Name: a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)
Uploaded: 2011-09-14T13:00:00
Description: Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

Эта работа финансировалась личным стипендии из Wellcome Trust, чтобы Беате Kampmann (056608, GRO77273).

1. Подготовка БЦЖ люкс репортер микобактерий акции <ol><li> Расти БЦЖ люкс (М. Bovis БЦЖ штамма Монреаль трансформированных плазмидой трансфер pSMT1) при встряхивании на 200rpm до середины логарифмической фазы в Миддлбрук 7H9 бульона, содержащего 0,2% глицерина, 0,05% между 80 и 10% АЦП обогащени...

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Бактерицидное анализов в контексте исследований проблемы туберкулеза, вакцина хорошо известны на животных моделях, где они применяются для отбора потенциально перспективных вакцин-кандидатов. Сокращение бактериальной нагрузки в различных функциях органов в качестве основной для ч?...

<table border="1"><tbody><tr><td> Реагент </td><td> Компания </td><td> Cat Нет </td></tr><tr><td> БЦЖ люкс </td><td> Различный </td><td> н / </td></tr><tr><td> Миддлбрук 7H11 агар </td><td> Becton Dickinson </td><td> 283810 </td></tr><tr><td> Миддлбрук 7H9 бульон </td><td> BD Biosciences </td><td> 271 310 (500 г) </td></tr><tr><td> Миддлбрук АЦП обогащения </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> Миддлбрук OADC дополнения </td><td> Becton Dickinson </td><td> 212240 </td></tr><tr><td> Твин-80 </td><td> Sigma Aldrich </td><td> 274364 </td></tr><tr><td> Глицерин </td><td> Sigma Aldrich </td><td> G5150 </td></tr><tr><td> Гигромицину B </td><td> Roche </td><td> 10843555001 (20 мл) </td></tr><tr><td> N-децил альдегид </td><td> Сигма </td><td> D7384-100G </td></tr><tr><td> Этанол (&gt; 99,7%) </td><td> VWR </td><td> 101077 </td></tr><tr><td> Стерильные PBS </td><td> В Доме </td><td> н / </td></tr><tr><td> RPMI-1640 с L-глютамин, 25 мМ HEPES </td><td> Сигма </td><td> R0883 </td></tr><tr><td> Стерильной дистиллированной воде </td><td> В Доме </td><td> н / </td></tr><tr><td colspan="3"> Оборудование </td></tr><tr><td> Труба люминометра (инъекционный порт обязательно) </td><td> Бертольд AutoLumat LB953 или однотрубные люминометра Сириус </td><td></td></tr><tr><td> Люминометр труб, 12 х 75 мм, без оправы пирекса </td><td> Corning от VWR </td><td> 99445-12 </td></tr><tr><td> Колб Эрленмейера с вентилируемыми колпачками, стерильные поликарбоната 250 мл </td><td> Corning через Fisher Scientific </td><td> 2150329 </td></tr><tr><td> BD Vacutainer сбору крови трубки (без консервантов гепарин) </td><td> BD </td><td> 367676 </td></tr><tr><td> 50мл Сокол трубы, конические </td><td> BD </td><td> 352077 </td></tr><tr><td> 7 мл Sterilin образца полипропиленовые трубы украшения </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin универсальные контейнеры (30 мл) </td><td> Лабораторный анализ ООО </td><td> 128C </td></tr><tr><td> Стеклянные бусы диаметром 2 мм </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10 мл пипетки и пипетки мальчика </td><td> любой </td><td></td></tr><tr><td> 1000μl и 200 мкл пипетки и фильтр советы </td><td> любой </td><td></td></tr><tr><td colspan="3"> Общее лабораторное оборудование: </td></tr><tr><td> Класс II безопасности кабинета кабинет </td><td></td><td></td></tr><tr><td> Холодильник (4 ° С) </td><td></td><td></td></tr><tr><td> Центрифуга </td><td></td><td></td></tr><tr><td> Раскачивание шейкер платформа (180 0) смешать труб </td><td></td><td></td></tr><tr><td> Орбитальный Шейкер инкубатор установлена ​​на уровне 37 ° С для посадки бактериальных культур </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Мы опишем альтернативный подход к перечислению микобактерий<em&gt; В пробирке</em&gt;, Который использует репортер-гена меткой микобактерий вместо колониеобразующих единиц (КОЕ). &quot;Выживание&quot; организмов, а в качестве принимающей ответ-маркеров измеряются одновременно, обеспечивая недорогой, универсальный и функциональная система для исследования хост / патогена взаимодействия в контексте туберкулеза.

Функциональные Всего Пробирная крови для измерения жизнеспособности микобактерий, используя Репортер-Gene меткой БЦЖ или M. Tb (БЦЖ Люкс / М. Tb Люкс)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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Immunology and Infection

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