The blank value is for control only (background signal) and must not be divided by the measured values. “Blanked” absorbance data for creating the calibration curve may cause error in the generated standard curve. Never subtract the BLANK E450 value from the other measurement values.
Pipetting errors during preparation of the SAN HQ standards (liquid transfer, mixing, residual liquid in the pipette tips)
Improper plate washing steps, including
residual liquid in the wells after washing
plate wells run dry after removal of liquid due to time delays during assay development
air or particles in the liquid-channels when using an automated plate washer
damage of the wells surface by pipette tips or needles from automated washers during washing
Well-to-well cross-contaminations (liquid drops on the plate cover foil)
8-well strips not properly fixed during plate washing and/or reading of the colored dye (might cause imprecise results due to insufficient washing and light scattering effects)
Preparation of dilutions (calibrators/ samples) within the antibody-precoated assay plate (might cause imprecise and inaccurate results)
Inaccurate and uneven well filling volumes during incubation steps (common source of errors when using multi-channel pipettes by inexperienced operators)
To isolate the cause, it is required to evaluate the measurement results from the controls. It could result from
Error within the initial dilution of SAN HQ standard and control.
Issues during setting up the assay: insufficient shaking speed, wrong dilution of detection antibody concentrate and/or enzyme conjugate, buffer residues within wells after washing (especially before adding the TMB substrate).