引用本文:	刘田,李硕,汪铭书,赵立宁,黄锦炉,吴艺琳,贾爱卿.抗杂交鳢(Channa argus×C. maculate)弹状病毒卵黄抗体制备及ELISA检测方法的建立.海洋与湖沼,2023,54(6):1756-1765.

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*抗杂交鳢(Channa argus×C. maculate)弹状病毒卵黄抗体制备及ELISA检测方法的建立*
刘田^1,2,3,4, 李硕², 汪铭书⁴, 赵立宁², 黄锦炉², 吴艺琳², 贾爱卿²
1.农业农村部微生态资源养殖利用企业重点实验室广东广州 511400;2.广东海大集团股份有限公司畜牧水产研究中心广东广州 511400;3.广东海大畜牧兽医研究院广东广州 511400;4.四川农业大学预防兽医研究所四川成都 611100

摘要:

为确定杂交鳢(Channa argus×C. maculate)弹状病毒卵黄抗体效价水平消减规律并验证抗体对病毒的中和能力, 采用杂交鳢弹状病毒灭活疫苗免疫蛋鸡, 使用醋酸-醋酸钠-辛酸法制备卵黄抗体, 建立间接ELISA检测方法以监测卵黄抗体产生过程抗体水平消减情况, 以中和试验评估卵黄抗体中和杂交鳢弹状病毒效果。结果显示, ELISA检测方法最佳条件为抗原包被浓度0.5×10^5.8 TCID₅₀/0.1 mL, 37 ℃包被2 h, 37 ℃封闭2 h。与大口黑鲈蛙虹彩病毒卵黄抗体、SPF蛋黄提取物、免疫前蛋黄提取物以及MBC空细胞均无交叉反应。以S/N(sample/negative) > 2.1且敏感性最高为标准确定阳性、阴性样品。卵黄抗体于首免后28 d产生效价, 四免后达到最高水平, 效价为1︰25 600, 平台期可持续40~60 d, 通过中和抗体检测结果证明平台期内抗体中和效价在免疫周期内最高, 约为4^6.3。通过ELISA检测方法对卵黄抗体产生规律进行监测, 通过中和检测方法评估卵黄抗体中和病毒效果。高滴度卵黄抗体具有被开发为新型抗杂交鳢弹状病毒生物制品的潜力, 杂交鳢弹状病毒卵黄抗体的制备及快速检测方法的构建为该病的防治提供了理论基础和技术手段。

关键词: 杂交鳢弹状病毒卵黄抗体间接ELISA 中和抗体

DOI：10.11693/hyhz20230500108

分类号:S942; Q955; S965

基金项目:年乡村振兴战略专项-农业科技发展及资源环境保护管理项目,2023KJ115号

附件

PREPARATION OF ANTI-HYBRID SNAKEHEAD RHABDOVIRUS IGY AND DEVELOPMENT OF INDIRECT ELISA

LIU Tian^1,2,3,4, LI Shuo², WANG Ming-Shu⁴, ZHAO Li-Ning², HUANG Jin-Lu², WU Yi-Lin², JIA Ai-Qing²

1.Key Laboratory of Microecological Resources and Utilization in Breeding Industry, Ministry of Agriculture and Rural Affairs, Guangzhou 511400, China;2.Animal Husbandry and Fisheries Research Center of Guangdong Haid Group Co., Ltd., Guangzhou 511400, China;3.Guangdong Haid Institute of Animal Husbandry and Veterinary, Guangzhou 511400, China;4.Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611100, China

Abstract:

To analyze the production regularity of anti-Hybrid snakehead rhabdovirus IgY and the effectiveness of neutralization antibody, laying hens were immunized with inactivated vaccine and then the indirect ELISA method was established for monitoring the production regularity of egg yolk antibody. Neutralization assay was also conducted to evaluate antibody neutralization. The optimal antigen coating concentration of ELISA assay was 0.5×10^5.8 TCID₅₀/0.1 mL with coating at 37 ℃ for 2 h, and blocking at 37 ℃ for 2 h. No cross reaction was found with anti-M. salmoides rhabdovirus IgY, SPF egg yolk extract, pre-immunized egg yolk extract and MBC cells. The positive and negative samples were determined by S/N (sample/negative) > 2.1 with the highest sensitivity. Anti-Hybrid snakehead rhabdovirus IgY was produced at 28 d after the first immunization, and reached the highest level after the fourth immunization with 1 : 25 600. And also the plateau phase was lasting for 40~60 d, during the plateau phase, the antibody neutralization titer reached to highest with 4^6.3. In this study, the production regularity of anti-Hybrid snakehead rhabdovirus IgY was monitored by indirect ELISA, and the effectiveness of neutralization antibody was valued by neutralization assay. The high titer IgY has the potential to be developed as a new type of biologic product against hybrid snakehead rhabdovirus. The preparation of anti-Hybrid snakehead rhabdovirus IgY and the construction of indirect ELISA method will provide theoretical basis and a technical mean for the prevention and treatment of this disease.

Key words: hybrid snakehead rhabdovirus immunoglobulin of egg yolk (IgY) indirect ELISA neutralizing antibody