ELISA failure can be attributed to many factors. Many errors can be avoided if the protocol is read and fully understood before starting the assay. On identifying assay failure, check the expiration dates of the individual reagents and ensure that all the reagents have been stored as indicated on the product label. Once this has been established, check for signs of instability or deterioration in reagent solutions, (e.g., precipitation or discoloration). All substrate solutions should be colorless. Whenever possible, use clean plastic disposable pipettes, tips, and containers for reagent preparation and storage. Avoid cross-contamination of kit reagents by changing pipette tips between addition of each standard, sample and reagent. Finally, ensure that specified incubation times and temperatures have been adhered to and that no substitution of kit reagents has occurred. To improve accuracy, it is recommended that samples and standards be run in duplicate.

Problem: Poor Precision

Possible Source	Test or Action
Incomplete washing of wells	Ensure that wash apparatus is working correctly
Inadequate aspiration of wells	Wells should appear dry after aspiration
Unequal mixing of reagents	Ensure adequate mixing
Pipetting error	Repeat assay

Problem: Poor Standard Curve

Possible Source	Test or Action
Improper dilution of highest standard, standard curve, or blank values*	Ensure use of appropriate diluent as blank value. Ensure accurate completion of 2-fold dilution series* **
Incomplete washing of wells	Ensure that wash apparatus is working correctly
Inadequate aspiration of wells	Wells should appear dry after aspiration
Unequal volumes added to wells	Check pipette function; recalibrate if necessary
Color Reagents A and B mixed too early*	Substrate Solution must be used within 15 minutes of mixing*
Pipetting error	Repeat assay

Problem: Inadequate Color Development

Possible Source	Test or Action
Inadequate volume of substrate added to wells	Check pipette function
Incomplete mixing of Color Reagents A and B*	Ensure that equal volumes of Color Reagents A and B are properly mixed*
Incorrect incubation times or temperature	Adhere to recommended incubation times and temperatures
Conjugate or color reagent failure	Check by mixing equal volumes of Color Reagents A and B and conjugate. Color should develop immediately*

Problem: Edge Effects

Possible Source	Test or Action
Uneven temperatures around work surface	Adhere to recommended incubation periods and temperatures
Inadequate fixing of plate cover, leading to evaporation	Ensure correct use of plate cover

Problem: Drift

Possible Source	Test or Action
Interrupted assay set-up or reagents not at room temperature	Assay set-up should be continuous. Have all standards/samples prepared appropriately before commencement of the assay

Problem: Unexpected Result

Possible Source	Test or Action
We as manufacturers take great care to ensure that our products are suitable for use with all valid samples, as designated in the product inserts. However, it is possible that in some cases high levels of interfering factors may cause unusual results.