4 STEPS OF INDIRECT ELISA (Enzyme Linked Immunosorbent Assay)
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2 سال پیش
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ELISA is an abbreviation of
ELISA is an abbreviation of enzyme linked immunosorbent assay and utilize the bond between an antibody and its specific antigen for it to work. There are 4 main types of ELISA:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
Today we will look closer at indirect ELISA which is carried out in the following manner:
1. First, components of a sample are bound to the floor of a micro well and then any unbound components are washed away.
2. Second, primary antibodies specific to the target antigen are added to the well and bind to the target antigen if it is present. Then the well is rinsed again to remove any unbound antibodies.
3. Third, secondary enzyme-conjugated antibodies are added which bind to the primary antibodies if these are present.
4. Finally, substrate for the enzyme which is linked to the secondary antibody is added and the enzyme converts this substrate into an observable signal.
This means that if the target antigen is present, the primary antibody can bind to it and then the enzyme-conjugated secondary antibody can bind to the primary antibody. This allows the enzyme-conjugated secondary antibody to then produce a signal once the substrate is added, producing a positive result. If the target antigens are not present however, this prevents the primary antibodies from binding to them which in turn makes it impossible for the enzyme-conjugated secondary antibody to bind to them. Then the enzyme can not react with the conjugate once it is added, causing a negative result. In other words, if a color change occurs, it is interpreted as a positive test result and if no color change occurs it is interpreted as a negative test result.
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
Today we will look closer at indirect ELISA which is carried out in the following manner:
1. First, components of a sample are bound to the floor of a micro well and then any unbound components are washed away.
2. Second, primary antibodies specific to the target antigen are added to the well and bind to the target antigen if it is present. Then the well is rinsed again to remove any unbound antibodies.
3. Third, secondary enzyme-conjugated antibodies are added which bind to the primary antibodies if these are present.
4. Finally, substrate for the enzyme which is linked to the secondary antibody is added and the enzyme converts this substrate into an observable signal.
This means that if the target antigen is present, the primary antibody can bind to it and then the enzyme-conjugated secondary antibody can bind to the primary antibody. This allows the enzyme-conjugated secondary antibody to then produce a signal once the substrate is added, producing a positive result. If the target antigens are not present however, this prevents the primary antibodies from binding to them which in turn makes it impossible for the enzyme-conjugated secondary antibody to bind to them. Then the enzyme can not react with the conjugate once it is added, causing a negative result. In other words, if a color change occurs, it is interpreted as a positive test result and if no color change occurs it is interpreted as a negative test result.
2 سال پیش
در تاریخ 1401/07/01 منتشر شده
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