ELISA is known as an enzyme-linked immunosorbent assay. It is a basic assay technique, that is carried out to detect and measure antibodies, hormones, peptides and proteins in the blood.
Antibodies are blood proteins produced in response to a specific antigen. It helps to examine the presence of antibodies in the body, in the case of certain infectious diseases.
It is a distinguished analysis compared to other antibody-assays as it yields quantitative results and separation of non-specific and specific interactions that take place through serial binding to solid surfaces, which is normally a polystyrene multiwell plate.
Principle of ELISA
It works on the principle that specific antibodies bind the target antigen and detect the presence and quantity of antigens binding. To increase the sensitivity and precision of the assay, the plate must be coated with antibodies with high affinity. ELISA can provide a useful measurement of antigen-antibody concentration.
Types Of ELISA
It can be classified into three types depending upon the different methods used for binding between antigen and antibodies, namely:
- It detects the presence of an antibody in a sample.
- The antigen is attached to the wells of the microtitre plate.
- A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen.
- The free primary antibodies are washed away and the antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody.
- All the free secondary antibodies are washed away. A specific substrate is added which gives a coloured product.
- The absorbance of the coloured product is measured by spectrophotometry.
- It helps to detect the presence of antigen in a sample.
- The microtitre well is coated by the antibody.
- The sample containing the antigen is added to the well and washed to remove free antigens.
- Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies.
- The enzyme-specific substrate is added to the plate to form a coloured product, which can be measured.
- It helps to detect antigen concentration in a sample.
- The microtitre wells are coated with the antigen.
- Antibodies are incubated in a solution having the antigen.
- The solution of the antigen-antibody complex is added to the microtitre wells. The well is then washed to remove any unbound antibodies.
- More the concentration of antigen in the sample, the lesser the free antibodies available to interact with the antigen, which is coated in the well.
- The enzyme-linked secondary antibody is added to detect the number of primary antibodies present in the well.
- The concentration is then determined by spectrophotometry.
Diseases That Can Be Diagnosed Using ELISA
- Pernicious anaemia
- Lyme disease
- Zika virus
- Carcinoma of the epithelial cells
Advantages Of ELISA
Following are some of the advantages of the ELISA technique:
- Results fetched from ELISA gives an accurate diagnosis of a particular disease since two antibodies are used.
- Can be carried out for complex samples as the antigen is not required to get purified to detect.
- It is highly responsive since direct and indirect analysis methods can be carried out.
- It is a rapid test, yields results quickly.
- Possible detection for ELISA ranges from the quantitative, semi-quantitative, standard curve, qualitative, calibration curve models etc.
- Easier to perform and uncomplicated process as compared to other assays which require the presence of radioactive materials.
Applications of ELISA
- The presence of antibodies and antigens in a sample can be determined.
- It is used in the food industry to detect any food allergens present.
- To determine the concentration of serum antibodies in a virus test.
- During a disease outbreak, to evaluate the spread of the disease, e.g. during a recent COVID-19 outbreak, rapid testing kits are being used to determine the presence of antibodies in the blood sample.