ELISA Test Steps

step 1

Antigens are absorbed to the bottom of the wells. This forms the capture layer for the ELISA test.

Rinsing:
The wells are rinsed to remove any unbound antigens.

step 2

A blocking buffer is added to coat any remaining surface of the wells to prevent non-specific binding. The antigens remain bound to the bottom of the wells.

Rinsing:
The wells are rinsed again to remove excess blocking solution.

step 1

Antigens are absorbed to the bottom of the wells. This forms the capture layer for the ELISA test.

Rinsing:
The wells are rinsed to remove any unbound antigens.

step 2

A blocking buffer is added to coat any remaining surface of the wells to prevent non-specific binding. The antigens remain bound to the bottom of the wells.

Rinsing:
The wells are rinsed again to remove excess blocking solution.

step 3

A sample, containing primary antibodies to the antigen, is added. If the target antigen is present, the primary antibodies bind to the antigens.

Rinsing:
The wells are rinsed to wash away unbound primary antibodies.

step 4

An enzyme-conjugated secondary antibody is added. This antibody binds to the primary antibody.

Rinsing:
The wells are rinsed to remove any unbound secondary antibodies.

step 5

A substrate solution is added. At this stage, there is no color change because the substrate has not yet interacted with the enzyme.

Rinsing:
No rinsing at this step; the substrate remains in the well to interact with the enzyme.

step 6

The substrate reacts with the enzyme on the secondary antibody, forming an enzyme-substrate complex. This reaction produces a color change, usually yellow or blue, depending on the substrate used (substrate changes color).

Rinsing:
No rinsing; the color change is the final readout.

in what order should you add the reagents to perform an Eliza test?

antigen sample, primary antibody, enzyme linked secondary antibody, substrate