Theoretical Framework and Experimental Validation of Multiplexed Analyte Quantification Using Cross-Reactive Affinity...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Analytical Chemistry

Background:

• Immunoassays rely on specific affinity reagents for accurate molecular quantification.
• Reagent cross-reactivity causes false positive signals, leading to inaccurate analyte quantification.
• Mitigating cross-reactivity is a significant unsolved challenge in molecular diagnostics.

Purpose of the Study:

• To develop a mathematical framework enabling accurate molecular quantification using multiple cross-reactive affinity reagents.
• To address the limitations posed by non-specific binding in diagnostic assays.
• To transform conventionally unused cross-reactive reagents into a tool for precise analyte quantification.

Main Methods:

• Utilized generalized binding equations and noise estimation.
• Developed a mathematical framework to manage and correct for cross-reactivity.
• Experimentally validated the approach using a 2-plex quantification assay.

Main Results:

• Demonstrated accurate 2-plex quantification of a small molecule despite the presence of a cross-reactive molecule.
• Achieved reliable quantification in 10% human serum, even with high concentrations of interfering substances.
• Established well-defined bounds of quantification for assessing assay quality and predicting performance limitations.

Conclusions:

• The proposed mathematical framework effectively mitigates the impact of cross-reactivity in affinity-based assays.
• This approach enables accurate molecular quantification using reagents previously considered unusable due to cross-reactivity.
• The developed schema enhances the reliability and quality assessment of molecular diagnostic results.