| Description |
The western honeybee (Apis mellifera) is the most frequent pollinator of flowering plants, making it exceptionally important for agriculture and biodiversity. However, bees are being threatened by various diseases, including bacterial infections. The bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most destructive disease of the honeybee brood. A traditional method for P. larvae detection is polymerase chain reaction (PCR), which allows for very sensitive analysis with high sample throughput. This then makes the DNA isolation and purification a possible source of complications. An adequate alternative to the conventional diagnostic methods are antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). These assays rely on the signal generated by enzymes, typically horseradish peroxidase. ELISAs are considered the gold standard of immunochemical methods for their high sensitivity, specificity even in complex matrices, and the ability to detect a wide range of analytes. Nevertheless, the conventional ELISA is not sensitive enough for the detection of early stages of the AFB. The recent progress in nanotechnology has provided various nanomaterials that can be used as sensitive labels in immunoassays to enhance their sensitivity. Particularly, photonupconversion nanoparticles (UCNP) are promising alternative luminescent labels in immunoassays. After the surface modification step, UCNPs can carry antibodies or streptavidin on their surface and thus be used as labels in various immunoassay formats, including the microtiter plate-based upconversion-linked immunosorbent assay (ULISA). We have developed a sandwich ULISA for the diagnosis of AFB, using UCNPs conjugated with antibody or streptavidin, and compared the results to the conventional ELISA. Specific polyclonal antibodies were prepared via immunization of New Zealand white rabbits and tested in a standard sandwich ELISA, which achieved an LOD of 6.5×104 CFU/mL. The UCNPs were modified using an Alkyne-PEG-neridronate linker, which allowed their bioconjugation with azide-modified antibody or streptavidin using copper-catalyzed click chemistry. The alkyne on one side of the linker was connected to the azide on the Ab or SA in a click-reaction, whereas the neridronate on the other side strongly coordinated to the UCNP surface. Firstly, the UCNPs were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Results from the TEM analysis have shown an average UCNP size of 58.5 nm. DLS was used to define the hydrodynamic diameter, which was 54.2 ± 1.7 nm before and 88 ± 2 nm after the conjugation with antibody; the streptavidin conjugate has reached the average size of 97 ± 3 nm. The synthesized labels were then employed in a sandwich ULISA, in which the bacteria Melissococcus plutonius, Paenibacillus alvei, and Brevibacillus laterosporus were used as negative controls. These bacteria are relevant as they also invade the honeybee brood. The results showed low cross-reactivity with Paenibacillus alvei and no crossreactivity with the other bacteria. The antibody-based UCNP conjugates have shown specific binding; however, the achieved LOD of 4.7×106 CFU/mL was slightly worse than in the case of ELISA. This may be due to the antibody being of polyclonal nature. When the polyclonal antibody contains a high percentage of IgG molecules not specific for the analyte, some of the nanoparticles might not show a specific binding due to the limited number of alkyne groups on the surface. On the other hand, the streptavidin-based labels achieved the LOD of 2.9×103 CFU/mL, which represents a 22-fold improvement in comparison with the ELISA. Finally, the potential for practical use of the ULISA was successfully demonstrated by the analysis of real samples of spiked larvae, adult bees, and bottom hive debris.
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