What Is ELISA Technique?
ELISA Technique is a basic testing technique, known as enzyme-linked immunosorbent assay (EIA: also called enzyme immunoassay), which is designed to detect and measure antibodies, hormones, peptides and proteins in blood.
Antibodies are blood proteins produced in response to a specific antigen. It helps to test the presence of antibodies in the body, in some infectious diseases.
The ELISA is a specific assay compared to other immunoassays because it provides quantitative results and distinguishes non-specific interactions occurring by subsequent binding to solid surfaces, typically polystyrene multiwell plates.
Types Of ELISA
ELISA assays can be divided into three types according to the different methods used for binding between antigens and antibodies, viz.
- Indirect ELISA – antigen is applied to a microtiter well
- Sandwich ELISA – Antibody is applied to the top of a microtiter well
- Competitive ELISA – An antigen-coated microtiter well is filled with a mixture of antigens and antibodies.
- Indirect ELISA detects the presence of antibodies in the sample.
- The antigen is conjugated to the wells of the microtiter plate.
- The antibody-containing sample is added to the antigen-coated wells for binding to the antigen.
- Naked primary antibodies are filtered and the antigen-antibody complex is determined by the addition of an enzyme-linked secondary antibody capable of binding to the primary antibody.
- Any secreted secondary antibodies are washed away. A specific substrate is added to give a colored product.
- The absorption of the substance in question is measured spectrophotometrically.
- Sandwich ELISA helps determine the presence of an antigen in a sample.
- The micro tire is well coated with antibody.
- The antigen-containing sample is added to the well and washed to remove free antigens.
- A second anti-enzyme antibody is then added, which binds to another epitope on the antigen. The wells are washed to remove any secondary resistance released.
- An enzyme-specific substrate is added to the plate to form a colored product that can be measured.
Principle of ELISA
The ELISA works on the principle that specific antibodies bind the target antigen to determine the presence and amount of antigen binding. Plates should be coated with high-affinity antibodies to increase the sensitivity and specificity of the assay. ELISAs can provide useful measures of antigen and antibody concentrations.
ELISA is one of the simplest blood tests that can be performed. It is quick, urgent and requires blood from the patient. The complete procedure of the ELISA is shown below.
- The antibody is attached to a hard surface polystyrene plate and attracts or binds to bacteria and other antibodies and hormones.
- In this antigen-antibody mixture, a microtitre labeled with saturated antigen followed by washing to remove free antibodies.
- A second antibody specific for the primary antibody is added which is usually conjugated to the enzyme.
- The secondary resistance associated with the free enzyme is removed by washing the plate.
- Finally, substrate is added. The substrate is metabolized by enzymes to form a colored product that can be measured spectrophotometrically.
- Pregnancy marker HCG protein is detected by ELISA. A combination of blood or urine sample and enzyme-linked purified hCG is added to the system. If HCG is not present in the test sample, only the enzyme involved binds to the solid surface.
- The larger the adsorbent, the larger the interface and the smaller the enzyme associated with the solid surface. These reactions are usually indicated by the color of the solution changing.