Unexpected or Inconsistent Assay Results (ELISA (Sigma Guide))

Question

Accepted Answer

Sample quantification values are outside expected biological range, controls fail to meet acceptance criteria, or results are not reproducible between runs despite similar experimental conditions. Common causes: 1) Omission of critical reagents or incorrect preparation of working solutions 2) Dilution errors in sample or standard preparation 3) Technique problems with reagent mixing or washing procedures 4) Inappropriate ELISA plate used (e.g. non-fluorescence compatible plates for fluorescence detection) 5) Blocking protein inadvertently included in coating solution, preventing proper antigen/antibody binding Solutions: 1) Verify reagents prepared correctly following manufacturer instructions; use preparation checklist; confirm reagents added in correct order 2) Double-check pipetting technique and all dilution calculations; use calibrated pipettes 3) Ensure proper mixing of all reagents before use; standardize wash step protocols with consistent timing and volumes 4) Use appropriate plate type for detection method: fluorescence-compatible black or white plates for fluorescence; clear plates for colorimetric; verify plate specifications 5) Omit blocking proteins (BSA, casein, milk) from coating solution; add blocking step only after coating and washing