| Popis |
Western honeybee is an important pollinator and therefore, an invaluable part of agriculture and biodiversity. Serious losses of honeybee colonies in recent years are attributed mostly to climate changes and various diseases. One of the most important microbial diseases of honeybees is European foulbrood (EFB), which is caused by pathogen Melissococcus plutonius. From the economic and environmental point of view, the prevention of EFB spreading is crucial to prevent losses of honeybee colonies. Therefore, the development of an effective method for the detection of M. plutonius is necessary, ideally in the point-of-care (POC) format with sensitivity high enough to detect the pathogen before the clinical symptoms develop. Amperometry and electrochemical impedance spectroscopy (EIS) are highly sensitive and robust approaches compatible with POC testing. Due to their low cost, portability and mass production capabilities, the electrochemical biosensors are typically based on screen-printed electrodes (SPEs). For proper functionality, the immunosensors require antibodies with high affinity and low cross-reactivity. Since there were no antibodies against M. plutonius available, we have prepared them in-house. Purified bacterial cell wall fraction was prepared for rabbit immunization. After 45 days, rabbit blood was collected and serum was prepared. Subsequently, the immunoglobulin G fraction was separated from the serum by liquid chromatography with protein G column. The final antibody in PBS was stored at -30 °C for further use. Functionality of the prepared antibodies was verified using enzyme-linked immunosorbent assay (ELISA). The sandwich assay provided a limit of detection (LOD) of 1.4×10^5 CFU·mL-1. The ELISA was used to detect M. plutonius in real samples of bees, larvae and bottom hive debris, which are the matrices where this bacterium is typically present in the case of EFB infection. For the electrochemical biosensing, the anti-Melissococcus antibody was immobilized on the surface of SPE and allowed specific capture of bacteria. Non-specific binding was evaluated by incubating the sensor with Paenibacillus alvei instead of M. plutonius. The label-free EIS allowed to detect M. plutonius, however, the level of non-specific binding was very high, which was limiting for real samples analysis. Thus, better performance was obtained with amperometric sandwich assay, where the antibodies were conjugated with horseradish peroxidase (HRP). The Ab-HRP conjugate was binding to surface-captured immunocomplex and provided oxidation of 3,3´,5,5´-tetramethylbenzidine (TMB) in presence of H2O2. The electrochemical detection of current was based on the reduction of the enzymatically oxidized TMB on working electrode. For pure bacterial culture in buffer, the LOD was 6.6×10^4 CFU·mL-1. After optimization of amperometric immunosensor, real samples of bees and larvae were tested with LODs 2.4×10^5 CFU·mL-1 and 7.0×10^5 CFU·mL-1, respectively. Time of analysis was only 2 hours compared to time-consuming laboratory assays such as ELISA. The analysis of real bee and larvae samples confirmed the suitability of the developed immunosensor for in-field M. plutonius detection.
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