ELISA Problems Troubleshooting Guide and Tips

 ELISA Washing Problems

• Make sure wells are filled equally with washing buffer.
• Ensure residual conjugate is removed from ELISA well.
• Ensure that the plate is level and does not allow wells to overflow.
• Do not Wash with pressure and pour washing solution beside the wells.
• Ensure your ELISA Washing Buffer and containers are not contaminated.
• Dry ELISA plate at end of wash by vigorously tapping on proper liquid absorbent.
• Find out how you should prepare ready to use ELISA washing buffer.
• If you use automatic ELISA Plate washer, Check the washer alignment daily and perform daily maintenance.
• Wash ELISA plates 3-5X according to your kit instruction
• If you need, allow 10-20 second soaking time.
• Check complete aspiration of ELISA well contents.

ELISA Problems Troubleshooting Guide and Tips

Fast TMB Colour development problems

• Check residue of washing is completely removed and be for adding TMB-Substrate plates have been dried properly.
• Ensure samples, controls and standards diluted correctly.
• Ensure there is a free space between pipette tips and top of pipette.
• Check TMB-Solution expiry date and colour change due to contamination.
• Do not incubate TMB more than manufacturer recommended time.
• Check contamination of tips.
• Use a proper cover or dark place to incubate TMB-Solution.
• Check your ELISA plate washer and your washing method.
• Check your TMB- Solution incubation temperature.
• Check your sample diluent contamination.

Check incubation time and temperature of your Enzyme conjugate solution.

ELISA Problems Troubleshooting Guide and Tips

Slow colour development problems

• Check the temperature of Lab, it should be 22-28C. Check your incubator temperature.
• Check incubation timing.
• Bring samples to room temperature before running the test.
• Check dilutions and ensure you don’t have HRP inactivation chemicals inside your reaction buffers.
• Bring your TMB-Substrate to room temperature and ensure it still works.
• Ensure pipette tips are fitted correctly and sample volume is correct.
• Check the expiry date of your kit and its components.
• Check your washing system pressure and ensure that the washing cycles are 3-5X.

Troubleshooting of ELISA High Background and High Negative Control Value

• Do not allow wells to overflow while you do ELISA plate washing.
• Check residue of washing is completely removed and be for adding TMB-Substrate plates have been dried properly.
• Re-run with fresh reagents.
• Use Unsuitable blocking buffer, Conjugate stabilizer, and other ELISA reagents!
• Ensure your sample dilution is according to your ELISA kit protocol.
• Check TMB-Solution expiry date and colour change due to contamination.
• Check your ELISA plate washer and your washing method.
• Check your TMB- Solution incubation temperature.
• Check your sample diluent contamination.
• Check incubation time and temperature of your Enzyme conjugate solution

ELISA Problems Troubleshooting Guide and Tips

False Positive Results

• Check your Sample diluent is not expired or contaminated.
• Check if your samples need to be diluted in HAMA blocking sample diluent or not.
• Ensure that your sample is not Hemolyzed, Lipemic, have much bilirubin.
• Make sure wells are filled equally with washing buffer.
• Ensure residual conjugate is removed from ELISA well.
• Ensure that the plate is level and does not allow wells to overflow.
• Do not Wash with pressure and pour washing solution beside the wells.
• Ensure your ELISA Washing Buffer and containers are not contaminated.
• Use fresh samples and run the test again.

ELISA Problems Troubleshooting Guide and Tips

ELISA Problems Troubleshooting Guide and Tips

Poor Reprehensibility or Bad Duplication

• Ensure your samples are diluted properly.
• Remove bubbles from your wells by slowly shaking.
• Ensure your samples are dispensed correctly.
• Remove residues and dry wells correctly.
• Wash more and add some soaking time to your washing steps.
• Avoid splashing samples and reagents.
• Use a new tip for each sample/control/reagent addition.
• Check if your samples need to be diluted in HAMA blocking sample diluent or not.
• Ensure that your sample is not hemolyzed, lipemic, have much bilirubin.