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Assay Optimization with Akrivis SmartPlates™

 

Using Z-TECT™ HS ELISA Secondary MonsterHRP™ Colorimetric Reagents only requires substituting the secondary reagent in an established ELISA assay.  However, due to the resulting large signal amplification, it is often necessary to calibrate and "tone down" the concentrations of capture and/or detection antibodies.  Although the various buffers provided in the Akrivis kits were formulated to ensure optimal performance of the Z-TECT™ HS ELISA Secondary MonsterHRP™ Colorimetric Reagents, overall assay performance will depend on the specific characteristics of each ELISA assay, and especially on any NSB-generating cross-reactivity between capture and detection antibodies.

Therefore Akrivis has developed SmartPlates™, which are pre-designed plate layouts for a [4x4x4] concentration array of the capture antibody (CAB, for sandwich assays), detection antibody (DAB) and Z-TECT™ HS ELISA Secondary MonsterHRP™ Colorimetric Reagent (SR), to expedite the process of minimizing NSB and maximizing SNR.

The concentrations of CAB, DAB and SR previously established by the user for the ELISA assay are called “Alpha Conditions”.  Then, in addition to these Alpha Conditions, the user chooses 3 additional sets of concentrations for CAB, DAB and Z-TECT™ SR (any concentrations can be chosen although lower concentrations are recommended) resulting in a [4x4x4] concentration array.

Only an example: any 3 concentrations of CAB/DAB/SR can be chosen, whether higher or lower than Alpha Conditions.

Only an example: any 3 concentrations of CAB/DAB/SR can be chosen, whether higher or lower than Alpha Conditions.

The user then runs the four SmartPlates™ described below: two SmartPlates™, #1 and #2, are run at a fixed antigen test concentration, [Ag], which is sufficient to generate a strong signal, and two SmartPlates™, #3 and #4, are run without any antigen to minimize NSB.  Assay optimization can be continued by successive SmartPlates™ iterations until the desired performance is achieved.

Larger descriptions and detailed instructions for running SmartPlates™ can be found in the brochure provided with each standalone reagent or kit.