A basic array technique called enzyme-linked immunosorbent assay that basically used to identify and measure the antibodies, proteins, peptides, and hormones in the blood. Its test result can provide us the information about the disease that may help in planning treatment. It compares with other antibody assays to provide quantitative results and separation of non-specific and specific interactions caused by continuous binding to solid surfaces, usually polystyrene multiwell plates.
Antigen (Ag)
A toxic molecule or any foreign matter that causes an immune response in the body is called an antigen. In immunology, an antigen term 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.
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.
Concept
This technique works on the principle of antigen-antibody binding. This technique falls under the category of labeled immunoassay. 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.
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 it was attracted to bacteria, other hormones, and antibodies.
- Antigen-coated microtiters 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 colored product that can be measured spectrophotometrically.
Application of ELISA
- You can measure thepresence of antibodies and antigens in asample.
- Usedin the food industry to detectthe presence offoodallergens.
- A virus test measurestheantibodyconcentration inserum.
- During an outbreak ofdisease, assessthe spread of thedisease. During therecentoutbreak of COVID-19,rapidtestkitshave beenused toconfirm thepresence of antibodies in bloodsamples.
Types of ELISA
There are three types of ELISA tests that are distinguished on the basis of methods used for binding between antibodies and antigens-
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
Indirect ELISA
- It detects whether an antibody is present in the sample or not.
- The antigen binds to the wells of the microtiter plate.
- A sample containing antibodies is added to the antigen-coated well and bound 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 colored product.
- Absorbance measurement of the colored product is done by spectrophotometer.
Sandwich ELISA
- 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.
- An enzyme-specific substrate is added to the plate to form a measurable color product.
Competitive ELISA
- It helps to measure the antigen concentration in a sample.
- The microtiter plate is coated with antigen.
- The antibody is incubated in a solution that contains antigens.
- Now add a solution of the antigen-antibody complex to the microtiter well and the wells are then washed away 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.
Diseases that can be diagnosed through ELISA
- Chickenpox
- Rotavirus
- AIDS
- Lyme diseases
- Pernicious anemia
- Zika virus
- Syphilis
- Toxoplasmosis
- Carcinoma of the epithelial cells
- Rocky Mountain spotted fever
- Shingles
Advantages of ELISA
- It yields results quickly as it is a rapid test.
- By using antibodies it even detects antigens at the picogram level.
- It gives an accurate diagnosis of particular diseases as it uses two antibodies.
- Can be done by use of any sample type like urine, saliva, tissues and cellular extract, plasma, and serum.
- It is uncomplicated and easier to perform as compared to other assays.
Disadvantages of ELISA
- Results should be obtained in a short time as the detection is based on substrate/enzyme reaction.
- Information is limited to the presence or amount of antigens.
Frequently Asked Question
Question 1: Write the principle on which ELISA works?
Answer:
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 measures of antibody-antigen concentration.
Question 2: Briefly discuss Sandwich ELISA.
Answer:
Sandwich ELISA
- 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 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.
- An enzyme-specific substrate is added to the plate to form a measurable color product.
Question 3: Write disadvantages of ELISA.
Answer:
Disadvantages of ELISA
- Results should be obtained in a short time as the detection is based on substrate/enzyme reaction.
- Information is limited to the presence or amount of antigens.
Question 4: Name the diseases diagnosed by ELISA.
Answer:
Diseases that can be diagnosed through ELISA
- Chickenpox
- Rotavirus
- AIDS
- Lyme diseases
- Pernicious anemia
Question 5: Explain the procedure of ELISA.
Answer:
ELISA is one of the easiest test 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 it was attracted towards bacteria, other hormones and antibodies.
- Antigen-coated microtiters 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 colored product that can be measured spectrophotometrically.