Positive Results in Blank Well

• Contamination of reagents. Use fresh reagents and pipette carefully.
• Splashing between wells. Use a plate sealer during incubations.
• Insufficient washing of plate. Check that plate washer is functioning correctly and that the correct numbers of washes are being done.

High Background Across Entire Plate

• Conjugate left on too long. Follow instructions for conjugate incubation.
• If using lyophilized conjugate, it was reconstituted incorrectly. Check reconstitution instructions.
• Substrate too old.
• Substrate reaction allowed to go to long before adding stop reagent. Reaction should be stopped as soon as a medium blue color has developed.
• Substrate incubation not done in the dark.
• Incubation temperature too high.
• Stop solution is contaminated. It should be a clear, colorless liquid.
• Plate not read soon after stop reagent is added. (Color may continue to develop at a slow rate even if stop reagent has been added.)
• Re-using plate sealers, reservoirs, or pipette tips causing contamination.
• Insufficient washing of plate.  Check that plate washer is functioning correctly and that the correct numbers of washes are being done.

Low Absorbance Values or No Absorbance Values

• Target expression in samples is low or not present. You may need to change to a more sensitive assay. Consult with technical service.
• Incubation times not long enough. Check that you are using the recommended length of time.
• Incubation temperature too low. Make sure to bring all reagents to room temperature prior to use.
• Stop solution not added.
• Plate washed after substrate incubation.
• Incorrect wavelength set on plate reader.
• Concentration of samples is too high and out of range for sensitivity of assay. Re-assess the assay you are using (consult with technical service) or dilute your samples. Remember to take into account the dilution factor when calculating results.

Inconsistent Absorbance Values for Replicates

• Pipetting inconsistent. Ensure pipettes are working correctly and are calibrated. Check that tips are the correct ones and are on far enough to create a good seal. Ensure you are not using a volume outside the range of the pipette. Watch to make sure tips are aspirating and delivering the correct amount of fluid.
• Standards and Reagents are not well mixed prior to adding to plate. Mixing ensures a consistent concentration across the plate.
• Well are allowed to dry out. Ensure that new plate sealers are used during all incubations and that they are adhering to all sides of the plate.
• Inadequate washing. Ensure correct amount of wash is being dispensed into all wells.
• Bottom of the plate is dirty, affecting the absorbance readings. Clean the bottom of the plate carefully before reading plate.
• Check reagents for any deterioration. Substrate should be clear and colorless.
• Check that storage conditions for all reagents are correct.
• Ensure that wash solution is made with deionized water.

Standard Curve was Flat

• Check that correct wavelength was used to read plate.
• Inadequate washing.
• Conjugate or substrate may be bad.
• Ensure that standards were made correctly.

Edge Effects

• Possible causes are evaporation in wells or inconsistent incubation temperature across the plate. Use a plate sealer that is adhered to all sides of the plate. Avoid incubating plate in an area with temperature fluctuations.

Poor Assay to Assay Results

• Insufficient washing. Check that the appropriate numbers of washes are being done. Ensure that the correct amount of wash solution is being dispensed into all wells. None of the dispense outlets are plugged.
• Variation in assay protocol. Ensure the same protocol is being followed each time.
• Standard degradation. Check standards and make fresh ones if necessary.
• Plate sealers reused. Use a fresh plate sealer for each step.
• Improper calculation of standard curve. Check math and recalculate curve. Refer to kit technical data sheet for help with curve equation.