ELISA Protocol

The enzyme-linked immunosorbent assay (ELISA) method is widely used to assay antibodies and antigens without fully comprehending the numerous vexing phenomena attributed to the principle, which utilizes the high binding affinity of proteins to solid surfaces such as micro plates and latex beads. There are four major types of ELISAs: direct ELISA, indirect ELISA, competitive ELISA and sandwich ELISA.


Flow Chart:






1) Prepare the ELISA plate: Coat the wells of a 96-well ELISA plate with the antigen of interest by adding a solution of the antigen to each well and incubating the plate overnight at 4°C.

2) Block the plate: After the antigen has been immobilized, add a blocking solution, such as 1% bovine serum albumin (BSA) or 5% non-fat milk, to each well to prevent non-specific binding of the detection reagent to the plate. Incubate the plate for 1-2 hours at room temperature.

3) Add the sample: Remove the blocking solution and add the sample containing the unknown antigen to the wells. Incubate the plate for 1-2 hours at room temperature.

4) Add the detection reagent: After the sample has been added, add a specific detection antibody that is conjugated to an enzyme to each well. This antibody will recognize and bind to the antigen of interest that has been immobilized on the plate. Incubate the plate for 1-2 hours at room temperature.

5) Add the substrate: Add a substrate that will be converted by the enzyme into a detectable signal, such as a colored product, to each well. Incubate the plate for 10-30 minutes at room temperature, protected from light.

6) Read the plate: Measure the absorbance of each well at a specific wavelength using a microplate reader. The absorbance will be proportional to the amount of antigen present in the sample.

It is important to include appropriate controls in the assay, such as negative controls (no antigen or antibody) and positive controls (known concentrations of antigen or antibody), to ensure the accuracy and specificity of the assay. The specific details of the ELISA protocol may vary depending on the type of ELISA and the specific reagents used.