It is essential for the assay that all of the HRP Conjugate Solution is removed before adding the TMB Substrate Solution. Make sure that the Wash Solution completely fills each well and is completely removed before the next filling.

Avoid contamination between wells.

Dispense the Stop Solution with the same speed as the TMB Substrate Solution to obtain the same incubation time in all of the wells.

Avoid non-homogenous samples. Ensure that all samples are sufficiently mixed and/or centrifuged before performing the assay. This is described in section 9: Collection and Preparation of Samples.

Note: In the procedure described in section 9:D,samples are not centrifuged. The protocol is intended for drug screening.