Sandwich ELISA is widely used in immunology and biochemistry to quantify the concentration of specific antigens in a sample because of its high specificity and sensitivity.
Here, a capture antibody is immobilized on a microplate to bind the target antigen from the sample. A detection antibody, which binds to a different epitope on the antigen, is then added. This detection antibody is usually linked directly or indirectly to an enzyme (HRP). Upon addition of a substrate (TMB), the enzyme catalyses a reaction that produces a measurable colour change. The intensity of the signal is proportional to the amount of antigen present in the sample and is quantified using a plate reader.
This protocol represents an example of a standard capture or sandwich ELISA using a biotinylated detection antibody and streptavidin-HRP indirect detection system with commonly used reagents and TMB (tetramethyl benzene) substrate.
Equipment Required
- Clear 96 well plate
- Multi-channel precision pipettor with disposable plastic tips
- Microplate reader
Reagents Required
- Coating buffer: 0.2 M sodium carbonate/bicarbonate, pH 9.4
- Capture antibody: Diluted in Coating Buffer
- Wash buffer: 0.1 M phosphate, 0.15 M sodium chloride, pH 7.2 containing 0.05% Tween 20
- Blocking buffer: 2% (w/v) Bovine Serum Albumin (BSA) in Wash Buffer
- Standard diluent: 2% (w/v) BSA in Wash Buffer.
- Detection antibody (biotinylated): Diluted in 1/5 strength standard diluent
- Enzyme conjugate: Streptavidin-HRP diluted in 1/5 strength standard diluent
- Substrate/ Stop solution: TMB substrate / 2M sulfuric acid
Preparation of Standards
A standard curve is typically prepared in the range of 0 to 1000 pg/mL, although it may extend up to 3000 pg/mL depending on the expected antigen concentration in the sample and the availability of standard protein. Do not pipette up and down when first adding the buffer; simply swirl and invert 5 times and let the standard sit for at least 10 mins to ensure complete constitution. Serial dilutions of the standard protein are usually made from the stock solution using the standard diluent. Fresh pipette tips should be used at each dilution step to avoid cross-contamination.
Preparation of wash Buffer
Let the wash buffer (from kit) reach room temperature and mix to dissolve any precipitated salts, dilute the concentrate in deionized water accordingly.
Preparation of Samples
If the antigen concentration in the sample is expected to exceed the highest standard concentration (>1000 pg/mL), the sample should be diluted appropriately using the standard diluent.
Procedure
Important: Do not allow the plate to dry at any point.
Note: If the plate is precoated with a capture antibody, start from step 4. Remove the 8-well stripes that will not be needed for the current run. Unused strips can be rebadged and stored at 2-8°C, for future use.
- Dilute the capture antibody to the appropriate concentration with a coating buffer allowing sufficient volume for 50-100 μl per well.
- Add the diluted capture antibody to the plate, cover and incubate for 2 hours with gentle agitation at RT or overnight at 4°C.
- Decant the solution and wash the plate with 200 μl per well wash buffer for 3 x 5 minutes each on a shaking platform and tap the plate on absorbent paper to remove excess liquids.
- Add 300 μl blocking buffer per well, cover the plate and incubate for 1 hour at room temperature. Alternatively block overnight at 4°C.
- Prepare the samples and standards. The volume per well should be the same as the capture antibody used in step 1.
- Discard the blocking buffer and add the samples and standards. Cover the plate and incubate for 1 hour at RT.
- Discard the solution and wash the plate with 200 μl per well wash buffer for 3 x 5 minutes each on a shaking platform and tap the plate on absorbent paper to remove excess liquids.
- Dilute the biotinylated detection antibody to the appropriate concentration. The volume per well should be the same as the capture antibody used in step 1.
- Add the diluted detection antibody to the plate, cover and incubate for 1 hour at RT.
- Decant the solution and wash the plate with 200 μl per well wash buffer for 3 x 5 minutes each on a shaking platform and tap the plate on absorbent paper to remove excess liquids.
- Dilute the enzyme conjugate to the appropriate concentration. The volume per well should be the same as the capture antibody used in step 1.
- Add the diluted enzyme conjugate to the plate, cover and incubate for 1 hour at RT.
- Discard the solution and wash the plate with 200 μl per well wash buffer for 6 x 5 minutes each on a shaking platform and tap the plate on absorbent paper to remove excess liquids.
- Add substrate solution to the plate. The volume per well should be the same as the capture antibody used in step 1.
- Incubate the plate at RT until the desired colour intensity is reached. Ideally a clear gradient will result for the standards.
- Add the equal amount of stop solution to terminate the reaction, the solution will turn yellow.
- If using TMB, measure the absorbance at 450 nm.
- Note: For other substrates use the appropriate detection technique.
Troubleshoot
| Problem | Problem Cause | Solution |
|---|---|---|
| Weak or no Signal | Low antigen concentration Inefficient capture or detection antibody |
Increase sample concentration or adjust dilution. Verify antibody activity, optimise concentrations |
| High background | Non-specific binding Cross-reactivity of detection antibody |
Improve blocking buffer, extend washing, reduce antibody concentration. Use more specific antibodies or validate with negative controls. |
| Irregular standard curve | Inconsistent pipetting | Use calibrated pipette, multi-channel pipette, mix reagents gently |
| Saturated Signal | Antigen too concentrated | Dilute samples to fall within standard curve range |
| Edge effects | Uneven plate coating or evaporation | Use plate sealers, incubate in humidity controlled chamber. |
Note: Ensuring consistent sample preparation, using validated reagents, and maintaining clean equipment are critical for producing reliable and reproducible results.
Limitations
Despite its widespread use, sandwich ELISA has several limitations.
- The assay is highly dependent on antibody quality, requiring two specific antibodies that recognize different epitopes on the target antigen; poor-quality or cross-reactive antibodies can lead to inaccurate results.
- The procedure is relatively time-consuming due to multiple incubation and washing steps, making it longer than some rapid detection methods.
- Assay performance can be affected by matrix effects, where components in complex samples such as serum or plasma interfere with antigen–antibody binding or enzymatic activity.
- The dynamic range is limited, as high antigen concentrations may lead to signal saturation, while low-abundance targets may require signal amplification for detection.
- The technique does not provide spatial or localization information, as it measures overall antigen concentration in solution rather than within tissues or cells.
Closing Statement:
Successful sandwich ELISA performance depends on maintaining consistent assay conditions throughout the procedure, including accurate standard preparation, careful pipetting, proper blocking, and thorough washing to minimize background noise and improve signal specificity. The use of matched capture and detection antibodies, together with optimized incubation conditions and appropriate sample dilution, is critical for achieving reliable antigen quantification within the assay’s dynamic range. Although factors such as matrix interference, signal saturation, and antibody cross-reactivity can affect assay accuracy, careful optimization and the inclusion of suitable controls ensure sensitive, reproducible, and interpretable results for immunological and biochemical analysis.