RU2012143991A - METHOD FOR DETERMINING NON-SPECIFIC STABILITY OF PATHOGENIC MICROORGANISMS TO ANTIBIOTICS ON THE BASIS OF MEASURING THE CATALYTIC ACTIVITY OF PHOSPHODYESTERASES, CRIMPING CYCLIC DYNOFINOSIS - Google Patents
METHOD FOR DETERMINING NON-SPECIFIC STABILITY OF PATHOGENIC MICROORGANISMS TO ANTIBIOTICS ON THE BASIS OF MEASURING THE CATALYTIC ACTIVITY OF PHOSPHODYESTERASES, CRIMPING CYCLIC DYNOFINOSIS Download PDFInfo
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- RU2012143991A RU2012143991A RU2012143991/10A RU2012143991A RU2012143991A RU 2012143991 A RU2012143991 A RU 2012143991A RU 2012143991/10 A RU2012143991/10 A RU 2012143991/10A RU 2012143991 A RU2012143991 A RU 2012143991A RU 2012143991 A RU2012143991 A RU 2012143991A
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- phosphodiesterase
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- gmp
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
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Abstract
Способ определения неспецифической устойчивости патогенных микроорганизмов к антибиотикам и факта присутствия бактериальных биопленок на основании измерения каталитической активности фосфодиэстераз, расщепляющих циклический дигуанозинмонофосфат, включающий:1) формирование иммунных комплексов между фосфодиэстеразой-мишенью и биотинилированным антителом или иным аффинным лигандом, модифицированным биотином и специфичным к некаталитическим доменам фосфодиэстеразы;2) аффинную очистку комплексов, сформированных фосфодиэстеразой-мишенью и биотинилированным антителом или иным аффинным лигандом, модифицированным биотином, при помощи парамагнитных частиц, содержащих нейтравидин или его аналоги, связывающие биотин;3) взаимодействиекомплексов фосфодиэстеразабиотинилированноеантитело, иммобилизованных на парамагнитных частицах, с комплексами, содержащими с-di-GMP в форме G-квадруплексов с интеркалированным красителем, сопровождающееся падением интенсивности флуоресценции по мере разрушения комплексов интеркалирующего красителя c-di-GMP;4) измерение падения флуоресценции при гидролизе c-di-GMP и разрушении комплекса c-di-GMP с интеркалирующим красителем с последующим количественным определением активности фосфодиэстеразы на основании калибровочных кривых, построенных с использованием известных количеств рекомбинантного фермента фосфодиэстеразы, идентичного исследуемой мишени.A method for determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms based on the measurement of the catalytic activity of phosphodiesterases that cleave cyclic diguanosine monophosphate, including: 1) the formation of immune complexes between the target phosphodiesterase and a biotinylated specific non-specific and specific affinity specific phosphodiesterase; 2) affinity purification of complexes formed by phosphodiesterase- with a target and a biotinylated antibody or other affinity ligand modified with biotin, with the help of paramagnetic particles containing neutravidin or its analogues that bind biotin; 3) the interaction of phosphodiesterase-biotinylated antibody complexes immobilized on paramagnetic particles with GM complexes containing G-groups with di with intercalated dye, accompanied by a decrease in fluorescence intensity as the complexes of the intercalating dye c-di-GMP are destroyed; 4) measurement of the fluorescence drop In the course of hydrolysis of c-di-GMP and destruction of the complex of c-di-GMP with an intercalating dye, followed by quantitative determination of phosphodiesterase activity based on calibration curves constructed using known amounts of the recombinant phosphodiesterase enzyme identical to the target under study.
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RU2012143991/10A RU2518249C1 (en) | 2012-10-16 | 2012-10-16 | Method of determining nonspecific resistance of pathogenic microorganisms to antibiotics based on measuring catalytic activity of phosphodiesterases cleaving cyclic diguanosine monophosphate |
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RU2012143991/10A RU2518249C1 (en) | 2012-10-16 | 2012-10-16 | Method of determining nonspecific resistance of pathogenic microorganisms to antibiotics based on measuring catalytic activity of phosphodiesterases cleaving cyclic diguanosine monophosphate |
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RU2694964C2 (en) * | 2017-12-19 | 2019-07-19 | ФЕДЕРАЛЬНОЕ АГЕНТСТВО ПО ТЕХНИЧЕСКОМУ РЕГУЛИРОВАНИЮ И МЕТРОЛОГИИ (Росстандарт) | Method of measuring catalytic activity (catalytic concentration) of enzymes using methods of mass spectrometry and spectrophotometry with preliminary identification of target analyte and determination of purity of substances |
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