蛙类脑膜脓毒性伊丽莎白菌多克隆抗体的制备及应用

张新艳; 曾占壮; 叶小军; 卓玉琛; 钟全福; 余培建

doi:10.19303/j.issn.1008-0384.2021.07.008

蛙类脑膜脓毒性伊丽莎白菌多克隆抗体的制备及应用

Preparation and Application of Rabbit Polyclonal Antibodies against Elizabethkingia meningosepticum on Frogs

摘要

摘要:
目的制备蛙类脑膜脓毒性伊丽莎白菌多克隆抗体，建立间接ELISA检测方法，进行病原菌在宿主体内的示踪研究，为病原菌的致病机理研究奠定基础。
方法将分离自患歪头病的棘胸蛙的脑膜脓毒性伊丽莎白菌（Elizabethkingia meningosepticum）RsB1151018NA甲醛灭活后，免疫新西兰大白兔制备兔抗血清。通过优化反应条件，建立脑膜脓毒性伊丽莎白菌间接ELISA的检测方法；兔抗血清多克隆抗体采用Protein A亲和层析柱分离纯化，以异硫氰酸荧光素（FITC）标记，标记后的抗体与感染RsB1151018NA的棘胸蛙不同脏器组织的冷冻切片反应，荧光显微镜观察，跟踪RsB1151018NA在棘胸蛙体内的迁移。
结果试验结果表明，制备的兔抗棘胸蛙脑膜脓毒性伊丽莎白菌多克隆抗体血清效价为1∶5.12×10⁵，纯化后的抗体效价为1∶1.6×10⁴，且建立的间接ELISA方法检测脑膜脓毒性伊丽莎白菌具有高度特异性，与其他水产常见病原菌没有交叉反应，检测灵敏度为1.0×10⁴ CFU·mL⁻¹。脑膜脓毒性伊丽莎白菌在棘胸蛙体内随血液循环而分布至全身，可侵袭肌肉、肝脏、肾脏、脾脏、肠道、心脏、眼和脑等组织器官，肾脏、眼和脑感染程度最严重，推测这三个组织器官是其靶器官；创伤感染是可能的感染途径之一。
结论建立的检测方法能够快速、灵敏、特异地检测脑膜脓毒性伊丽莎白菌，对蛙类歪头病的防控和早期诊断可提供理论参考依据；另外利用免疫荧光检测病原菌在宿主体内的迁移，研究病原的致病机理及发病过程等，能为防治药物的研发和防治方法的确定提供理论依据。

Abstract:
Objective Rabbit polyclonal antibodies against Elizabethkingia meningosepticum (Em) were prepared to establish an indirect ELISA detection to trace the pathogenic attack in organs of frogs (Rana spp).
Method The strain RsB1151018NA of Em from a diseased R. spinosa was isolated, formalin-inactivated, and injected into a rabbit for the serum preparation. An optimized indirect ELISA using the rabbit polyclonal antibodies was developed for tracing RsB1151018NA. The rabbit immunoglobulin G was purified on a protein A column. The purified antibodies were marked with FITC and further purified on a desalting column Sephadex G-25 for subsequently tracing the pathogen in organs of the inoculated frogs.
Results The obtained rabbit polyclonal antibodies showed a titer up to 1 ∶ 5.12×10⁵, and 1 ∶ 1.6×10⁴ after purification. The indirect ELISA assay on the polyclonal antibody was specific and sensitive in detecting Em without cross-reaction on other bacteria. It had a detection limit of 1.0×10⁴ cfu·mL⁻¹. According to the in vivo tracing test, RsB1151018NA distributed throughout the entire frog body by blood circulation with the most serious infections in the kidney, eyes, and brain.
Conclusion The optimized indirect ELISA detection of Em was rapid, sensitive, and specific in tracing the bacterial infection on R. spp. It was considered applicable for early diagnosis of the “crooked-head disease” and breeding of healthy frogs for food and medicine.

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