The presented study is focused on the impact of binding zone location on immunochromatographic test strips on the analytical parameters of multiplex lateral flow assays. Due to non-equilibrium conditions for such assays the duration of immune reactions influences significantly the analytical parameters, and the integration of several analytes into one multiplex strip may cause an essential decrease of sensitivity. To choose the best location for binding zones, we have tested reactants for immunochromatographic assays of lincomycin, chloramphenicol, and tetracycline. The influence of the distance to the binding zones on the intensity of coloration and limit of detection (LOD) was rather different. Basing on the data obtained, the best order of binding zones was chosen. In comparison with non-optimal location the LODs were 5–10 fold improved. The final assay provides LODs 0.4, 0.4 and 1.0 ng/mL for lincomycin, chloramphenicol, and tetracycline, respectively. The proposed approach can be applied for multiplexed assays of other analytes.

A new scheme of reagents interaction for lateral flow immunoassay (LFIA) is proposed, which combines the features of competitive and sandwich assay and provides highly sensitive detection of low-molecular-weight analytes. Namely, the antigen in the sample interferes with the formation of the antibody (on the membrane)–hapten-protein–antibody (on the nanoparticle-marker) complex, competing with hapten-protein conjugate in both reactions. The proposed scheme was modelled using COPASI software, with a prediction of limit of detection (LOD) decrease by one order of magnitude compared to the standard competitive LFIA. This feature was experimentally confirmed for the detection of chloramphenicol (CAP) in honey. When tested in spiked honey, the visual LOD was 50 ng/mL for the common scheme and 5 ng/mL for the proposed scheme. Instrumental LOD was 300 pg/mL (1.2 µg/kg in conversion per sample weight of honey) in the standard scheme and 20 pg/mL (80 ng/kg in conversion per sample weight of honey) in the proposed scheme.

Maximum permissible levels of mycotoxins in baby food may be 1% of those in ordinary food. Therefore, highly sensitive methods of mycotoxin control are in demand. To detect such low amounts, expensive instrumental methods are commonly used. Advantages of immunochromatographic analyses are their low cost and simple sample preparation; however, their sensitivity needs to be increased to contend with instrumental methods. A scheme for competitive immunochromatography with indirect labelling was implemented and developed for the detection of mycotoxin zearalenone (ZEA). Two separate reagents were used for the assay, namely free specific antibodies and antispecies antibodies conjugated with gold nanoparticles. This made it possible to simultaneously increase the sensitivity of the assay and the reliability of measurements. The instrumental detection limit of ZEA in baby food was 5 pg/mL (100 pg/g). Thus, the sensitivity attained is comparable with liquid chromatography characteristics. The duration of the analysis was 17 min.

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.

A new scheme of immunochromatographic assay was developed for the highly sensitive detection of low-molecular-weight analytes. This scheme includes the following two steps: the formation of complexes of free specific antibodies with an antigen and their detection by anti-species antibodies conjugated to gold nanoparticles as the label. This scheme was tested with mycotoxin T-2 toxin in maize extracts. The use of specific antibodies and a label as two individual components made it possible to independently vary their concentrations with a simultaneous decrease in the detection limit and an increase in the color intensity. The assay did not require additional reagents and manipulations. The instrumental and visual detection limits of the designed test system were 0.1 and 5.0 ng/mL, respectively (2 and 90 ng per gram of analytes), which are two orders of magnitude lower compared to conventional immunochromatography using the same reagents.

Detection of antibiotics in the blood is necessary for characterizing their common or individual pharmacokinetics. This has increased the need in rapid detection techniques, such as lateral flow immunoassay, for the on-site control of antibiotics. The present study characterized factors influencing the analytical parameters of lateral flow immunoassay to increase its sensitivity for detecting tetracycline in human serum samples. Assay sensitivity was increased by altering the concentrations of immunoreagents and surfactant and the number of interaction stages in the assay with indirect labeling a specific antibody. The optimal assay conditions reduced the limit of visual detection of tetracycline from 100 to 10 ng/mL. The developed assay allowed us to detect tetracycline in both two-fold diluted and undiluted human serum samples within 15 min. Our results suggest that the developed assay can be used to screen patients under antibiotic treatment.