Quantification of diphtheria toxin-mediated ADP-Ribosylation in a solid-phase assay
Quantification of diphtheria toxin-mediated ADP-Ribosylation in a solid-phase assay
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DOI：
10.1373/clinchem.2007.085365
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发表时间：
2007-09-01
影响因子：
9.3
通讯作者：
Fuchs, Hendrik
中科院分区：
文献类型：
--
作者：
Bachran, Christopher;Sutherland, Mark;Fuchs, Hendrik
背景：由于疫苗接种计划的减少，白喉感染的数量在过去十年中有所增加。白喉毒素（DT）由白喉棒状杆菌表达，并且负责白喉的致死性。DT通过真核细胞延伸因子2（eEF 2）的ADP-核糖基化抑制细胞蛋白质合成。没有用于定量DT酶活性的体外系统。方法：以生物素标记的NAD'为底物，将固相结合的组氨酸标签eEF 2与毒素进行ADP核糖基化，然后用链霉亲和素-过氧化物酶检测转移的生物素标记的ADP核糖。DT酶活性与吸光度相关。我们测量了用链霉亲和素-琼脂糖沉淀后ADP-核糖基化的eEF 2的量。结果：该方法检测到DT的酶活性为30 ng/L，相当于5 mU/L ADP-核糖基化活性。在100 ng/L时也检测到假单胞菌外毒素A（PE）活性。我们用DT或PE的催化结构域和肿瘤特异性配体组成的嵌合毒素验证了测定。这些嵌合毒素在1000 ng/L时显示信号增加。热灭活DT和霍乱毒素，ADP-核糖基G-蛋白没有表现出任何信号increasing.Conclusions：该测定可能是一个常规的诊断检测DT活性和高度特异性抑制剂DT的发展的基础。(c)2007年美国临床化学协会
Background: Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADPribosylation by DT.Methods: Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD' as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System.Results: The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase.Conclusions: The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT. (c) 2007 American Association for Clinical Chemistry