Elisa Troubleshooting

Common Problems

• Using mismatched tips and pipettes.
• Handling tips directly without gloves, which can introduce contaminants.
• Uneven attachment of tips in multichannel pipettes, leading to variations in pipetting volumes.

Solution

• Use tips recommended by the pipette manufacturer, especially for small volumes, to reduce the risk of pipetting error.
• Always wear plastic gloves when handling tips to prevent contamination.
• Ensure that the wash buffer and other reagents are stored in reagent reservoirs to avoid contamination.

Minimize Errors

• Ensure all tips are securely attached in multichannel pipettes to avoid uneven distribution of reagents across the microplate wells.
• Visually check volumes to minimize pipetting errors. Check pipetting technique regularly to ensure precision and accuracy.
• Only recycle tips after thorough washing, and promptly discard any damaged tips, especially those used with enzyme conjugates or detection reagent.
• Use a calibrated microplate reader to ensure accurate measurements across different wavelengths. Ensure protocols are followed precisely to avoid inconsistencies.

Training

• Proper training in pipetting techniques is essential for maintaining accuracy and consistency in ELISA assays, leading to reliable results. Training should include proper use of a plate washer to ensure efficient washing procedure steps without cross-contamination. 

Strong or prolonged conjugate reactions:

Ensure the conjugate is used at the recommended dilution and stop the reaction promptly with a stop buffer to prevent overdevelopment of the signal. This maintains specificity.

Stale substrate or stop solutions:

Use fresh substrate solution and stop solutions. The stop solution should be clear, not yellow, and should be added immediately after the substrate incubation.

Reaction not stopped:

Stop the reaction to prevent continuous color development, which can cause high background signals.

Delayed plate reading:

Read the plate immediately after adding the stop solution to avoid further color development. Using a plate reader promptly can help reduce errors due to signal variations and ensure optimal sensitivity.

Contaminated glassware or plastics:

Use clean, sterilized glassware and fresh plastics to prevent contamination that could lead to false signals.

Incorrect incubation temperatures:

Conduct incubations at the correct room temperature or specified incubation temperature and verify that incubators are properly set and functioning. Incorrect temperatures can lead to high background or weak signals. Proper use of the incubator is crucial to prevent edge effects and ensure uniform results.

Non-specific antibody binding:

Include a blocking step with a suitable blocking buffer, such as 5-10% serum from the same species as the secondary antibody or bovine serum. Ensure wells are pre-processed to prevent non-specific attachment of the detection antibody.

Insufficient washing or blocking:

Increase the number and duration of washing steps. Use protein blockers like BSA, casein, or gelatin to prevent non-specific binding. Add detergents like Tween-20 to wash buffers to ensure complete removal of unbound reagents.

High antibody concentrations:

Decrease the concentration of the primary or secondary antibody. Perform titrations if necessary to find the optimal concentration that minimizes background without sacrificing signal strength. Accurate antibody dilutions are crucial for achieving optimal results.

Early preparation of substrate solutions:

Mix substrate solutions immediately before adding them to the plate to prevent premature reactions and high background.

Contaminated reservoirs, plate sealers, pipette tips, or buffers:

Use fresh plastics and prepare fresh buffers for each step to avoid contamination with HRP, which could lead to background noise. Ensure plate sealers are used to prevent evaporation and cross-contamination between wells.

Reagent Handling:

• Add reagents in the correct order as per the protocol to ensure proper reaction sequencing.
• Prepare reagents accurately from the stock solution, and repeat the experiment as needed to confirm findings. Proper storage of reagents is essential to maintain their activity.

Antibody Concentration:

• If antibody concentration is too low, increase the primary or secondary antibody concentration. Proper preparation of the capture antibody and detection reagent is crucial for signal detection.
• Incubate overnight at 4°C for better results. Consider using antibody concentration kits to optimize conditions.

Antibody Compatibility:

• Ensure primary and secondary antibodies are compatible and match in species to prevent signal loss due to cross-reactivity. Selecting appropriate antibody pairs is essential for immunoassays.
• For poor adhesion, use ELISA-validated plates like tissue culture plates and extend coating duration to overnight at 4°C. Make sure the sample walls are well-coated for optimal binding.

Sandwich ELISAs:

• Confirm that capture antibody and detection antibodies target different epitopes to avoid competitive binding, which can reduce signal.
• Use a different antibody pair or consider an alternative assay type if needed to enhance signal.

Standards and Samples:

• Ensure the standard is prepared correctly. Use a new vial if the standard is expired or degraded, as degraded standards can cause weak signals.
• If the target protein is not in the detectable range, perform serial dilutions and spike the sample to check for interference.

Buffer Considerations:

• Ensure buffers are azide-free or wash sufficiently to avoid inhibiting HRP activity, which could result in weak or no signal.

Identifying the Issue:

• Samples may contain no or below detectable levels of the analyte, leading to weak signals.
• Analyte concentrations may be higher than the highest standard point, causing out-of-range readings on the microplate reader.

Solutions for Low Analyte Levels:

• Use a higher sample volume to improve detection by increasing the concentration of the analyte within the detectable range. This is particularly important when detecting small molecules or peptides.
• Seek advice for suitable protocol modifications, such as increasing incubation times or changing incubation temperatures.

Solutions for High Analyte Concentrations:

• Dilute the samples and reanalyze to bring them within the detectable range of the assay. Ensure proper calculations are made when diluting samples to maintain accuracy. Check calculations carefully during curve dilutions to ensure accurate quantification.
• Properly sealing the microtiter plate during incubation is essential to prevent evaporation and maintain sample integrity.