Complete Signal Absence (ELISA Development)

Question

Accepted Answer

No detectable signal across the entire plate when signal is expected Common causes: 1) Reagents added in incorrect order or improperly prepared 2) HRP contamination with azide 3) Insufficient antibody concentration used 4) Degraded or mishandled standard 5) Buffer containing FCS used to reconstitute antibodies 6) Capture antibody failed to bind to plate 7) Contaminated buffers Solutions: 1) Repeat assay and review protocol carefully 2) Check all calculations and prepare fresh buffers and standards 3) Use fresh reagents free from contamination 4) Increase antibody concentration 5) Use new vial of standard handled according to directions 6) Requalify reagents if FCS was used 7) Use proper ELISA plates (not tissue culture plates) 8) Dilute capture antibody in PBS without additional protein 9) Make fresh buffers