EnzymeLinked Immunosorbent Assay (ELISA) (original) (raw)
Enzyme-Linked Immunosorbent Assay (ELISA)
Last Updated : 18 Dec, 2025
Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used laboratory technique to detect and measure substances such as antibodies, antigens, proteins, or hormones in a sample (like blood, urine, or saliva). It is a basic analytical technique used to identify and quantify antibodies, proteins, peptides, and hormones present in the blood. The results of an ELISA test provide important information about possible diseases and can help in planning appropriate treatment.
ELISA is often compared with other antibody-based assays because it provides quantitative results and helps distinguish between specific and non-specific interactions. This is achieved through controlled binding to solid surfaces, typically polystyrene multi-well plates, which allow accurate detection and measurement of the target molecules.
**Types of ELISA
Three types of ELISA tests are distinguished based on the methods used for binding between antibodies and antigens-
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
**Direct ELISA
Direct ELISA is an immunological technique used to detect the presence of an antigen (a specific protein) in a sample using an enzyme-linked antibody.
- The sample that contains the antigen is added to a microplate well, and the antigen attaches to the well surface.
- A blocking agent is added to cover empty spaces to prevent non-specific binding.
- A single, enzyme-linked primary antibody is added, and it binds directly to the antigen.
- A substrate is added, and the enzyme reacts with the substrate, and colour develops.
**Indirect ELISA
It detects whether an antibody is present in the sample or not, and the antigen binds to the wells of the microtiter plate. Absorbance measurement of the coloured product is done by a spectrophotometer.
- A sample containing antibodies is added to the antigen-coated well and binds to the antigen.
- The free primary antibody is washed away, and the antigen-antibody complex is detected by adding the secondary antibody that is bound to the enzyme capable of binding to the primary antibody.
- All the free secondary antibodies will be washed away, and a specific substrate is added to give a coloured product.
**Sandwich ELISA
A capture antibody is coated on the plate. T antigen binds to it. A second detection antibody (enzyme-linked) binds to the antigen, forming an antibody–antigen–antibody “sandwich”.
- It helps to detect antigens in samples.
- Here microtiter well is coated with antibodies
- Add a sample that contains antigen to the wells and wash it to remove the free antigen.
- Now, an enzyme-bound secondary antibody that binds to a different epitope on the antigen is added. Wash the wells to remove all free secondary antibodies.
**Competitive ELISA
It helps to measure the antigen concentration in a sample, and the microtiter plate is coated with antigen. The antibody is incubated in a solution that contains antigens.
- Add a solution of the antigen-antibody complex to the microtiter well, and the wells are then washed to remove the unbound antibodies.
- The higher the antigen concentration in the sample, the less free antibody is available to interact with the well-coated antigen.
- The enzyme-bound secondary antibody is added to detect the number of primary antibodies present in the well.
- Now the concentration can be determined through spectrophotometry.
Characteristics of ELISA
Characteristics of ELISA are given below:
- ****Antigen (Ag)**- A toxic molecule or any foreign matter that causes an immune response in the body is called an antigen. In immunology, the term antigen originally referred to substances that generate antibodies. Antigens can be proteins, peptides, lipids, or nucleic acids.
- ****Antibody (Ab)**- An antibody is a blood protein that is produced in our body by the immune system in response to and to counteract a specific antigen. Antibodies are also called immunoglobulins.
- This technique works on the principle of antigen-antibody binding.
- This technique falls under the category of labelled immunoassay. An immunoassay basically depends upon how the interaction occurs between the antigen (Ag) and antibody (Ab). Now, an immunocomplex will be formed, which consists of an Ag-Ab complex.
- The ELISA technique is basically used to identify and measure the antibodies, proteins, peptides, and hormones in the blood.
- This technique is very sensitive and specific in nature, in which the specific antigens or antibodies will bind to their homologous antigens or antibodies.
**Principle
It works on the principle that particular antibodies will bind the target antigens, and they will detect the absence or presence and quantity of binding antigens. The plate we are using should be coated with antibodies with high affinity to increase the precision and sensitivity of the assay. ELISA can provide a correct and useful measure of antibody-antigen concentration.
**Procedure of ELISA
ELISA is one of the easiest tests that can be done quickly and rapidly, and it requires any type of sample from the patient. The entire process includes :
- Antibodies bind to polystyrene sheets, which are solid surfaces, and they are attracted to bacteria, other hormones, and antibodies.
- Antigen-coated microtiter plates are filled with this antigen-antibody mixture, and free antibodies are removed by washing.
- A secondary antibody specific to the primary antibody is added, which normally binds to the enzyme.
- Remove free enzyme-bound secondary antibodies by washing the plate.
- Finally, the board is added. The substrate is converted by an enzyme into a coloured product that can be measured spectrophotometrically.
**Application of ELISA
Applications of ELISA are given below:
- You can measure the presence of antibodies and antigens in a sample.
- Used in the food industry to detect the presence of food allergens.
- A virus test measures the antibody concentration in serum.
- During an outbreak of disease, assess the spread of the disease. During the recent outbreak of COVID-19, rapid test kits have been used to confirm the presence of antibodies in blood samples.
**Diseases that can be diagnosed through ELISA
ELISA is used to detect a wide range of diseases, including****:**
- **Viral diseases- HIV/AIDS, Hepatitis B & Hepatitis C, Dengue fever, COVID-19, Zika virus, Chikungunya, and Influenza viruses.
- **Bacterial diseases- Tuberculosis (TB), Helicobacter pylori infection, Typhoid (Salmonella typhi), Lyme disease (Borrelia burgdorferi), and Syphilis.
- **Parasitic Diseases- Malaria, Leishmaniasis, Toxoplasmosis, and Trichinellosis.
- **Autoimmune Diseases- Rheumatoid arthritis, Systemic lupus erythematosus (SLE), and Celiac disease.