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

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中图分类号：S852.616.3 文献标志码：A 文章编号：0366-6964（2025）06-2906-11

Establishment of Double-antibody Sandwich ELISA Method Based on the Specifiec Nanobody against Clostridium Perfringens β Toxin

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 halflife，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 successfullyobtained withahalf-lifeof 2.152h ，anaffinity constant of O.961 2and good stability within

. Based on CPB-VHH，a specific double-antibody sandwich ELISA was established with the critical value of O.183，the sensitivity of 0.977ng⋅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.

Keywords： Clostridium perfringens （C. perfringens）; β toxin （CPB）；nanobody;ELISA ∗ Corresponding author： FAN Ruiwen，E-mail： ruiwenfan 163.com

产气荚膜梭菌（Clostridium perfringens）是一种对动物和人类都具有致病性的革兰阳性厌氧菌，普遍存在于土壤和周边环境中，通过分泌多种毒素引起人和畜禽多种肠道疾病。（剩余17123字）