基于产气荚膜梭菌β毒素纳米抗体双抗夹心ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2025.06.033

摘要/Abstract

摘要：

产气荚膜梭菌(Clostridium perfringens)分泌的β毒素(CPB)可引起家畜和野生动物发生肠炎和肠毒血症以及人类严重的食物中毒。因此，开发有效的检测方法对于鉴定该菌株的感染至关重要。本研究通过建立产气荚膜梭菌CPB纳米抗体文库和噬菌体展示技术淘选其特异性纳米抗体(命名为CPB-VHH)，并对该纳米抗体半衰期、亲和性及温度稳定性进行评估。同时，分别以CPB-VHH为捕获抗体和羊驼抗CPB多克隆血清为检测抗体，建立双抗夹心ELISA方法，并对该方法临界值、灵敏性、交叉反应及准确性进行评价。结果显示，本研究筛选获得一种针对CPB的特异性纳米抗体CPB-VHH，其半衰期为2.152 h，亲和性常数为0.961 2，在37℃以内的稳定性较好；以该抗体为基础，建立一种检测CPB的双抗夹心ELISA方法，临界值为0.183，灵敏度为0.977 ng·mL^-1，准确率为92%，且与产气荚膜梭菌CPA、CPB、ETX重组蛋白、产单核细胞李氏杆菌(L. monocytogenes)、金黄色葡萄球菌(S. aureus)、火鸡沙门菌(Salmonella)、大肠杆菌(Escherichia coli)无交叉反应，特异性良好。本研究建立的双抗夹心ELISA方法为B型和C型产气荚膜梭菌感染提供一种诊断方法，同时也为涉及CPB定量的疫苗研发及该毒素致病性研究提供指导。

关键词: 产气荚膜梭菌, β毒素(CPB), 纳米抗体, ELISA

Abstract:

Clostridium perfringens (C. perfringens) secretes β toxin (CPB) that can cause enteritis and enterotoxemia in livestock and wild animals, as well as severe food poisoning in humans. Therefore, the development of effective detection method is crucial for inhibiting the infection with C. perfringens. Here, the specific nanobody (named as CPB-VHH) was obtained by establishing a CPB nanobody library and panning through phage display technology, and the half-life, affinity and temperature stability were evaluated. Using CPB-VHH as the capture antibody and alpaca anti-CPB polyclonal serum as the detection antibody, a double-antibody sandwich ELISA method was established and evaluated. The results showed that CPB-VHH was successfully obtained with a half-life of 2.152 h, an affinity constant of 0.961 2 and good stability within 37 ℃. Based on CPB-VHH, a specific double-antibody sandwich ELISA was established with the critical value of 0.183, the sensitivity of 0.977 ng·mL^-1, accuracy of 92%, and without cross reaction with CPA, CPB, ETX recombinant protein of Clostridium perfringens, L. monocytogenes, S. aureus, Salmonella and Escherichia coli, which showed the good specification. The results suggested that the double-antibody sandwich ELISA method established in this study provided a diagnostic method for C. perfringens type B and C infections, and also provided guidance for vaccine development involving CPB quantification and research on the pathogenicity of β toxin.

Key words: Clostridium perfringens (C. perfringens), β toxin (CPB), nanobody, ELISA

中图分类号:

S852.616.3

贾琼, 高帅鹏, 修艳宇, 任泓睿, 张书茵, 杨皓宇, 范瑞文. 基于产气荚膜梭菌β毒素纳米抗体双抗夹心ELISA方法的建立[J]. 畜牧兽医学报, 2025, 56(6): 2906-2916.

JIA Qiong, GAO Shuaipeng, XIU Yanyu, REN Hongrui, ZHANG Shuyin, YANG Haoyu, FAN Ruiwen. Establishment of Double-antibody Sandwich ELISA Method Based on the Specifiec Nanobody against Clostridium Perfringens β Toxin[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2906-2916.

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反应体系 Reaction system	加样体积/μL Volume	扩增程序 PCR program
2×Es Taq MasterMix	25	$\left.\begin{array}{r}95{ }^{\circ} \mathrm{C} \quad 5 \mathrm{~min} \\95{ }^{\circ} \mathrm{C} \quad 30 \mathrm{~s} \\55{ }^{\circ} \mathrm{C} \quad 20 \mathrm{~s} \\72{ }^{\circ} \mathrm{C} \quad 1 \mathrm{~min} \\72{ }^{\circ} \mathrm{C} \quad 10 \mathrm{~min} \\4{ }^{\circ} \mathrm{C} \quad \text { forever }\end{array}\right\} 32 \text { cycles }$
CPB-F	2
CPB-R	2
模板	5
ddH₂O	16
合计	50

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