ELISA - Enzyme-linked Immunosorbent Assay

An Enzyme-Linked Immunosorbent Assay (ELISA) is used to quickly and accurately measure unknown concentrations of antibodies and antigens. Commonly used in diagnostics and research purposes.

There are four major types of ELISAs are:

1. Direct ELISA

2. Indirect ELISA

3. Sandwich ELISA

4. Competitive ELISA

Required Material:

• PBS

• Bicarbonate/ Carbonate coating buffer

• Blocking solution

• Wash solution

• Diluent buffer

• Antibodies (Capture antibody and detection antibody conjugated to Biotin)

• Samples, standards, and controls

• Streptavidin-HRP

• Stop solution

• ELISA plate reader (650 nm and 450 nm)

• TMB solution

Protocol:

Direct ELISA:

1. Dilute the antigen to a final concentration of 10µg/ml in PBS or another carbonate buffer. Coat the wells of a PVC microtiter plate with the antigen by pipetting 100µl of the antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4℃ or 2 hours at room temperature.

2. Wash the plate three times with wash solution.

3. Block the plate by adding 200µl blocking buffer.

4. Cover the plate and incubate for at least 2 hours at room temperature, or overnight at 4℃.

5. Wash the plate twice with wash solution.

6. Add 100µl of the antibody, diluted at the optimal concentration.

7. Cover the plate with adhesive plastic and incubate for 2h at room temperature.

8. Wash the plate five times with wash solution.

9. Add 100µl of the substrate solution per well for 30 minutes in the dark.

10. After sufficient colour development add 50µl of stop solution.

11. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

Indirect ELISA:

1. Dilute antigen to a final concentration of 1-20μg/ml using PBS or Bicarbonate/carbonate coating buffer. Coat the wells of a PVC microtiter plate with the antigen by pipetting 50µl of the antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4℃ or 2 hours at room temperature.

2. Wash the plate three times with wash solution.

3. Block the plate by adding 200µl blocking buffer.

4. Cover the plate and incubate for at least 2 hours at room temperature, or overnight at 4℃.

5. Wash the plate three times with wash solution.

6. Add 100µl of diluted primary antibody to each well and cover the plate for 2 hours at room temperature.

7. Wash the plate four times with wash solution.

8. Add 100µl of conjugated secondary antibody, diluted to the optimal concentration.

9. Cover the plate and incubate for 2 hours at room temperature.

10. Wash the plate five times with wash solution.

11. Add 100µl of the substrate solution per well for 30 minutes in the dark.

12. After sufficient colour development add 50µl of stop solution.

13. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

Sandwich ELISA:

1. Coat the wells of a PVC microtiter plate with the capture antibody at a concentration of 1-10µg/ml in carbonate/bicarbonate buffer (pH 7.4). Seal the plate and incubate overnight at 4℃ or 2 hours at room temperature.

2. Wash the plate three times with wash solution.

3. Block the plate by adding 200µl blocking buffer.

4. Cover the plate and incubate for at least 2 hours at room temperature, or overnight at 4℃.

5. Add 100µl of appropriately diluted samples to each well. For accurate quantitative results, always compare signals of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy. Incubate for 90 minutes at 37℃.

6. Wash the plate twice with wash solution.

7. Add 100µl of diluted detection antibody to each well and cover the plate for 2 hours at room temperature.

8. Wash the plate four times with wash solution.

9. Add 100µl of secondary antibody conjugated, diluted at the optimal concentration.

10. Cover the plate with adhesive plastic and incubate for 1 - 2 hours at room temperature.

11. Wash the plate five times with wash solution.

12. Add 100µl of the substrate solution per well for 30 minutes in the dark.

13. After sufficient colour development add 50µl of stop solution.

14. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

Competitive ELISA:

1. Coat microtiter plate wells with 100µl of the antigen solution, at a concentration of between 1-10µg/ml in coating buffer.

2. Cover the plate and incubate overnight at 4°C.

3. Wash the plate three times with wash solution.

4. Block the plate by adding 200µl blocking buffer.

5. Cover the plate and incubate for at least 2 hours at room temperature, or overnight at 4℃.

6. Prepare the antigen-antibody mixture by adding 50μl of antigen to 50 μl of antibody for each well in the assay (use a range of antigen concentrations appropriately diluted in wash buffer).

7. Add 100µl of the mixture to each well. Incubate for 1 hour at room temperature.

8. Wash three times with wash solution.

9. Add 100µl enzyme-conjugated secondary antibody to each well and incubate for 1 hour at room temperature.

10. Wash three times with wash solution.

11. Add 100µl of the substrate solution per well for 30 minutes in the dark.

12. After sufficient colour development add 50µl of stop solution.

13. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

Results:

From an ELISA test there are three different types of data generated:

• Quantitative: With quantitative data, the results are interpreted by comparing them to a standard curve, which allows the concentrations of antigens in different samples to be precisely determined.

• Qualitative: Will either confirm or deny whether there is a presence of a particular antigen in a sample.

• Semi-quantitative: The intensity of a signal can differ directly based on antigen concentration. ELISA data can be used to compare the relative levels of antigens within an assay sample.