No Signal for Standard Curve with Normal Zero Standard (ELISA (Competitive))

Question

Accepted Answer

All standard concentrations show no signal (high OD in competitive format similar to zero standard), while the zero standard (no competitor) shows normal maximum binding signal. Common causes: 1) Standard curve pipetting errors resulting in no analyte addition 2) Incorrect serial dilution procedure producing uniform zero concentration 3) Standard concentration far exceeds assay dynamic range causing complete inhibition at all points 4) Standard stock solution degraded or improperly reconstituted Solutions: 1) Verify the standard curve was pipetted and diluted properly with documented procedure 2) Check calculation of dilution factors and physically verify volumes 3) Decrease the starting standard concentration to fall within assay dynamic range 4) Prepare fresh standard from new stock and verify reconstitution protocol