基于酶联免疫吸附测定法的索氏梭菌致死毒素检测系统的建立。
Development of ELISA based detection system for lethal toxin of Clostridium sordellii.
机构信息
Biotechnology Division, Defence Research & Development Establishment, Gwalior, India.
出版信息
Indian J Med Res. 2013 Jun;137(6):1180-7.
BACKGROUND & OBJECTIVES: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii.
METHODS
The catalytic domain (1.6 kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin.
RESULTS
The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody.
INTERPRETATION & CONCLUSIONS: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples.
背景与目的
梭状芽胞杆菌及其毒素与动物和人类疾病有关。梭状芽胞杆菌产生两种蛋白毒素（致死毒素和出血毒素）。由于其高毒性，致死毒素受到了更多的关注。本研究旨在建立一种梭状芽胞杆菌致死毒素的夹心 ELISA 检测方法。
方法
采用 PCR 扩增梭状芽胞杆菌致死毒素的催化结构域（1.6 kb），克隆至 pQE30 UA 载体，转化至大肠杆菌 SG13009。优化表达条件，并在天然条件下使用 Ni-NTA 亲和层析对重组蛋白进行纯化，通过 SDS-PAGE 和 Western blot 进行鉴定。用弗氏完全佐剂和弗氏不完全佐剂（FCA 和 FIA）在 BALB/c 小鼠和兔中对纯化的重组蛋白进行免疫，制备多克隆抗体。优化夹心 ELISA 检测方法用于检测致死毒素。
结果
在 IPTG（异丙基硫代半乳糖苷）诱导 4.0 h 时，达到最大重组蛋白表达量 0.5 mM。用弗氏完全佐剂和弗氏不完全佐剂免疫的小鼠和兔产生的多克隆抗体效价高达 1:512000。制备的抗体具有高度敏感性，在 1:4000 稀释度下，对小鼠（捕获）和兔（揭示）抗体的检测限为 0.3 ng/ml。
解释与结论
建立了一种基于 ELISA 的梭状芽胞杆菌致死毒素检测系统。该检测系统特异性强，与其他梭状芽胞杆菌毒素无交叉反应。它将有助于检测临床和环境样本中的梭状芽胞杆菌致死毒素。
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