After adding the TMB SUBS why are the standards not darkening, and/or why are the O.D. readings low?

• Less enzyme-conjugate added to the diluent. When adding the enzyme be sure to rinse pipette tip in the diluent to remove all of the enzyme coated inside pipette tip. • Contents of the wells are not incubated for the time directed in the insert for the appropriate VitaKitTM. • Always use distilled water for washing the wells. • Ensure your lab follows proper storage instructions ex: (store at 4C in a fridge). • Kit may have expired, check expiry date. • There may have been contamination. Always use aseptic techniques when opening and removing reagents from containers. Keep the plate covered except when adding reagents, washing. Always use different pipette tips for each reagent, calibrator, control and sample. When pipetting do not allow the pipette tip to touch any of the reagents already in the well.