High signal and false positives usually come from insufficient plate washing, not stopping the reaction, too much detection reagent, contaminated substrate or ineffective blocking.

Out-of-range values occur when the analyte is below or above the standard curve, or from insufficient washing and incorrect dilutions. Dilute or concentrate samples and re-check your dilution calculations.

High variation comes from pipetting errors, non-homogeneous samples, uneven washing or agitation, edge effects and dirty plates. Calibrate pipettes, mix samples, agitate evenly and equilibrate reagents to room temperature.

High background is caused by inadequate washing, cross-reactivity, non-specific binding, too much detection reagent or ineffective blocking. Increase washes, optimise blocking and titrate antibodies.

No signal can result from a missing detection antibody or avidin-HRP, azide in the wash buffer, target below the detection limit, short incubation, or reagents not at room temperature.

A poor standard curve results from a degraded or poorly reconstituted standard, pipetting errors, poor mixing, incomplete aspiration or an unsuitable curve fit. Remake the curve and check calculations.