Optimizing Sandwich Type Assays

INTRODUCTION - TRADITIONAL ASSAY

 

Zimmer and Peacock are focused on biosensor development and manufacturing, which we do for our customers, clients and collaborators.

 

One of the most common types of assays we are asked to development and manufacturing are immuno-assays. In the adjacent image we show a 'typical' immunoassay, where  a secondary antibody labelled with a reporter molecule/probe binds and gives a signal proportional to the analyte/antigen.

 

Zimmer and Peacock supports this type of assay and the utilisation in DNA, RNA, protein and whole cell, etc  detection.  

 

We are often asked to implement the assay in a lower cost disposable cartridge assay. This requirement means that when Zimmer and Peacock are brought onto a program we often simplify the assay so that the client can have  a lower cost of goods when going to market.

SIMPLIFYING THE ASSAY

 

A way to simplify an assay is to have the reporter probe solubilized in the sample as shown in the adjacent image.  In the image we show that as the antigen binds it effectively blocks the surface and the signal decreases proportionally to the binding.

 

It is often assumed that this simplified assay leads only to a signal decrease as the molecule/cell of interest is bound, but in the discussion below we illustrate that this is not necessarily the case and in fact whether the simplified assay leads to a signal decrease or a signal increase is linked to four factors:

 

  • the polarity of the applied voltage
  • the polarity of the reporter molecule.
  • the polarity of the capture molecule.
  • the polarity of the analyte.

SIGNAL DECREASE

In this scenario the analyte of interest has the same charge relative to the reporter molecule and so we have shown that the signal decreases with binding due to mutual electrostatic repulsion.

SIGNAL INCREASE

In this scenario the analyte of interest has the opposite charge relative to the reporter molecule and so we have shown that the signal increases with binding due to mutual electrostatic repulsion.


CONCLUSION

 

When developing an assay where cost is important to the commercial success keeping the assay simple is of course the best policy. Zimmer and Peacock have implemented assay simplification strategies for multiple-clients over the years and allowed a simpler less expensive product.

RECOMMENDATIONS

 

The table below are parameters and chemicals  we would recommend a developer tries when attempting to simplify an assay. Please contact Zimmer and Peacock and discuss how to implement this table below into your development.

 

 

 

Parameter Charge on reporter ion Comment
 Hexaammineruthenium (III) chloride Positive  This reporter ion undergoes a reduction so works best with electrodes that are polarised towards a negative voltage.
Hexaammineruthenium (II) chloride Positive  This  reporter ion undergoes an oxidation so works best with electrodes that are polarised towards a positive voltage.
Potassium hexacyanoferrate (III) Negative This reporter ion undergoes a reduction so works best with electrodes that are polarised towards a negative voltage.
Potassium hexacyanoferrate (II) Negative This reporter ion undergoes an oxidation so works best with electrodes that are polarised towards a positive voltage.
Ferrocene Neutral This  reporter ion undergoes an oxidation so works best with electrodes that are polarised towards a positive voltage.
Anthraquinone Neutral This  reporter ion undergoes a reduction so works best with electrodes that are polarised towards a negative  voltage.

www.zimmerpeacock.com 2017