Chemiluminescence immunossay troubleshooting

Chemiluminescence Immunoassay Troubleshooting

ProblemCauseSolution
1. Weak or no signala. 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 backgrounda. 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 samplesa. 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 rangea. 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 platea. 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.