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

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.

Immunochromatographic assay for miroestrol and deoxymiroestrol, its cross‐reactivity, and application in Pueraria mirifica (white Kwao Krua) analysis

“…Increasing the concentration ratio can increase the cross‐reactivity of analogues. However, increasing the immune reaction time can lower the cross‐reactivity 33 . In this study, the immune reaction time of ICA was shorter than that of ELISA; however, the cross‐reactivity decreased.…”

“…Interactions between mAb‐12G11 and the AuNPs may impact the mAb conformation; thus, electrostatic conjugation may alter the cross‐reactivity of ICA. Other factors that affect the cross‐reactivity include the format of immunoassay, duration of the immune reaction, and the concentration ratio of the antibody and the modified hapten (hapten–carrier protein conjugate) 33 . Increasing the concentration ratio can increase the cross‐reactivity of analogues.…”

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen‐binding antibody produced via Escherichia coli

“…Indeed, intracellular factors, such as ionic strength and oxidative state, may have an effect on binding reactivity. Previously, cross‐reactivities were altered based on the assay format, the ratio of immunoreactant concentrations, and the length of the antigen–antibody reaction in a given assay format 27 …”

A Low-Cost, 3D-Printed Biosensor for Rapid Detection of Escherichia coli

“…Figure 6 A showed specificity with the antibody-coated into WE, which presented a current drop (ΔI) lower than the cutoff value of 7.5 μA obtained by SWV for the lowest E. coli concentration detected. The cross-reactivity could be due to shared epitopes of the surface proteins between the E. coli strains that led to a reduced specific binding affinity toward whole cell detection [ 10 ]. Shorter DNA fragments of TOP10 can be amplified by the primer pairs that are specific to DH5alpha ( Figure 6 B).…”