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What is an ELISA Kit?

 

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An ELISA (Enzyme Linked Immuno Sorbent Assay) Kit contains all materials and reagents which are used to perform a quantitative determination of an antigen of interest. This could be for example antibodies, proteins and viruses or low-molecular substances like hormones, pesticides and toxins. The ELISA method belongs to the group of immunoassays and is based on the principle of an enzymatic colour reaction linked to an antibody. Only few materials and instruments are needed to analyse samples via ELISA. In comparison to more extensive chromatographic methods like GC-MS or HPLC it offers a cheap alternative.

 

How does an ELISA Kit work?

Every well is pre-coated with antibodies specific to the antigen of interest

 

 

The ELISA Kit contains a 96-well microtiter plate. The bottom of every well has been pre-coated with antibodies specific to the antigen of interest (1).

 

  

 

 

Antigen bound by the pre-coated antibody

 

 

Within the first step, standards or samples are added to the wells. If the antigen of interest is present, it will be bound by the fixed antibodies (2).

After this it is of importance to wash the microtiter plate to remove unbound elements, otherwise the measurement could get distorted.

 

 

 

 

The antigen ist bound between two different antibodiesDuring the second step, another Biotin-conjugated polyclonal antibody (named Detection Reagent A in the ELISA Kit) is added to the antibody-antigen complex. It is important that the Biotin-conjugated antibody is able to recognize different epitopes (binding sites) in contrast to the first one fixed to the bottom of the well. In this manner, a so-called “Sandwich Complex” is formed by the antigen between two antibodies (3).
 

Subsequently the plate should be washed again to remove the unbound antibody-enzyme conjugates.

 

 

 

 

 

The HRP enzym is bound to the "Sandwich-Complex" to enable a colour reactionAfterwards an enzyme (HRP =horse radish peroxidase) which is needed for the following colour reaction is bound to the “Sandwich Complex”, by adding the Biotin-binding Avidin (chicken egg white protein), conjugated with HRP (named Detection Reagent B in the ELISA Kit), to the wells (4).

This intermediate step via Avidin-Biotin-conjugation provides an amplification of the signal and is mainly used by detection of minimal amounts of the compound of interest. To ensure that the results are not corrupted by unbound enzyme-conjugates, the wells have to be washed carefully before starting the colour reaction by addition of TMB (TMB = 3, 3’, 5, 5’- tetramethylbenzidin).

TMB is a colourless substrate which shows a dark blue colour in the presence of peroxidases. By the addition of acid (for example sulphuric acid) after a certain time, the enzyme (HRP) is denatured and thus the reaction is stopped. Because the pH-value is changed thereby, TMB turns from blue to yellow.

The intensity of the colour (absorbance) can be measured photometrically at an absorption maximum of 450 nm using a microplate reader. A precise quantification of the compound of interest in the sample is possible by comparison of the measured absorbance to a previously generated standard curve.