FAQs and Troubleshooting

Indeterminate results

Indeterminate results may be related to the immune status of the individual being tested, but may also be related to a number of technical factors:

  • Longer than 16 hours from blood draw to incubation at 37°C.
  • Storage of blood outside the recommended temperature range (22°C ± 5°C). 
  • Insufficient mixing of blood collection tubes.
  • Incomplete washing of the ELISA plate.

If technical issues are suspected with the collection or handling of blood samples, repeat the entire QF-CMV test with new blood specimens. Repeating the ELISA testing of stimulated plasmas can be performed if any procedural deviation with the ELISA test is suspected. Indeterminate results (from low Mitogen values) would not be expected to change on repeat unless there was an error with the ELISA testing.

Clotted plasma samples

Should fibrin clots occur with long-term storage of plasma samples, centrifuge samples to sediment clotted material and facilitate pipetting of plasma.

ELISA troubleshooting

Non specific Color Development
POSSIBLE CAUSE SOLUTION
Incomplete washing of the plate Wash the plate at least 6 times with 400 μl/well of wash buffer. More than 6 washing cycles may be required depending on the washer being used. A soak time of at least 5 seconds between cycles should be used.
Cross-contamination of ELISA wells Take care when pipetting and mixing sample to minimize risk.
Kit/components have expired Ensure that the kit is used before the expiry date. Ensure reconstituted Standard and Conjugate 100x Concentrate are used within three months of the reconstitution date.
Enzyme Substrate Solution is contaminated Discard substrate if blue coloration exists. Ensure clean reagent reservoirs are used.
Mixing of plasma in centrifuge Ensure that plasma samples are carefully harvested from above gel without pipetting up and down, taking care not to disturb material on the surface of the gel.
High Background
POSSIBLE CAUSE SOLUTION
Incomplete washing of the plate Wash the plate at least 6 times with 400 μl/well of wash buffer. More than 6 washing cycles may be required depending on the washer being used. A soak time of at least 5 seconds between cycles should be used.
Incubation temperature too high Incubation of the ELISA should be performed at room temperature (22°C ± 5°C).
Kit/components have expired Ensure that the kit is used before the expiry date. Ensure reconstituted standard and Conjugate 100x Concentrate are used within 3 months of the reconstitution date.
Enzyme Substrate Solution is contaminated Discard substrate if blue coloration exists. Ensure clean reagent reservoirs are used.
Low optical density readings for standards
POSSIBLE CAUSE SOLUTION
Standard dilution error Ensure dilutions of the Kit Standard are prepared correctly as per the QF-CMV ELISA Package Insert.
Pipetting error Ensure pipets are calibrated and used according to manufacturer’s instructions.
Incubation temperature too low Incubation of ELISA should be performed at room temperature (22°C ± 5°C).
Incorrect plate reader filter used Plate should be read at 450 nm with a reference filter between 620 and 650 nm.
Reagents are too cold All reagents, with the exception of the Conjugate 100x Concentrate, must be brought to room temperature prior to commencing the assay. This takes approximately 1 hour.
Kit/components have expired Ensure that the kit is used before the expiry date. Ensure reconstituted Standard and Conjugate 100x Concentrate are used within 3 months of the reconstitution date.
Nonlinear standard curve and duplicate variability
POSSIBLE CAUSE SOLUTION
Incomplete washing of the plate Wash the plate at least 6 times with 400 μl/well of wash buffer. More than 6 washing cycles may be required depending on the washer being used. A soak time of at least 5 seconds between cycles should be used.
Standard Dilution Error Ensure dilutions of the kit standard are prepared correctly as per the QF-CMV ELISA Package Insert.
Poor mixing Mix reagents thoroughly by inversion or gentle vortexing prior to their addition to the plate.
Inconsistent pipetting technique or interruption during assay setup Sample and standard addition should be performed in a continuous manner. All reagents should be prepared prior to commencing the assay.
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