Molecular interference in antibody-antigen interaction studied with magnetic force immunoassay

Area of Science:

• Biotechnology
• Biochemistry
• Molecular Biology

Background:

• Molecular interferences pose challenges in antibody-antigen based biotechnologies like immunoassays.
• Surfactants are used to enhance immunoassay performance but can impact protein interactions, with mechanisms often unclear.

Purpose of the Study:

• To investigate molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay.
• To quantify the influence of surfactant concentration on antibody-antigen binding kinetics and protein conformation.

Main Methods:

• Utilized a sandwich immunoassay with magnetic particle labels and a magnetic tweezers setup to apply force.
• Quantified force-dependent dissociation curves to analyze binding properties.
• Employed circular dichroism spectroscopy to assess protein conformational changes.

Main Results:

• Identified two distinct cTn-dependent bond types: weak (non-specific) and strong (specific binding).
• Observed a two-fold increase in the dissociation rate constant for strong bonds with increasing surfactant concentration.
• Circular dichroism revealed surfactant-induced conformational changes in cTn, but not in antibodies.

Conclusions:

• The nonionic surfactant alters cTn conformation, potentially explaining the increased dissociation rate of specific binding.
• The magnetic tweezers methodology effectively probes surfactant influence on protein interactions in immunoassays.
• This approach aids in understanding and mitigating molecular interferences in biotechnological assays.