ELISA Test: Principle, Types, and Uses

Enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), is the most widely used type of immunoassay.  ELISA is a rapid test for detecting the presence and amount of either antigen or antibody in a sample.

The indirect ELISA is used to screen patients for detecting or quantifying antibodies (Ab) against viruses, bacteria, and other antigens (Ag). For example, detection of HIV antibodies, antibodies against rubella virus, etc., are done using indirect ELISA. Direct ELISA is used to detect hormones (such as human chorionic gonadotropin (HCG) in some pregnancy tests and luteinizing hormone (LH) in ovulation tests), drugs, and viral and bacterial antigens.

ELISA  is so named because the test technique involves using an enzyme system and immunosorbent. ELISA consists of antibodies bonded to enzymes; the enzymes remain able to catalyze a reaction, yielding a visible end product. The term enzyme-linked refers to an enzyme’s covalent binding to an antibody.

The use of enzymes as the label has several advantages.

1. The enzyme itself is not changed during ELISA. It can catalyze the reaction of many substrate molecules, greatly amplifying the reaction and enhancing detection.
2. Enzyme-conjugated antibodies are stable and can be stored for a relatively long time.
3. The formation of a colored end product allows direct observation of the reaction or automated spectrophotometric reading.

Salient Features of ELISA Test 

ELISA test is being increasingly used in the detection of antigen (the infectious agent) or antibody due to its simplicity and sensitivity. It is as sensitive as radioimmunoassay (RIA) and requires only microlitre quantities of test reagents. It has now been widely applied in the detection of a variety of antibodies and antigens such as hormones, toxins, and viruses.

1. ELISA test has high sensitivity and specificity
2. The result of quantitative ELISA tests can be read visually
3. A large number of tests can be done at one time
   ELISAs are designed specifically for screening large numbers of specimens at a time, making them suitable for use in surveillance and centralized blood transfusion services.
4. Reagents used for ELISA are stable and can be distributed in the district and rural laboratories but as ELISAs require sophisticated equipment and skilled technicians to perform the tests, their use is limited to certain circumstances.

Materials needed in ELISA Testing

1. Pipettes, washer system, ELISA plate reader:  Readers, washers, and pipettes are available as manual or automated systems. One of the main factors affecting equipment selection is the number and types of test samples being run.
   1. ELISA Readers: Readers need to have an appropriate filter (650 nm and 450 nm).
   2. Pipette: These are available as fixed as well as adjustable volume as well as single-channel and multi-channel.
   3. Washing system: It can be a manual system that washes one row or column at a time or semi-automated system that washes one strip or plate at a time or a fully automated system that can process multiple plates
2. Reagents needed for the testing- included in the kit (coated plates, sample diluents, controls, wash concentrate, conjugate, substrate, stop solution)
   • Coated plates: The 96-well plates are made of polystyrene and are coated with either inactivated antigen or antibody. The function of the plate has to hold the immobilized either antigen or antibody. Antigens or antibodies present in the sample will bind to the plate. This coating acts as the binding site for the antibodies or antigens in the sample.
   • Controls: Negative and positive controls are provided in each kit. The controls help to normalize or standardize each plate. Controls are also used to validate the assay and to calculate sample results. Controls might be pre-diluted and ready to use. (Please refer to the kit for specific instructions).
   • Conjugates: ELISA conjugates are enzyme-labeled antibodies that react specifically to plate-bound sample analytes. Unbound conjugates are washed away after incubation and before the addition of substrate.
   • Wash Concentrate: It acts as a buffered solution containing detergent to wash unbound material from the plate. (Not all test kits have wash concentrate; in that case, distilled water can be used for washing; please refer to kit insert for specific instructions)
   • Stop solution: It stops the enzyme-substrate reaction and color development.

Principle of ELISA Test 

Most ELISA methods developed for the detection of antigen or antibody consist of the use of the corresponding antibody or antigen in question which is firmly fixed on a solid phase, such as a plastic surface of a polyvinyl plate or polystyrene tube. Such systems are also called solid-phase immunosorbent assays (SPIA). A test sample is added to the microtitre plate, if there is the presence of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized Ab or Ag). Later enzyme-labeled antibody is added to the reaction mixture, which will combine with either the test antigen or the Fc portion of the test antibody. The enzyme system consists of;

1. An enzyme:  horse radish peroxidase, alkaline phosphatase which is labeled or linked, to a specific antibody.
2. A specific substrate:

• o-Phenylenediamine dihydrochloride for peroxidase
• P Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase

A substrate is added after the antigen-antibody reaction. The enzyme catalyzes (usually hydrolyses) the substrate to give a color endpoint (a yellow compound in the case of alkaline phosphatase). The intensity of the color is proportional to the amount of antibody or antigen present in the test sample, which can be quantified using an ELISA reader.

LAM ELISA is used for the measurement of lipoarabinomannan (LAM), a major surface antigen of Mycobacterial tuberculosis, from the urine and/or sputum of suspected tuberculosis patients. The analytical sensitivity of this assay is 50-100 folds higher compared to conventional ELISA.

Types of ELISA

Direct ELISA

The direct ELISA detects the presence of antigen in a sample. A microtiter well is first coated with a homologous antibody specific to the sought antigen. This is followed by coating the well with a blocking agent to prevent nonspecific binding of components. The sample being assayed is added to the well. If the antigen is present, it will react with the antibody coating the well; if none is present, no reaction occurs.

In this form of ELISA, the enzyme-linked (conjugate) antibody is also specific for the antigen being sought. When added to the well, it binds to the antigen if present. After allowing time for the enzyme-linked antibody to react with the antigen, the well is washed to remove any unbound enzyme-linked antibody (which would produce a false positive when the substrate is added). Upon addition of substrate, a color change is evidence of the enzymatic conversion of substrate to its product, which indicates the presence of the antigen.

Indirect ELISA

The indirect ELISA detects the presence of antibodies in a sample. In this type of ELISA, the microtiter well is coated with antigen-specific to the antibody being sought and a blocking agent. The sample being assayed is added to the well, and if the antibody is present, it will react with the antigen coating the well; if none is present, no reaction occurs.

In this case, the enzyme-linked antibody is an antihuman immunoglobin antibody. When added to the well, it binds to the antibody in the sample, if present. As with the direct ELISA, the unbound enzyme-linked antibodies are washed away to prevent a false positive result. Enzyme substrate is added, and a color change indicates a positive test.

Uses

ELISA test has wide applications in all fields of Microbiology. It is used to screen or quantify antigens or antibodies in patient specimens.

1. Bacteriology
   1. Direct ELISA test is used to detect group A streptococcal antigen in throat swabs, exotoxins of C. difficile in the stool, and botulinum toxin in a food sample.
   2. Antibodies detection tests are available to diagnose Rocky Mountain spotted fever (caused by Rickettasia rickettsii).
2. Virology
   1. Direct ELISA test is used to detect viral antigens in various specimens of the patient. ELISA test is commonly used to detect P24 antigen (HIV diagnosis), HBsAg (diagnosis of hepatitis B), influenza virus antigen detection, and rota-virus antigen detection.
   2. Indirect ELISA test detects specific viral antibodies against hepatitis C virus, Chikungunya virus, Zika virus, human T-cell lymphotropic virus, and HIV in the patient’s serum.
3. Mycology: Antigen detection test is available for the diagnosis of histoplasmosis. This test detects the presence of histoplasma antigen in the patient’s tissue sample.
4. Parasitology: In parasitology, direct ELISA is used to detect Giardia antigen and protein-specific antigen of P. falciparum. Indirect ELISA is available for detecting IgM antibodies against Trypanosoma brucei antigen (for diagnosing African trypanosomiasis) and antigen against Taenia solium.

References

1. Alhajj, M., Zubair, M., & Farhana, A. (2023). Enzyme Linked Immunosorbent Assay. In StatPearls. StatPearls Publishing.
2. Hayrapetyan, H., Tran, T., Tellez-Corrales, E., & Madiraju, C. (2023). Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods in molecular biology (Clifton, N.J.), 2612, 1–17. https://doi.org/10.1007/978-1-0716-2903-1_1
3. Aydin S. (2015). A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4–15. https://doi.org/10.1016/j.peptides.2015.04.012