Direct ELISA Protocol Using Immobilized Recombinant Proteins

Background

Enzyme-Linked Immunosorbent Assay (ELISA) is a popular analytical biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample. The direct ELISA, one of the simplest forms of ELISA, involves immobilizing the antigen directly onto the plate and then detecting it with an enzyme-conjugated antibody specific to the antigen. This method is particularly useful when dealing with recombinant proteins, as it allows for a straightforward assessment of the protein's presence and concentration. Variations of this protocol also enable screening of antibody libraries against a particular immobilized protein antigen target.

Materials Needed

• 96-well microtiter plate
• Recombinant protein antigen
• Enzyme-conjugated primary antibody specific to the recombinant protein
• Coating buffer (e.g., carbonate-bicarbonate buffer, pH 9.6)
• Blocking buffer (e.g., 5% non-fat dry milk in PBS or 1-5% BSA in PBS)
• Wash buffer (PBS with 0.05% Tween 20)
• Substrate solution for the enzyme
• Stop solution (e.g., 1M sulfuric acid, or 2N H₂SO₄)
• Plate reader capable of reading the substrate’s absorbance

Protocol

1. Coating the Plate:
   • Dilute the recombinant protein antigen in a coating buffer.
   • Add 100 μL of the antigen solution to each well of the 96-well plate.
   • Cover the plate and incubate overnight at 4°C or for 2 hours at 37°C.
2. Blocking:
   • Remove the coating solution and wash the wells three times with wash buffer.
   • Add 200 μL of blocking buffer to each well to prevent non-specific binding.
   • Incubate for 1 hour at room temperature.
3. Antibody Incubation:
   • Dilute the enzyme-conjugated primary antibody in blocking buffer according to the manufacturer's instructions.
   • Remove the blocking buffer and add 100 μL of the antibody solution to each well.
   • Incubate for 1 hour at room temperature.
4. Washing:
   • Remove the antibody solution.
   • Wash the wells four to five times with wash buffer to remove any unbound antibody.
5. Substrate Reaction:
   • Add 100 μL of substrate solution to each well.
   • Incubate at room temperature until a sufficient color develops (typically 10-30 minutes).
   • Add 50 μL of stop solution to each well to halt the reaction.
6. Reading the Plate:
   • Measure the absorbance of each well using a plate reader set to the appropriate wavelength for the enzyme's substrate.

Tips and Tricks

• Optimize Antigen Concentration: Start with a range of antigen concentrations to find the optimal amount that provides the best signal without background issues.
• Required Controls: Use appropriate negative controls in order to accurately assess binding. This typically includes a set of wells coated with only a coating buffer (no antigen) and wells that skip the primary antibody incubation step. These are essential for accurately assessing background signals and non-specific binding.
• Temperature Control: Perform all incubations at a consistent temperature to avoid variations in enzyme activity and binding efficiencies.
• Plate Coating: Ensure the plate is coated evenly. Inconsistent coating can lead to variable assay results.
• Optimize Antibody Incubation: If signals are weak, try a longer antibody incubation (e.g., 2 hours at RT or overnight at 4°C) to increase signal sensitivity.
• Washing Steps: Be thorough but gentle during the washing steps to avoid washing away the bound antigen or antibody.
• Substrate Choice: Choose a substrate that provides a strong signal and has a good dynamic range for your specific enzyme.

Troubleshooting

• High background: Increase washing steps or blocking time, or choose a different blocking buffer.
• Low signal: Check antibody dilutions, increase incubation time.
• Edge effects: Equilibrate plates to room temperature before use, minimize air movement around the plate, or avoid using outside wells.