Possible causes

Solutions

High Background

Insufficient washing

·         Wash 5x and increase soak time during washing steps

TMB Solution prepared to early

·         Prepare TMB solution immediately before use

Substrate incubation carried out in the light

·         Incubation steps should preferably be carried out in the dark

Plate measured too long after stop solution added

·         Measure plate within 10 minutes after stop solution was added

Low signal

Color reaction not long enough

·         Increase color reaction time

Biotinylated Tracer degraded

·         Only use freshly prepared tracer

Wash buffer contains azide

·         Avoid using sodium azide in your solutions

Insufficiently reconstituted ST, AB or BT

·         Be sure to completely dissolve lyophilized powder in buffer for all components

Edge effects

Temperature or humidity differences across plate

·         Equilibrate all components to room temperature before beginning

Plate sealer not applied correctly

·         Check sealer for wrinkles

Stacking multiple plates for incubation

·         Do not stack plates, incubate them side by side

Poor replica values

Bubble in wells

·         Eliminate bubbles before measuring or use reverse pipetting technique

Dirty well bottoms

·         Clean well bottoms with a soft tissue before measuring

Pipetting error

·         Calibrate pipettes on a regular basis. Ensure that pipette tips are attached airtight

Poor mixing

·         Mix plate thoroughly after pipetting steps using plate shaker or by swirling by hand for several seconds

High signal in all standards

Standard degraded

·         Only use freshly prepared standard

Samples too high or low compared to standards

Not in optimal dilution range

·         Dilute or concentrate samples to be in linear range of standard curve

Standard curve shifted

ST - AB - BT ratios not correct

·         Follow protocol precisely

High signal across plate

Plate measured too long after stop solution added

·         Measure plate within 10 minutes after stop solution was added