- It is the abbreviated form of Enzyme-linked immunosorbent assay or EIA.
- It is similar in principle to Radio immune assay (RIA) but instead of radioactive substance it uses an enzyme.
- Chromogenic substrate is generated by reacting an enzyme conjugated with the antibody and a colorless substrate.
- Chromogenic substrate is a colored reaction product.
- Various enzymes have been employed in ELISA which includes alkaline phosphatase, horseradish peroxidase and beta-galactosidase.
- The sensitivity of this assay is similar to RIAs and has various advantages being safer and less costly.
- This method for measuring antigen-antibody reaction is becoming increasingly used in the detection of antigen (infectious agent)or antibody for its simplicity and sensitivity.
- It requires only microliter quantities of test reagents.
- It is used for detection of a variety of antibody and antigens such as hormones, toxins, and viruses.
- The results of quantitative ELISA tests can be read visually.
- A large number of tests can be done at a single time.
- Reagents that are used in this method are stable and can be distributed in district and rural laboratories.
- Most methods developed for detection of antigen or antibody consists of use of corresponding antibody or antigen in question.
- This mixture get firmly fixed on solid phase like plastic surface of polyvinyl plate or polystyrene tube, inside the well of a micro dilution tray or outside of spherical plastic or metal bead.
- For this, the methods are also called solid phase immune-sorbent assays (SPIA).
- The enzyme system consists of
i) An enzyme horseradish peroxidase and alkaline phosphatase which is labeled or linked to a specific antibody or antigen.
ii) A specific substrate such as O-phenyl-diamin-dihydrochloride is used for peroxidase and p-nitrophenyl phosphate for alkaline phosphatase.
iii) These are added after the antigen-antibody reaction takes place.
iv) The enzyme catalyzes or hydrolyses the substrate to give a color end point i.e. yellow compound in case of alkaline phosphatase.
v) The amount of bound antigen or antibody is indicated by the intensity of the color.
- There are numerous variants of ELISA. They are as follows:
A) Indirect ELISA
- Antibody can be detected or quantitatively determined with an indirect ELISA.
- Primary antibody (Ab1) containing serum or some other sample is added to an antigen coated microtiter well.
- They are allowed to react with the antigen attached to the well.
- The free antibody Ab1 is washed away if present.
- Then, presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody (Ab2) that binds to the primary (Ab1) antibody.
- Again, the free antibody Ab2 is washed away if present in that mixture.
- After then, a substrate is added which is specific for the enzyme.
- Specialized spectrophotometric plate readers are used to measure the amount of colored reaction product.
- These plate readers can measure the absorbance of all of the wells of a 96-well plate in seconds.
- This method is used to detect the presence of serum antibodies against HIV that causes AIDS.
- The recombinant envelope and core proteins of HIV are adsorbed as solid phase antigens on microtiter wells.
- Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins.
- Within 6 weeks of infection, the serum antibodies to HIV can be detected by this method.
- Several parasitic infections can also be diagnosed by this method.
B) Double antibody technique for detection of antigen or sandwich ELISA
- This method is used for detection of antigens.
- Here, the antibody (rather than antigen) is immobilized in the microtiter well.
- The sample containing the antigen is added and allowed to react with the immobilized antibody.
- This mixture is incubated for 2 hours at 370C.
- Then, the well is washed to remove excess of unbound antigen.
- After that a second enzyme linked antibody specific for a different epitope on the antigen is added.
- This mixture is allowed to react with the bound antigen with the incubation for one hour at 370C.
- The free enzyme linked antibody is washed away if present in excess which is followed by addition of a substrate.
- A colored reaction product is obtained which is measured as in indirect ELISA.
- This technique is useful in detection of hepatitis B antigen, hormones, toxin and HCG.
C) Competitive ELISA
- This method is also used for measuring the amounts of antigen.
- In this technique, antibody is first incubated in solution with a sample containing antigen.
- This mixture of antibody-antigen is added to a microtiter well coated with an antigen.
- The more antigens present in the sample, the less free antibody will be available to bind to the antigen coated well.
- The excess of mixture is then washed away after proper incubation for appropriate time.
- An enzyme conjugated secondary antibody (Ab2) is added after the washing process is completed.
- The Ab2 is specific for the isotype of the primary antibody bound to the well which can be used to determine the amount of primary antibody bound to the well.
- The other processes are similar as in indirect ELISA.
- In this method, the higher the antigen in the original sample the lower the absorbance we obtained.
- This method is used for detection of bluetongue virus antibodies in Illamas and wild ruminants.