| Problem | Cause | Solution |
| 1. Weak or no signal | a. HRP enzyme conjugate was not added. | a. Add HRP enzyme conjugate in the correct volume. |
| b. Improper dilution of reagents. | b. Check pipettes and confirm correct preparation. |
| c. Incubation temperature is too low. | c. Control the room temperature to the recommended range, and bring substrate to room temperature. |
| d. Wells left to dry out. | d. Ensure wells are not left without reagent or wash solution for extended periods. |
| e. Delay in reading plate. | e. Make sure to read plates between 5 and 20 minutes after dispensing the substrate. |
| 2. High background | a. Concentration of the detector antibody is too high. | a. Use recommended dilution for the conjugate. |
| b. Contaminated wells. | b. Seal the plates when incubation with the plate sealer provided. |
| c. Incubation times were too long. | c. Strictly follow the incubation time in the procedure. |
| d. The substrates are contaminated. | d. Check the colour (should be colourless). |
| 3. High variation in standards and samples | a. Error in pipetting. | a. Ensure pipettes are calibrated and ensure there isn’t any bubbles. |
| b. Insufficient plate washing. | b. Ensure plates are washing uniformly, and possibly use an automated plate washer. |
| c. Cross plate contamination. | c. Ensure pipette tips do not touch plates and use new pipette tips when adding new reagents. |
| d. Samples are non-homogeneous. | d. Make sure samples are thoroughly mixed. |
| 2. Sample readings are not within range | a. Sample contains an analyte concentration that is too low to be detected. | a. A higher volume of sample may be needed, or analyte may not be present in sample. |
| b. The samples contain a high concentration of analyte. | b. Dilute the sample, and check for interfering proteins. |
| 5. High background across plate | a. Incubation temperature is too high. | a. Incubate at temperature specified. |
| b. Incubation times too long. | b. Decrease incubation period to time specified. |
| c. Wrong dilution of conjugate is used. | c. Adjust dilution to the recommended. |
