A Guide to Building a Direct Sandwich ELISA
Written by Dr. Reshma Patil
What is an ELISA?
The enzyme-linked immunosorbent assay (ELISA) is an analytical biochemistry technique designed to detect and quantify soluble substances such as peptides, proteins, antibodies, and hormones. This guide provides a detailed overview of how to successfully build and troubleshoot an ELISA, covering essential materials, protocols, and troubleshooting tips.
ELISAs are quick and simple to carry out since they are designed to handle large sample volumes effectively. These highly specific and sensitive assays can detect concentrations as low as 0.01 nanograms of antigen or antibody per milliliter. During an ELISA, antigen-antibody complexes are immobilized on a solid surface. An enzyme is covalently attached to one of the molecules in the complex, and the addition of an enzyme-specific substrate results in a colored reaction product that can be quantified.
When selecting the right type of ELISA for your experiment, it is important to consider the options, such as direct, indirect, sandwich, and competitive ELISA (Figure 1). Among these, the sandwich ELISA method provides unparalleled sensitivity and specificity for detecting target analytes.
Figure 1: Illustration of five different ELISA methods
Direct Sandwich ELISA
A direct sandwich ELISA detects an antigen by capturing it between two specific antibodies: a capture antibody attached to the plate and a detection antibody conjugated to an enzyme.
Materials for Generating a Direct Sandwich ELISA
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Microplate: Typically, a 96-well plate, either high-binding or medium-binding, depending on the assay.
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Capture Antibody: A monoclonal, polyclonal, or recombinant antibody that binds to your specific protein of interest.
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Coating Buffer: Usually carbonate-bicarbonate buffer (pH 9.6) or PBS (pH 7.4) for protein adsorption.
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Sample and Standard Solutions: Antigens or other target proteins. Common types of samples for ELISA are serum, plasma, cell supernatants, urine, saliva, tissue homogenates, and cerebrospinal fluid.
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Blocking Buffer: Bovine serum albumin (BSA), non-fat dry milk, or commercial blocking solutions to prevent non-specific binding.
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Detection Antibody: A monoclonal, polyclonal, or recombinant antibody that binds to a different epitope on the target antigen than your capture antibody and is enzyme-conjugated (e.g., horseradish peroxidase, HRP).
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Substrate Solution: TMB (3,3',5,5'-Tetramethylbenzidine) for HRP or a suitable substrate for other enzymes.
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Stop Solution: Typically, 1N sulfuric acid (for TMB).
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Washing Buffer: PBS or Tris-buffered saline (TBS) with Tween-20 (0.05%).
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Pipettes and Tips: For accurate volume measurements.
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Plate Reader: For detecting absorbance or fluorescence.
Step-by-Step Protocol
Figure 2: Process of direct sandwich ELISA
Step 1: Coating the Plate
Begin by coating the wells of a microtiter plate with a capture antibody at a concentration of 1-10 µg/mL in a carbonate/bicarbonate buffer (pH 9.6). After applying the antibody, incubate the plate overnight at 4°C to allow the antibody to adsorb to the wells.
Step 2: Blocking
After removing the coating solution, block the remaining protein-binding sites by adding a blocking buffer (e.g., 1% BSA in PBS) to each well. Incubate the plate for 1-2 hours at room temperature or overnight at 4°C to prevent non-specific binding. Remove the blocking buffer and wash with PBS 3-5 times to help remove any unbound blocking agent. This is key to reducing the likelihood of non-specific binding during subsequent steps.
Why blocking?
Coating the ELISA plate is a passive binding process where biomolecules are immobilized onto the well surface. Without blocking, antigens or detection antibodies could bind non-specifically to the plate, leading to high background and low sensitivity. Blocking saturates free binding sites, preventing non-specific interactions. Using a higher blocking volume (200 µl) compared to the reaction volume (100 µl) ensures comprehensive coverage.
Step 3: Adding Samples and Standards
Add 100 µL of the diluted sample (e.g., plasma, serum, or cell lysates) to each well. Ensure the samples are appropriately diluted in a dilution or blocking buffer, with concentrations within the expected dynamic range of the assay (typically between 1-100 ng/mL). Incubate the plate for 90 minutes at 37°C or overnight at 4°C.
What is the importance of ELISA controls?
Including both positive and negative controls is essential to verify the accuracy of the assay. Positive controls confirm that the assay worked properly, while negative controls can alert you to false positives or non-specific binding.
Step 4: Washing
Between each step, particularly after incubation with the sample and antibodies, wash the plate several times with PBS to remove any unbound substances.
Step 5: Detection Antibody Addition
Add 100 µL (typically 0.1-1 µg/mL) of the diluted conjugated detection antibody to each well and incubate for 2 hours at room temperature. Be sure to wash the plate 3-5 times with PBS to remove unbound detection antibodies.
Step 6: Substrate Addition
After washing, add the enzyme substrate to each well. The substrate reacts with the enzyme conjugated to the detection antibody, resulting in a measurable color change. For example, TMB is commonly used with HRP, and the reaction is stopped by adding sulfuric acid after 15-30 minutes.
Step 7: Reading the Results
Use a microplate reader to measure the optical density of each well at the appropriate wavelength (e.g., 450 nm for TMB). The intensity of the color change is directly proportional to the concentration of the antigen in the sample, which can be quantified by comparing it to a standard curve using the known concentrations of antigen standards (Figure 3).
Figure 3. A representation of sandwich ELISA format and calibration curve
Tips to remember:
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Handle samples with care: Avoid repeated freeze-thaw cycles, as these can degrade sensitive proteins and affect assay outcomes. Be consistent with the methods used for sample collection and processing.
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Bring all reagents to room temperature before use: When running an ELISA, it is important to bring all of the reagents to room temperature unless the protocol specifically states not to. This will help to ensure that the binding kinetics are consistent between assays, as well as keep temperature-sensitive components in solution.
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Practice proper pipetting technique: Best practice recommendations include selecting an appropriate pipet for the volume to be added, dispensing liquids with the pipet tips held at an angle and not touching the bottom of the wells, and always changing the pipet tips between different samples or standards to avoid cross-contamination.
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Stick to the protocol: For reproducible results, it is vital to stick to the protocol, whether provided by the kit or your in-house method. This includes being consistent with how samples are prepared, always using the optimized assay conditions, and adhering to incubation times and temperatures. As a general rule, it is suggested that timings do not vary by more than +/- 5 minutes per hour of incubation.
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Incorporate Controls and Reference Standards: Controls are used for data analysis and verifying assay performance. It is recommended that controls be positioned in such a way as to highlight any plate drift (e.g., columns 1 and 12 of a 96-well plate are often used as control wells, with the positive control being in wells A1-D1 and E12-H12 and the negative control in wells E1-H1 and A12-D12).
Troubleshooting Common Issues
1: High background signal
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Possible Cause: Non-specific binding due to insufficient blocking.
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Solution: Increase the concentration of the blocking agent or extend the blocking time.
2: Low signal or no signal
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Possible Cause: Insufficient coating of the target protein or antibody.
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Solution: Ensure proper coating concentrations and allow adequate incubation time.
3: Inconsistent results
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Possible Cause: Variability in sample handling or incubation times.
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Solution: Standardize sample handling procedures and adhere strictly to timing for all steps.
4: High variability between wells
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Possible Cause: Uneven pipetting or inconsistent well filling.
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Solution: Use calibrated pipettes and ensure thorough mixing of solutions before dispensing.
Conclusion
Building an ELISA can be straightforward with the right materials and attention to detail. This versatile assay continues to be an invaluable tool in research and clinical laboratories. By following the step-by-step protocol outlined in this guide and implementing the tips and troubleshooting strategies, you can achieve reliable, reproducible results with your sandwich ELISA.
Proteintech offers a wide range of products for ELISA, including:
- ELISA kits for the detection of protein cytokines, growth factors, and chemokines
- Over 19,000 primary antibodies validated for ELISA
- Expert-validated matched antibody pairs
References
- He, X., & Wu, G. (2021). Development of multiplex ELISA for simultaneous detection of multiple biomarkers. Analytical Chemistry, 93(18), 6775-6782.
- Reitz, R. (2020). Advances in ELISA methodologies for quantitative protein analysis. Journal of Immunological Methods, 482, 112-128.
- Cui, Y et al. (2015). Heavy chain single domain antibodies to detect native human insoluble epoxide hydrolase. Analytical and Bioanalytical Chemistry, 407.
- Crowther, JR., (2009). Methods in Molecular Biology, the ELISA Guidebook. Second Edition. Humana Press, a part of Springer Science + Business Media, LLC
- Van Weemen, BK., & Schuurs, AH. (1971). Immunoassay using antigen-enzyme conjugates. FEBS Letters, 15(3), 232-236.
