Troubleshooting Tactics
ELISA Test Format
- Low ODs for the Calibrators
Possible cause Resolution Reagent temperature Use reagents at room temperature. Use of plate sealers Do not use during any of the incubations. Wells overdrying Reduce time between wash step and addition of next reagent or use a 96-well washer instead of a strip washer. Incorrect substrate volume Use correct volume of substrate. Plate reading Read plate immediately after adding stop solution.Confirm that the plate reader is set up to the correct wavelength. Incubation time Incubate for the recommended times. Reagent order Ensure that the reagents are added in the order indicated in the package insert. Calibrator volume Ensure that the correct volume of calibrators was used. - Controls Out of Range
Possible cause Resolution Control or calibrator storage Store controls or calibrators in ice during use. Incubation times or temperatures Follow protocol for precise incubation times. Incubation steps Do not skip the sample incubation step. Plate washing and drying Do not allow plate to overdry after wash step. Assay temperature Ensure that the assay is performed at the recommended temperatures. Curve fit Ensure that the plate reader is set to use a point-to-point calibration fit. - Results Drift Across the Plate
Possible cause Resolution Time elapsed while loading the plate Load samples within 20 minutes. Use a transfer plate to minimize delays. Have all reagents ready in advance to avoid interruptions. Do not skip the 10 minute sample incubation step. - Poor Precision of Replicates
Possible cause Resolution Plate washing step Ensure that plate is washed 4 times per step and that the plate washer is functioning properly. Do not use DI water or tap water to wash plates. Use the wash buffer included with the kit. Pipetting Verify that volumes and pipette calibrations are correct. Contamination Ensure that proper pipetting technique is used to avoid cross contaminating wells while pipetting. - General Precaution
Ensure that all automated ELISA instrumentation is adequately validated to run the PLAC assay prior to use.