Handling Detection Limits of Multiplex Lateral Flow Immunoassay by Choosing the Order of Binding Zones

Changes in the limits of detection (LODs) for a multiplex lateral flow immunoassay (LFIA) caused by different locations of the binding zone on the test strips were studied. Due to the non-equilibrium conditions of the immune reactions in LFIAs, their analytical parameters are susceptible to the binding constants of antigen-antibody reactions and assay duration. Consequently, the integration of several tests into one multiplex assay can cause a significant worsening of the sensitivity. In this study, we propose a simple methodology for the determination of the best arrangement of binding zones, which takes into account the binding constants for immunoreagents. LFIAs of four mycotoxins, namely, aflatoxin B1, deoxynivalenol, T-2 toxin, and ochratoxin A, were integrated into a multiplex test strip. An enzyme-linked immunosorbent assay was applied to determine the equilibrium and kinetic constants of the immunoreactants for each analyte. It was found that the arrangement of binding zones with a descending order of the equilibrium association constants was optimal and provided both lower detection limits and a more uniform coloration. The selected position of the binding zones allowed decreasing the LODs down to 2 and 27 times for ochratoxin A and deoxynivalenol, respectively. The proposed approach can be applied to multiplex LFIAs for different analytes.