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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 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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