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.

In this article, we describe an immunochromatographic test system developed for rapid serodiagnostics of cattle brucellosis using two markers: Gold nanoparticles (GNPs) and quantum dots (QDs). The test system was compared with immunochromatographic serodiagnostics systems that use only one marker. The approbation of the test system was conducted on samples of cattle sera with low, but diagnostically significant titers of specific antibodies. We show that when two conjugates are used, the intensity of the detectable signal increases by 2–3 times compared with the test system using the QD conjugate and by more than nine times compared with the system using the GNP conjugate.

A highly sensitive lateral flow immunoassay (LFIA) for imidacloprid, a widely used neonicotinoid insecticide, has been developed. The LFIA realizes the indirect coupling of anti-imidacloprid antibodies and gold nanoparticle (GNP) labels directly in the course of the assay. For this purpose, the common GNPs conjugate with anti-imidacloprid antibodies and are changed into a combination of non-modified, anti-imidacloprid antibodies, and the GNPs conjugate with anti-species antibodies. The given approach provides the possibility of selecting independent concentrations of GNPs and anti-imidacloprid antibodies to obtain the influence of minimal imidacloprid concentrations in the samples on the formation of detected, labeled immune complexes. A comparative study of imidacloprid LFIAs with common and indirect antibody–label coupling was implemented. The second variant reduced the limit of detection (LOD) of imidacloprid 20 times, reaching 0.2 ng/mL and 0.002 ng/mL for visual and instrumental detection, respectively, thus surpassing the existing LFIAs for imidacloprid. The developed highly sensitive LFIA was tested for imidacloprid detection in freshly squeezed fruits and berries without any additional sample preparation. The imidacloprids revealed were in the range of 75–97% for grape, 75–85% for orange, and 86–97% for apple samples. The time of the testing was 15 min.

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten–biotin conjugate.

Finding optimal conditions for competitive lateral flow immunoassay is a controversial task. The content of specific antibodies labeled by nanoparticles should be simultaneously high to reach intense signals and low to register an influence on the signals for minimal concentrations of the target analyte. We propose to use two kinds of complexes of gold nanoparticles in the assay, with antigen–protein conjugates and with specific antibodies. The first complex interacts both with immobilized antibodies in the test zone and with antibodies on the surface of the second complex. In this assay, the coloration is enhanced by the binding of two-colored preparations in the test zone, whereas the antigen in the sample inhibits both the binding of the first conjugate with the immobilized antibodies and with the second conjugate. This approach is realized for the detection of insecticide imidacloprid (IMD), an important toxic contaminant connected with the recent global death of bees. The proposed technique expands the working range of the assay, that is, in accordance with its theoretical analysis. The reliable change of coloration intensity is achieved for a 2.3-times-lower concentration of the analyte. The limit of IMD detection is 0.13 ng/mL for tested solutions and 1.2 µg/kg for initial honey samples. The combination of two conjugates doubles the coloration in the absence of the analyte. The developed lateral flow immunoassay is applicable for five-fold-diluted honey samples without extraction, does not require additional stages (all reagents are pre-applied to the test strip), and is implemented in 10 min.

An immunochromatographic test system has been developed for the simultaneous rapid multiplex serodiagnostics of bovine brucellosis, tuberculosis, and leukemia. The test system is based on the use of a conjugate of gold nanoparticles with the chimeric protein Cysteine-A/G and three analytical zones with immobilized pathogen antigens: Brucella abortus lipolysaccharide, recombinant proteins MPB64 and MPB83-MPB63 of Mycobacterium bovis, and recombinant protein p24 of the bovine leukemia virus. Prototypes of the test system were tested on 98 samples of sera from healthy and infected animals. The diagnostic sensitivity of the developed test system was 92% for brucellosis, 92% for tuberculosis, and 96% for leukemia. False positive test results were not observed.