Enzyme Linked Immunosorbent Assay is to bind a known antigen or antibody to the surface of a solid phase carrier, then uses an enzyme-labeled (conjugated) antibody or antigen to incubate with it, and develops color through a chromogenic material. Its color depth is proportional to the content of the substance to be tested, which can be observed with the naked eye.
ELISA is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. ELISA assays are generally carried out in 96-well plate or 384-well plate to allow multiple samples to be measured in a single experiment.
Procedure
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Coating
Immobilize antigens to the surface of polystyrene microplate wells directly or indirectly.
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Blocking
Add irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
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Detection
Incubate with antigen-specific antibodies that affinity bind to the antigens.
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Readout
Add substrate and measure the signal produced by the enzyme-substrate reaction.
Fig. 1 The basic flow chart for ELISA.
Types
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Direct ELISA
The antigen is immobilized in the well of an ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP.
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Indirect ELISA
The antigen is adsorbed to a well in an ELISA plate. An antigen-specific binding antibody was added and incubated briefly. Add an antibody to detect the first binding antibody which is usually conjugated to an enzyme.
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Sandwich ELISA
Sandwich ELISA requires the use of matching antibody pairs (capture and detection antibodies). Thus, each antibody is specific for a different and non-overlapping region or epitope of the antigen. The matching antibody pairs are specifically tested in sandwich ELISA to ensure that they detect different epitopes to obtain accurate results.
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Competitive ELISA
Competitive ELISA relies on the competitive reaction between the sample antigen and antigen bound to the wells of microtiter plate with the primary antibody.
Features
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Give an accurate diagnosis of a particular disease as two antibodies are used.
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Carry out for complex samples since the antigen is not required to get purified to detect.
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Highly responsive because direct and indirect analysis methods can be performed.
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Rapid test and quick results.
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Detection methods range from the quantitative, semi-quantitative, standard curve, qualitative, calibration curve models, etc.
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Easier to perform and simple process as compared to other assays which require the presence of radioactive materials.
Applications
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Determine the presence of antibodies and antigens in a sample.
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Detect any food allergens present in the food industry.
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Determine the concentration of serum antibody in a virus test.
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Diagnose diseases, such as Ebola, Pernicious anaemia, AIDS, Rotavirus, Lyme disease, Syphilis, Toxoplasmosis, Zika virus and Carcinoma of the epithelial cells.
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STEMart provides you with a variety of ELISA equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for ELISA, please feel free to contact us.