Product Name or Catalog Number :
 
 
Troubleshooting Guide: ELISA
 
 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