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Bishal Paul posted an Question
August 05, 2021 • 03:10 am 30 points
  • CSIR NET
  • Life Sciences

Discuss the conclusion of direct elisa and indirect elisa? explain

Discuss the conclusion of direct ELISA and indirect ELISA

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  • Dr. kirti

    Thank you for posting as a separate question... here I list out application of Direct Elisa...

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  • Priya sarda best-answer

    What is ELISA? Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result. An antibody is a type of protein produced by an individual’s immune system. This protein type has specific regions that bind to antigens. An antigen is a protein that can come from some foreign source and, when bound to an antibody, induces a cascade of events through the body’s immune system. This interaction is utilized in ELISA testing and allows for identifying specific protein antibodies and antigens, with only small amounts of a test sample. ELISA testing is used to diagnose HIV infection, pregnancy tests, and blood typing, among others. Both direct and indirect ELISAs begin with the coating of antigen to the ELISA plates. The first binding step involves adding antigen to the plates, which is incubated for one hour at 37 degrees C or can be incubated at 4 degrees C overnight. Once the incubation step is completed, the next step is to wash the plates of any potential unbound antibody and block any unbound sites on the ELISA plate using agents like BSA, ovalbumin, aprotinin, or other animal proteins. This second step is important because it prevents the binding of any non-specific antibodies to the plate and minimizes false-positive results. After adding the buffer, the plate is rewashed, and a selected enzyme-conjugated primary detection antibody is added. The plate is further incubated for one hour. Direct ELISA In a direct ELISA, the primary detection antibody binds directly to the protein of interest. Next, the plate is rewashed to remove any unbound antibody and followed by the addition of a substrate/chromophore, such as alkaline phosphatase (AP) or Horseradish Peroxidase (HRP) to the plate, which results in a color change. The color change of the sample occurs by either the hydrolysis of phosphate groups from the substrate by AP or by the oxidation of substrates by HRP. The advantages of using direct ELISA include eliminating secondary antibody cross-reactivity, and due to fewer steps, it is rapid compared to indirect ELISA. Its disadvantages include its low sensitivity compared to the other types of ELISA and its high cost of reaction Indirect ELISA The steps of the indirect ELISA are identical to the direct ELISA, except for an additional wash step and the types of antibody added after the buffer is removed. Indirect ELISA requires two antibodies, a primary detection antibody that sticks to the protein of interest and a secondary enzyme-linked antibody complementary to the primary antibody. The primary antibody is added first, followed by a wash step, and then the enzyme-conjugated secondary antibody is added and incubated. After this, the steps are the same as the direct ELISA, which includes a wash step, the addition of substrate, and detection of a color change. The indirect ELISA has a higher sensitivity when compared to the direct ELISA. It is also less expensive and more flexible due to the many possible primary antibodies that can be used. The only major disadvantage with this type of ELISA is the risk of cross-reactivity between the secondary detection antibodies

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