ELISA and its type

  • 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.

Principle

  • 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.

 

Techniques

  • 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.

 

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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.

 

Image result for direct elisa                     Image result for direct elisa

 

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.

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ELISA and its type

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