Changing Cross-Reactivity for Different Immunoassays Using the Same Antibodies: Theoretical Description and Experimental Confirmation

Dmitriy V Sotnikov,Boris B Dzantiev,Elena A Zvereva,Anatoly V Zherdev,Sergei A Eremin

doi:10.3390/app11146581

Dmitriy V Sotnikov, Boris B Dzantiev + Show 3 more

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https://doi.org/10.3390/app11146581

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Abstract

Many applications of immunoassays involve the possible presence of structurally similar compounds that bind with antibodies, but with different affinities. In this regard, an important characteristic of an immunoassay is its cross-reactivity: the possibility of detecting various compounds in comparison with a certain standard. Based on cross-reactivity, analytical systems are assessed as either high-selective (responding strictly to a specific compound) or low-selective (responding to a number of similar compounds). The present study demonstrates that cross-reactivity is not an intrinsic characteristic of antibodies but can vary for different formats of competitive immunoassays using the same antibodies. Assays with sensitive detection of markers and, accordingly, implementation at low concentrations of antibodies and modified (competing) antigens are characterized by lower cross-reactivities and are, thus, more specific than assays requiring high concentrations of markers and interacting reagents. This effect was confirmed by both mathematical modeling and experimental comparison of an enzyme immunoassay and a fluorescence polarization immunoassay of sulfonamides and fluoroquinolones. Thus, shifting to lower concentrations of reagents decreases cross-reactivities by up to five-fold. Moreover, the cross-reactivities are changed even in the same assay format by varying the ratio of immunoreactants’ concentrations and shifting from the kinetic or equilibrium mode of the antigen-antibody reaction. The described patterns demonstrate the possibility of modulating immunodetection selectivity without searching for new binding reactants.

Highlights

The possibility of obtaining antibodies that can bind very different compounds has led to the intense development of immunoanalytical systems
In the course of a competitive immunoassay with a modified antigen, three compounds interact simultaneously: the antigen-binding sites of antibodies (Ab), an unlabeled antigen in the tested sample (Ag), and a modified competing antigen (Ag*). This leads to the formation of two types of complexes having the compositions AbAg and AbAg*
For enzyme-linked immunosorbent assay (ELISA), Ag* is an antigen immobilized on the solid phase, and the registered signals are determined by the formation of the AbAg* complexes

Summary

Introduction

The possibility of obtaining antibodies that can bind very different compounds has led to the intense development of immunoanalytical systems. Various forms of immunoassay are successfully used in medical diagnostics, quality and safety control of consumer products, environmental monitoring, etc. The advantages of immunoanalytical methods include simple sample preparation and assay implementation, highly productive testing, and the absence of the need for sophisticated equipment and highly trained operators. Depending on the tasks to be solved and the available equipment, various analysis formats can be realized with different labels and instrumentation. These formats of immunoassays include enzyme immunoassay, immunoagglutination, lateral-flow and through-flow membrane immunoassays, systems that detect the intensity or polarization of fluorescence, electrochemical immunosensors, etc. These formats of immunoassays include enzyme immunoassay, immunoagglutination, lateral-flow and through-flow membrane immunoassays, systems that detect the intensity or polarization of fluorescence, electrochemical immunosensors, etc. [8,9]

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