How do I set up a Sandwich ELISA?

The Sandwich ELISA quantifies target antigen concentration between capture antibody (coated on to the well plate) and detection antibody. The target antigen needs to have at least two non-overlapping antigenic sites with the ability of binding to antibodies. The capture and detection antibodies can be monoclonal or polyclonal. Generally, the detection antibody is monoclonal as it imparts accuracy, whereas polyclonal antibodies are used as capture antibody since they could pull down the maximum amount of target sample for binding to it. The following are the instructions to set up a sandwich ELISA.

• Coat the well of the microtiter plate with the capture antibody. Allow it for overnight binding on to the well plate at 4°C. To increase the sensitivity, the unbound antibody could be washed away with PBS.
• Block the protein binding-sites with 5% non-fat dry milk in PBS for 1-2 h at 37°C. Wash the wells with PBS.
• Add the diluted target samples to the coated wells. Always run the experiment in triplicates, along with positive control and blank. Allow at least 90 min for binding antigen with the capture antibody at 37°C. Wash the wells with PBS.
• Add detection antibody and incubate for at least 90 min at room temperature (R.T). Wash the plate with PBS.
• Add enzyme-bound secondary antibody and incubate it for 60 min at R.T. Wash the plate with PBS.
• Depending upon the enzyme used, add chromogen for detection. For example, P-Nitrophenyl-phosphate (pNPP) substrate for alkaline phosphatase enzyme. Incubate the pNPP for 15-30 min and read at 405nm after stopping the reaction using 2N NaOH.

The Sandwich ELISA quantifies target antigen concentration between capture antibody (coated on to the well plate) and detection antibody. The target antigen needs to have at least two non-overlapping antigenic sites with the ability of binding to antibodies. The capture and detection antibodies can be monoclonal or polyclonal. Generally, the detection antibody is monoclonal as it imparts accuracy, whereas polyclonal antibodies are used as capture antibody since they could pull down the maximum amount of target sample for binding to it. The following are the instructions to set up a sandwich ELISA.

• Coat the well of the microtiter plate with the capture antibody. Allow it for overnight binding on to the well plate at 4°C. To increase the sensitivity, the unbound antibody could be washed away with PBS.
• Block the protein binding-sites with 5% non-fat dry milk in PBS for 1-2 h at 37°C. Wash the wells with PBS.
• Add the diluted target samples to the coated wells. Always run the experiment in triplicates, along with positive control and blank. Allow at least 90 min for binding antigen with the capture antibody at 37°C. Wash the wells with PBS.
• Add detection antibody and incubate for at least 90 min at room temperature (R.T). Wash the plate with PBS.
• Add enzyme-bound secondary antibody and incubate it for 60 min at R.T. Wash the plate with PBS.
• Depending upon the enzyme used, add chromogen for detection. For example, P-Nitrophenyl-phosphate (pNPP) substrate for alkaline phosphatase enzyme. Incubate the pNPP for 15-30 min and read at 405nm after stopping the reaction using 2N NaOH.