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Saturday, December 28, 2019

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

Introduction:
Eliza is very sensitive immunological technique which is usually used to Detect or measure the concentration of antigen or antibodies in solution.The term was Coined by Engvall and Pearlman in 1971.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation
A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It can also be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).

BASIC PRINCIPLE OF ELISA:

Most ELISA methods developed for the detection of antigen or antibody consist of use of corresponding antibody or antigen in question which is firmly fixed on solid phase, such as plastic surface of polyvinyl plate or polystyrene tube. Such systems are also called Solid Phase Immunosorbent Assay (SPIA).
Test sample is added in the microtitre plate, if there is presence of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized Ab or Ag). Later enzyme labelled antibody is added in the reaction mixture, which will combine with either test antigen or Fc portion of test antibody.
The enzyme system consists of;
1.     An enzyme:  horse radish peroxidase, alkaline phosphatase which is labelled or linked, to a specific antibody.
2.     A specific substrate:
§  o-Phenylenediamine dihydrochloride for peroxidase
§  P Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase
Substrate is added after the antigen-antibody reaction. The enzyme catalyses (usually hydrolyses) the substrate to give a color end point (yellow compound in case of alkaline phosphatase). The intensity of the color is proportional to the amount of  antibody or antigen present in the test sample, which can be quantified using ELISA reader.


Salient Features of ELISA Test 
1.     ELISA test has high sensitivity and specificity
2.     The result of quantitative ELISA tests can be read visually
3.     A large number of tests can be done at one time ELISAs are designed specifically for screening large numbers of specimens at a time, making them suitable for use in surveillance and centralized blood transfusion services.
4.     Reagents used for ELISA are stable and can be distributed in district and rural laboratories but as ELISAs require sophisticated equipment and skilled technicians to perform the tests, their use is limited to certain circumstances.

Most Commonly used ELISA are:
Types of ELISA on the basis of binding structure between the Antibody and Antigen.
Ø Direct ELISA
Ø Indirect ELISA
Ø Sandwich ELISA
Ø Competitive ELISA

Direct ELISA: 
A direct ELISA (enzyme-linked immunosorbent assay) is a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample

Indirect ELISA

Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that the secondary antibody may bring about.
PROCEDURE OF INDIRECT ELISA
Ø Coat the micro titer plate wells with antigen.
Ø Block all unbound sites to prevent false positive results.
Ø Add sample containing antibody (e.g. rabbit monoclonal antibody) .
Ø Wash the plate, so that unbound antibody is removed.
Ø Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).
Ø Wash the plate, so that unbound enzyme-linked antibodies are removed.
Ø Add substrate which is converted by the enzyme to produce a colored product.
Ø Reaction of a substrate with the enzyme to produce a colored product.


SANDWISH ELISA

The Sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) is a sensitive and robust method which measures the antigen concentration in an unknown sample. The antigen of interest is quantified between two layers of antibodies: the capture and the detection antibody. These antibodies must bind to non-overlapping epitopes on the antigen. Either monoclonal or affinity-purified polyclonal antibodies can be used as capture and detection antibodies, depending on cost, the dynamic range and the sensitivity of the final assay.

PROCEDURE OF SANDWICH ELISA:
Ø Prepared a surface to which a known quantity of antibody is bound.
Ø Add the antigen-containing sample to the plate.
Ø Wash the plate, so that unbound antigen is removed.
Ø Add the enzyme-linked antibodies which are also specific to the antigen.
Ø Wash the plate, so that unbound enzyme-linked antibodies are removed.
Ø Add substrate which is converted by the enzyme to produce a colored product.
Ø Reaction of a substrate with the enzyme to produce a colored product.


COMPETITIVE ELISA:
​​​Also known as inhibition ELISA or competitive immunoassay, this assay measures the concentration of an antigen by detection of signal interference. Each of the
previous formats can be adapted to the competitive format. The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody. The reference antigen is pre-coated on a multi-well plate. The sample is pre-incubated with labeled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.



Application of ELISA

      Presence of antigen or the presence of antibody in a sample can be evaluated.
      Determination of serum antibody concentrations in a virus test.
      Used in food industry when detecting potential food allergens.
      Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc

References:
Ø Anathanarayan and Paniker’s ,Textbook of Microbiology,10th edition, Publications by University press(India)Private limited 2017,Page No.116-118.
Ø Prescott,Harley and Klein’s Microbiology,Seventh edition,published by McGraw-Hill education(Asia) 2008,Page no.877-779.




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