Poor Standard Curve Discrimination (ELISA Development)

Question

Accepted Answer

Standard curve achieved but shows low or flat curve with poor discrimination between concentration points Common causes: 1) Insufficient streptavidin-HRP 2) Capture antibody did not bind well to plate 3) Insufficient detection antibody 4) Plate not developed long enough 5) Incorrect procedure followed 6) Improper calculation of standard curve dilutions Solutions: 1) Check streptavidin-HRP dilution and titrate if necessary 2) Use proper ELISA plates (not tissue culture plates) 3) Dilute capture antibody in PBS without additional protein 4) Check detection antibody dilution and titrate if necessary 5) Increase substrate solution incubation time 6) Use recommended brand of substrate solution 7) Return to general ELISA protocol and eliminate modifications 8) Check calculations and make new standard curve