Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.
Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.
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用纳米叶裂酸酸盐酶测定法快速定量艰难梭菌谷氨酸脱氢酶和毒素B（TCDB）。
DOI：
10.1021/acs.analchem.1c05206
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发表时间：
2022-06-14
影响因子：
7.4
通讯作者：
Jeuken, Lars J. C.
中科院分区：
文献类型：
--
作者：
Adamson, Hope;Ajayi, Modupe O.;Gilroy, Kate E.;McPherson, Michael J.;Tomlinson, Darren C.;Jeuken, Lars J. C.
艰难梭菌感染(CDI)是一种主要的与医疗保健相关的感染，发病率和死亡率都很高，是一种经济负担。目前还没有一种独立的医疗点检测(POCT)足以在没有疾病的艰难梭菌携带的情况下识别真正的CDI，因此迫切需要一种方法来确保及时、适当的治疗。在这里，两种类型的结合蛋白，亲和分子和纳米体，针对艰难梭菌的两个生物标志物，谷氨酸脱氢酶(GdH)和毒素B(TcdB)，被结合在Nanobit(NanoLuc二元技术)分裂荧光素酶检测中。对检测方法进行了优化，并考察了其性能控制参数。缓冲液中TcdB分析的检测下限(LOD)为44fm，对数范围为4-5log，信号增益为1300倍，是迄今为止观察到的最好的纳米比特分析方法。在粪便样品基质中，GDH和TcdB检测的灵敏度(LOD分别为4.5和2 pm)和结果时间(32分钟)与目前的商业侧流POCT相似，但Nanobit检测没有洗涤步骤，通过TcdA检测临床相关的TcdB，并且是定量的。将其发展为POCT可能进一步提高敏感性，并为快速诊断真正的CDI提供迫切需要的超灵敏TcdB试验。NanoBiTBiP(带结合蛋白的纳米比特)系统在生产和检测性能方面比以抗体为结合元件的纳米比特检测系统更具优势。我们希望这种方法和途径也能普遍适用于其他生物标志物。
C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.
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影响因子：
4.6
作者：
Gite S;Archambault D;Cappillino MP;Cunha D;Dorich V;Shatova T;Tempesta A;Walsh B;Walsh JA;Williams A;Kirby JE;Bowers J;Straus D
通讯作者：
Straus D
艰难梭菌毒素 B 检测的超灵敏电化学免疫分析的简单方法
影响因子：
12.6
作者：
Fang, Yi-Shan;Chen, Shu-Yi;Wang, Ju-Fang
通讯作者：
Wang, Ju-Fang
影响因子：
7.4
作者：
Hall, Mary P.;Kincaid, Virginia A.;Dart, Melanie L.
通讯作者：
Dart, Melanie L.
影响因子：
6.4
作者：
Carter GP;Chakravorty A;Pham Nguyen TA;Mileto S;Schreiber F;Li L;Howarth P;Clare S;Cunningham B;Sambol SP;Cheknis A;Figueroa I;Johnson S;Gerding D;Rood JI;Dougan G;Lawley TD;Lyras D
通讯作者：
Lyras D
影响因子：
46.9
作者：
Elledge SK;Zhou XX;Byrnes JR;Martinko AJ;Lui I;Pance K;Lim SA;Glasgow JE;Glasgow AA;Turcios K;Iyer NS;Torres L;Peluso MJ;Henrich TJ;Wang TT;Tato CM;Leung KK;Greenhouse B;Wells JA
通讯作者：
Wells JA