Enzyme-Linked Immunosorbent Assays (ELISA) are used to measure an unknown concentration of antigen or antibody. The test is quick and precise. In the sandwich ELISA test, a purified antigen is applied to the bottom of a well in a 96-well plate. The target antibody is then added and binds to the antigen, followed by a labeled secondary antibody which binds to the target. Unbound products are removed between each step by washing. Finally, a colorimetric substrate reacts with the label carried by the secondary antibody. To obtain quantitative results, a standardized and purified antibody can be used for comparison to determine the absolute amount of protein in the sample studied. Alternatively, if the concentration of an unknown antigen is expected, the sandwich can be reversed, with an antibody attached to the bottom of the wells, the added antigen binds to the antibody, and a labeled secondary antibody is used to detect the amount of antigen.
Material
- 96-well plate
- Target antibody sample
- Standard calibrated target antibody
- Purified antigen that targets the antibody that will bind to it
- Labeled secondary antibody that will bind to an antibody target that will bind to the target antibody
- Coating buffer (50 mM Sodium Carbonate, pH 9.5)
- Wash buffer (10mM Tris, 1M Sodium Chloride, pH 7.2, 0.05% (v / v) Kathon)
- Blocking buffer (10 mM Tris, pH 7.2, 10% (w / v) D-Gluconic Acid, 5% (w / v) BSA)
- Antibody diluent (PBS)
- Colorimetric substrate
- Stop solution
- ELISA plate reader
Antigen Coating
1. Prepare a 1 μg / ml dilution of the purified antigen, using the coating buffer as diluent.
2. Add 100 µl of the diluted antigen to as many wells as necessary on the plate.
3. If using a standard curve, 33 wells will be required for 11 dilutions tested in triplicate. Wells will also be required to test at least 2 dilutions of the sample.
4. Add 100 µl of coating buffer (without the antigen) to a set of wells for the standard curve and the sample as a negative control.
5. Incubate the plate 3 hours at room temperature or 4 ° C overnight.
6. Cover each well with washing buffer and gently remove the liquid by inverting the plate. Wash the plate 3 times and dry on absorbent paper.
Blocking Step
1. Add 375 µl of blocking buffer to each well.
2. Incubate 2 hours at room temperature, or 4 to 24 hours at 4 ° C.
3. Remove the blocking buffer and tap the plate. Blocked plates can be stored with a protective film at 4 ° C for up to two weeks.
Unknown antibody and standard curve
1. To prepare a standard curve, dilute the control antibody to 1 pg / ml in PBS. Prepare serial dilutions ranging from 1 pg / ml to 0.0039 µg / ml. Include one well with only PBS (blank) as a negative control. Add 100 µl of each dilution to the coated wells. Each dilution (including the blank) must be added in triplicate.
2. Dilute the antibody sample to appropriate dilutions in PBS. Add 100 µl of each dilution of the test sample to the coated wells. Include a blank as a negative control.
3. Incubate the plates one hour at room temperature.
4. Wash the plates 3 times with washing buffer and dry.
Labeled secondary antibody
1. Dilute the labeled secondary antibody to 0.5 pg / ml in PBS. Add 100 µl of the diluted antibody to each well.
2. Incubate the plate for 60 minutes at room temperature.
3. Wash the plate 5 times with the washing buffer and dry.
Antibody detection and plaque reading
Immediately before use, prepare the colorimetric reagent, add 100 µl to each well. Incubate until the color develops, then stop the reaction with the appropriate solution. For example, if an HRP conjugate is used as a labeled secondary antibody, prepare a 1: 1 mixture of TMB solution and peroxide solution. Add 100 µl of the mixture to each well and incubate for approximately 15 minutes until it turns blue. Stop the reaction adding 100 µl of 2N sulfuric acid to each well.
1. Read the plate at appropriate wavelength. For the HRP, read the plate at 450 nm.
2. Plot the absorbance of standard antibody dilutions in relation to their concentrations and draw a line of best fit.
3. Compare the absorbance of the test sample to the standard curve to determine the concentration. Multiply the values by appropriate dilution factor.
Troubleshooting:
1. Control gives a positive result - Check if there is contamination of the substrate solution, the secondary conjugated antibody, or the controls.
2. No staining of positive control or samples - Check expiration dates, concentrations, reagent storage conditions.
3. Very weak staining of positive controls or samples - Check the dilutions of conjugated secondary antibody and substrate concentration.
4. Test sample staining but no the positive controls - Check the expiration dates and storage conditions of positive controls.
5. The coloring is present but the absorbance is lower than expected - Check the wavelength setting.
References:
Using Antibodies: A Laboratory Manual. Harlow & Lane. Cold Spring Harbor Laboratory Press. 1999.
Introduction to Antibodies, 2nd Edition. Chemicon International.