An indirect ELISA test, which usually commercially produced 96-we lystyrene-coat

Question

An indirect ELISA test, which usually commercially produced 96-we lystyrene-coated takes place in plate, has five main steps: (a) Each of the well of the (antigen) that is tested for presence of a microbial plate is coated with the sample virulence factor). product (usually a secreted or (b) Excess antigen molecules are washed off and another protein (such as BSA or gelatin) is added to the wells to completely coat any of the polystyrene surface not coated with antigen. (c) An antibody specific for the tested amtigen is added in a solution. If the plate well is coated with the right antigen, the antibody will bind to the antigen affixed to the plate. (d) After excess antibodies are washed off, anti-antibodies (secondary antibodies) labeled with the enzyme are added to each well. (e) After excess secondary antibodies are washed off, the enzyme substrate is added to each well. The reaction between the enzyme and its substrate produces products that cause a visible color change. In this ELISA test, presence of pathogenic E coli in avian samples (e.g., bird feces) is detected by testing for presence of a secreted virulence factor (the Tsh autotransporter adhesin).

Explanation / Answer

Antibody can be detected or quantitatively determined with an indirect ELISA.Serum or some other sample
containing primary antibody (Ab1) is added to an antigen- coated microtiter well and allowed to react with the antigen attached to the well. After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody. Any free Ab2 then is washed away, and a substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds.
Indirect ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection.

Reagents:-

Buffer solutions:-Antigen dissolved in PBS or other carbonate buffer

Blocking Reagents:-BSA(Bovine serum albumin)

Wash Buffer:-PBS

Primary Antibody:Test sample

Secondary Antibody:-Enzyme conjugated

Enzyme and Substrate used for detection:-many different types of enzymes have been used for detection, horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two widely used enzymes employed in ELISA assay.

ALP substrate:For most applications pNPP (p-Nitrophenyl-phosphate) is the most widely used substrate.The yellow color of nitrophenol can be measured at 405 nm after 15-30 min incubation at room temperature.

HRP chromogens: The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a hydrogen donor which changes color during reaction.

Other substrates:TMB (3,3’,5,5’-tetramethylbenzidine),OPD (o-phenylenediamine dihydrochloride),ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt).

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