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Preparation of affinity surfaces for clinical diagnostics
Kalaninová, Zuzana ; Pompach, Petr (advisor) ; Žáček, Petr (referee)
Botulinum toxins are group of protein venoms produced by Clostridium bacteria. These toxins belong to the most toxic substances causing paralytic disease called botulism. The endopeptidase activity of the toxins leads to the blockage of neurotransmitters release to neural junctions. Since the first symptoms of botulism are observed within 48 hours and toxin remains active for months, botulinum toxins are considered as potential bio- terrorism agents. For successful treatment, fast assays for detection of the toxin presence and type are highly required. This work presents a new method that could be potentially used for determination of botulotoxin in various matrices. The method is based on the detection of products of botulinum toxin A (BoNT/A) endopeptidase activity by affinity surfaces prepared by ambient ion soft landing and MALDI mass spectrometry. For immobilization and subsequent enrichment of biotinylated products of BoNT/A activity, proteins with high affinity to biotin - avidin, neutravidin and streptavidin - were used. The presented method, which combines affinity surfaces with MALDI mass spectrometry is sensitive, robust and fast and might be potentially used for detection of BoNT/A activity. Key words: ambient ion soft landing, neutravidin, botulinum toxin A, BoNT/A, MALDI/TOF mass...
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Preparation of affinity surfaces for mass spectrometry
Kalaninová, Zuzana ; Pompach, Petr (advisor) ; Kučera, Lukáš (referee)
One of the most potent toxic substances of biological origin known are botulinum toxins - nerve toxins produced by strains of Clostridium bacteria. They affect neuro-muscular connections where they cleave proteins of the SNARE complex, block neurotransmitter release, and thus stop signal transmission. This leads to flaccid paralysis, the main symptom of a disease called botulism. Due to their extremely low lethal dose and high toxicity, BoNTs take a place on the list of agents of bioterrorism. Despite numerous methods available to this date, the mouse bioassay remains the gold standard for the detection of BoNTs. Regardless of the ethical aspects of these experiments, the time they require is also inconvenient. In this work, a unique combination of protein surfaces and detection of active BoNTs through their endopeptidase activity and mass spectrometry, EndoPep-MS, is introduced. Functionalized surfaces were prepared by ambient ion soft landing - a technique utilizing a beam of hyperthermal ions (energy range 1-100 eV) as a tool for controlled modification. Two approaches to the detection of botulinum toxin A1 were applied. One of them, based on avidin- biotin interaction, used a neutravidin-modified surface for enhancement of biotinylated peptides (substrate peptide for BoNT/A and peptide products...
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Preparation of affinity surfaces for clinical diagnostics
Kalaninová, Zuzana ; Pompach, Petr (advisor) ; Žáček, Petr (referee)
Botulinum toxins are group of protein venoms produced by Clostridium bacteria. These toxins belong to the most toxic substances causing paralytic disease called botulism. The endopeptidase activity of the toxins leads to the blockage of neurotransmitters release to neural junctions. Since the first symptoms of botulism are observed within 48 hours and toxin remains active for months, botulinum toxins are considered as potential bio- terrorism agents. For successful treatment, fast assays for detection of the toxin presence and type are highly required. This work presents a new method that could be potentially used for determination of botulotoxin in various matrices. The method is based on the detection of products of botulinum toxin A (BoNT/A) endopeptidase activity by affinity surfaces prepared by ambient ion soft landing and MALDI mass spectrometry. For immobilization and subsequent enrichment of biotinylated products of BoNT/A activity, proteins with high affinity to biotin - avidin, neutravidin and streptavidin - were used. The presented method, which combines affinity surfaces with MALDI mass spectrometry is sensitive, robust and fast and might be potentially used for detection of BoNT/A activity. Key words: ambient ion soft landing, neutravidin, botulinum toxin A, BoNT/A, MALDI/TOF mass...
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