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.