No signal/absorbance in any well

Stop Solution

Substrate TMB was contaminated by stop solution prior to addition to wells.

Avoid contaminating Substrate TMB with Stop Solution by labelling any containers used.

The assay procedure has been done incorrectly.

Always make sure that the stop solution is added after the Substrate TMB. The Stop Solution causes the enzyme-substrate reaction to reach the colorimetric end-point.

Enzyme conjugate solution

No enzyme conjugate stock solution was added to the Enzyme Conjugate Buffer.

Dilute Enzyme conjugate according to the Directions for Use.

Wash Buffer

Wash buffer containing Sodium Azide has been used instead of the wash buffer provided in the kit.

We recommend the use of Wash Buffer included in the kit. The Wash Buffer in the kit is optimized for use with Mercodia ELISA kits. Washing solution from other companies may contain Sodium Azide, which can deactivate the enzyme.