ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY

ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY

14.      ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY
ELISA or Enzyme-Linked Immunosorbent Assay include an enzyme-linked antibody and when react with colourless substrate produce the coloured product and that type of substrate known as the chromogenic substrate. ELISA can detect -antigen or antibody concentration within the sample, both qualitatively and quantitatively. The quantity or concentration of unknown antigen or antibody is determined by plotting the Optical density of unknown on the standard curve generated by the known concentrations of antigen and antibody versus their respective optical density at that known concentrations. 
14.1.    Direct ELISA
In this type of ELISA, firstly, we need to immobilize the antigen on the 96-well microtiter plate, after that enzyme-linked antibody specific to that antigen which we want to identify is added and incubated, after incubation unbound enzyme-linked antibody is washed by washing buffer, next to this step colourless substrate is added, as a result, colour product is formed and the presence of colour defines the presence of the desired antigen but it can be confirmed by taking the optical density of the reaction at particular lambda. It is a qualitative method for the identification of the presence of the antigen.


14.2.    Indirect ELISA
Through this method, both qualitative and quantitative detection of antibody can be done. In this method antigen coated 96-well microtiter plate is used. The antigen is specific for a particular antibody whose presence we need to identify in our sample. It shows the presence of that antigen in the animal body from which antibody is isolated.
Firstly we isolate serum or other samples which contain antibody from the body of the subject. This antibody is known as a primary antibody. The Primary antibody is added to the antigen-coated 96-well microtiter plate and incubated which allows the primary antibody to react with antigen immobilized on the microtiter plate. After that unbound antibody is washed with the help of washing buffer. After that secondary enzyme-linked antibody is added to the 96-well microtiter plate, which binds to Fc region of the primary antibody. After incubating, the unbound secondary antibody is washed with the buffer. 

After this step substrate is added and incubated for some time, as a result, colourful product is formed and the optical density of the product is taken by the ELISA plate reader. Colour defines the presence of the desired antigen but it gets confirmed by taking the optical density of the reaction at particular lambda and the quantity or concentration of unknown antibody is determined by plotting the optical density of unknown antibody on the standard curve generated by the known concentrations of primary antibody versus their respective optical density at that known concentrations. This method is commonly used to determine the presence of human immunodeficiency virus (HIV) by detecting the antibody against HIV. ELISA can be carried out within 6 weeks of HIV infection.
14.3.    Sandwich ELISA
Through this method, both qualitative and quantitative detection of antigen can be done. In this method, the antibody-coated 96-well microtiter plate is used. The antibody is specific for particular antigen whose presence we need to identify in our sample. It shows the presence of that antigen in the animal body from it is isolated.
Firstly, we isolate the sample which contains antigen from the body of the subject. The antigen of unknown concentration is added to the antibody-coated 96-well microtiter plate and incubated which allows antigen to react with primary antibody immobilized on the microtiter plate, antibody bind to an epitope present on an antigen. To get prevent from cross reactivation we use mAb. After that unbound antigen is washed with the help of a buffer. 

After that secondary enzyme-linked antibody (mAb) is added to the 96-well microtiter plate, which binds to the different epitope on the same antigen and incubated which facilitates the binding of the secondary antibody to the same antigen. As incubation complete, unbound secondary antibody is washed with the washing buffer. After this step substrate is added and incubated for some time, as a result, the colourful product is formed and the optical density of the product is taken by the ELISA plate reader. Colour defines the presence of desire antigen but it gets confirmed by taking the optical density of the reaction at particular lambda and the quantity or concentration of unknown antigen to determine by plotting the optical density of unknown antigen on the standard curve generated by the known concentrations of antigen versus their respective optical density at that know concentrations. This method is called a sandwich because antigen is sandwiched between primary and secondary antibody. It is most commonly used to determine the concentration of cytokine.
14.4.    Competitive ELISA
In this type of ELISA, the sample containing antigen is incubated with antibody and the formation of the Ag-Ab complex takes place. After that, this complex is added to the 96-well microtiter plate coated with the same immobilized antigen which is present in the sample and the remaining free antibody binds with immobilized antigen. Very less antibody remains free to bind with immobilized antigen and now washing is done to remove unbound antibody. After that secondary enzyme-linked antibody is added and incubated which allows interaction between primary and secondary antibody. When incubation is done, the unbound secondary antibody is washed out by washing buffer, after that substrate is added. As a result colour product is produced, If the lower the concentration of antigen in sample higher the final result found and if higher the concentration of antigen then responsible to produce the low final result. It is like RIA.


14.5.    In ELISA, most commonly use the enzyme, all are chromogenic
14.5.1.    Horseradish peroxidises - It is isolated from Horseradish plant, the substrate of Horseradish peroxidase is 3.3', 5'5' tetraethyl benzedrine TMB. This enzyme converts its colourless substrate into the coloured product in the presence of H2O2, in turn also produces water.
14.5.2.    Alkaline phosphatase - It is isolated from calf intestine, the substrate of alkaline phosphatase is P-nitrophenyl phosphate (pNPP), pNPP is colourless and highly soluble. Due to the action of this enzyme on pNPP its converts into p-nitrophenol, it gives yellow colour at 460 nm.
14.5.3.    β – galactosidase-It is isolated from E.coli. This enzyme converts β-D-galactoside into galactose and alcohol in presence of water.
14.5.4.    Urease – It is isolated from Jack bean, It converts (NH2)2CO into 2NH4OH and CO2 in presence of water.
Chemiluminescence gives more sensitive result then the chromogenic substrate, sensitivity increased by them is 200 fold. As an example, luminol gets oxidized by HRP in the presence of H2O2 produce light. Chromogenic substrate give yes and no phenomenon by the production of colour and can be seen by the naked eye but if we use fluorogenic and chemiluminogenic substrates its visualization of results require special equipment, not seen by naked eye, but they give the highly sensitive result.


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