratory Technology by Ramnik Sood (2006)