李硕, 陈静妮, 赵立宁, 黄春萍, 黄锦炉, 王贵平, 仲颖. 抗大口黑鲈蛙虹彩病毒卵黄抗体的制备及其间接ELISA检测方法的建立[J]. 南方水产科学. DOI: 10.12131/20230148
引用本文: 李硕, 陈静妮, 赵立宁, 黄春萍, 黄锦炉, 王贵平, 仲颖. 抗大口黑鲈蛙虹彩病毒卵黄抗体的制备及其间接ELISA检测方法的建立[J]. 南方水产科学. DOI: 10.12131/20230148
LI Shuo, CHEN Jingni, ZHAO Lining, HUANG Chunping, HUANG Jinlu, WANG Guiping, ZHONG Ying. Preparation of anti-largemouth bass ranavirus egg yolk antibody and establishment of indirect ELISA method[J]. South China Fisheries Science. DOI: 10.12131/20230148
Citation: LI Shuo, CHEN Jingni, ZHAO Lining, HUANG Chunping, HUANG Jinlu, WANG Guiping, ZHONG Ying. Preparation of anti-largemouth bass ranavirus egg yolk antibody and establishment of indirect ELISA method[J]. South China Fisheries Science. DOI: 10.12131/20230148

抗大口黑鲈蛙虹彩病毒卵黄抗体的制备及其间接ELISA检测方法的建立

Preparation of anti-largemouth bass ranavirus egg yolk antibody and establishment of indirect ELISA method

  • 摘要: 大口黑鲈 (Micropterus salmoides) 蛙虹彩病毒 (Largemouth bass ranavirus, LMBV) 是我国大口黑鲈养殖中的常发病原,主要引起大口黑鲈病毒性溃疡病,制约其养殖业的健康发展。为探究卵黄抗体在防控LMBV中的潜在作用,利用LMBV灭活疫苗免疫蛋鸡,制备了抗LMBV卵黄抗体;通过筛选捕获物浓度、包被条件和封闭条件,建立了抗LMBV卵黄抗体的间接酶联免疫吸附测定 (ELISA)检测方法;使用构建的方法,评估了LMBV灭活疫苗免疫蛋鸡后卵黄抗体的效价消减规律。结果表明,1‰ (φ) β-丙内酯4 ℃作用72 h可完全灭活LMBV。间接ELISA反应中,每孔包被105 TCID50灭活病毒,37 ℃孵育2 h后,使用5% (φ) 牛血清白蛋白37 ℃封闭2 h,可有效降低阴性对照组卵黄抗体的背景值;此外,所建立的检测方法与杂交醴弹状病毒卵黄抗体、未免疫的蛋黄提取物和空白细胞不存在交叉反应,特异性较高。LMBV灭活疫苗免疫蛋鸡约48 d可产生特异性卵黄抗体,免疫后58 d效价升高至峰值(1∶12 800),峰值水平可持续至免疫后128 d。所构建的LMBV卵黄抗体检测方法,可实时监测LMBV灭活疫苗免疫蛋鸡后的特异性卵黄抗体效价水平的变化规律,确定LMBV卵黄抗体的高效免疫程序及高免蛋收集持续期,为LMBV卵黄抗体产品的开发、应用及LMBV的防治提供了新思路。

     

    Abstract: Largemouth bass ranavirus (LMBV) is a major pathogenic agent in largemouth bass culture in China, which mainly causes viral canker disease and restricts the healthy development of largemouth bass culture. In order to explore the potential role of egg yolk antibody in the prevention and control of LMBV, we immunized the LMBV inactivated vaccine to laying hens and prepared the egg yolk immunoglobulin against LMBV (Anti-LMBV IgY). Besides, we established an indirect enzyme linked immuno sorbent assay (ELISA) method for detecting the titer of anti-LMBV IgY by screening trapping concentration, encapsulation condition and sealing condition. Then we evaluated the titer of anti-LMBV IgY at different time points post LMBV inactivated vaccine immunization by using the established method. The results show that 1‰ β-propanolactone could inactivate LMBV at 4 ℃ for 72 h completely. For indirect ELISA, being coated with 105 TCID50 inactivated virus, incubated at 37 ℃ for 2 h, and blocked with 5% bovine serum albumin at 37 ℃ for 2 h could reduce the background values of negative control effectively. In addition, no cross reaction had been detected between inactivate LMBV with other egg yolk extract or anti-LMBV IgY with cells, indicating that the indirect ELISA method had high specificity. Finally, we detected specific anti-LMBV IgY titer at 48 d post LMBV inactivated vaccine immunization; the titer increased to a peak of 1:12800 after 58 d post immunization, and last up to 128 d. To sum up, the indirect ELISA method in this study can real-time monitor the titer level of anti-LMBV IgY, determine the efficient immunization program and the duration of high-immunity egg collection, facilitate the development and application of LMBV egg yolk antibody products, and provide a potential solution for the prevention and treatment of LMBV.

     

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