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Sandwich ELISA steps
A sandwich ELSIA gets its name from the ‘sandwich’ that is formed with two antibodies and an antigen during the process. Sandwich ELISA steps are:

Step 1: Immobilization of the capture protein
In a sandwich ELISA the capture protein is an antibody, and the target is the antigen. Therefore, the first ELISA step is to immobilize the capture antibody onto the plate. Many ELISA kits are available with the capture protein pre-immobilized.

Step 2: Wash off any un-adsorbed capture protein from the well surface
Washing steps with detergents reduce off-target hydrophobic interactions between proteins and remove unbound capture proteins from the plate.

Step 3: Block any unbound sites on the plate
The hydrophobic interactions between proteins are mediated by charge or hydrophobicity. This means they can be mitigated to some extent by blocking and washing steps.

Proteins are added to the plate, which adsorb to the open sites on the plate’s surface. Proteins such as Bovine Serum Albumin (BSA), Casein, or aprotinin are commonly used to block in an ELISA assay.

These proteins will adsorb to the plate, preventing the target protein from accessing these sites later on. Lowering the incidence of non-specific binding in this way results in lower background noise.

Step 4: Wash away any un-adsorbed blocking proteins from the well
A second washing step removes any unbound blocking proteins.

Step 5: Incubate with the sample (serum, urine, saliva, or spiked research solution)
In this step, the specific recognition between antibody and antigen takes place. In a sandwich ELISA, the antibodies are adsorbed to the plate, and bind to the target antigen in the sample fluid. This requires an incubation step to allow the binding kinetics to reach equilibrium.

Step 6: Wash away the incubation fluid
This is a crucial washing step that often requires multiple washes to ensure that any unbound antigen is washed away. After this step, the only antigens that should remain in the wells are those attached to capture antibodies.

Step 7: Incubate with detection antibody
The detection antibody in an ELISA is conjugated to a certain label such as a flurophore (for fluorimetry), or an enzyme (for colorimetric detection or chemiluminescence). The detection antibody ‘recognizes’ a different epitope on the target antigen than the capture antibody. Sandwich ELISA assays therefore result in a ‘sandwich’ of capture antibody-antigen-detection antibody.

Step 8: Wash away unbound detection antibody
Step 9: Apply substrate for chemical colorimetric or chemiluminiscent reactions, or apply incident light for fluorescent reactions, and quantify the signal
Depending on the ELISA method used, the amount of fluorescence, luminescence, or intensity of the color indicates how much target antigen was captured from the sample.