鸭疫里氏杆菌多种血清型间接ELISA检测方法的建立

【目的】鸭疫里氏杆菌(Riemerella anatipestifer，RA)是一种重要的禽病病原，分为21个血清型。但一直缺乏一种针对多种血清型广泛适用的抗体检测方法。前期的研究表明，外膜蛋白A (Outer membrane protein A，OmpA)广泛存在于多种血清型的RA菌株中，是一种重要的免疫原性蛋白，并且其基因序列在RA血清型之间具有高度的保守性，提示其可以作为RA感染血清抗体检测的靶点分子。以重组蛋白OmpA建立间接酶联免疫吸附试验检测RA的抗体。【方法】通过诱导表达条件的摸索及蛋白纯化，获得适用于ELISA包被的重组OmpA抗原。通过Western-blot证明重组蛋白OmpA是否与RA多种血清型发生免疫学反应。进行方阵试验以确定ELISA抗原的最佳包被浓度、被检测血清的反应浓度。重复性、特异性和敏感性试验检查该方法的实用性。【结果】实验证实加入1%乙醇的诱导培养基有利于重组蛋白的可溶性表达。Western-blot结果表明，重组蛋白OmpA可以与1、2、6、10、11、13、14和17型多种RA主要流行血清型有良好的免疫反应性。经方阵试验确定抗原的最佳包被浓度为8 mg/L，待检血清的最佳稀释度为1:160。所建立检测方法具有良好的重复性、特异性和敏感性。【结论】实验建立的鸭疫里氏杆菌多种血清型间接ELISA检测方法可以用于免疫后抗体消长以及感染性抗体的检测。

Development of indirect ELISA for detecting antibodies against multi-serotypes of Riemerella anatipestife

Objective] Riemerella anatipestifer (RA) is an important poultry pathogen. Considering the twenty-one serotypes, it still lacked a method for detecting multi-serotypes antibodies. Previous studies showed that the Outer membrane protein A (OmpA), widely existing in multi-serotypes of RA strains, is a protein with immunogenicity. Its encoding gene is highly conserved among the genomes of RA of different serotypes, which indicates that OmpA can be used as a target molecule for serum antibody detection of RA infection. An indirect ELISA method to detect the antibody caused by RA infection was developed with the recombinant OmpA as the coating antigen. Methods] The recombinant OmpA was purified after optimizing the prokaryotic expression conditions, and its immuno reactivity was tested by Western-blot with sera obtained from ducks infected by different serotypes of RA. Furthermore, the optimum testing conditions of ELISA were determined by phalanx test. Reproducibility, specificity and sensitivity tests were performed to estimate the practicability. Results] The recombinant protein would be more soluble by adding 1% of ethanol in the medium. As analysed by Western-blot, the purified protein is immunogenic among 1, 2, 6, 10, 11, 13, 14, 17 serotypes of RA. Phalanx test results showed the optimal concentration of coated antigen is 8 mg/L, and the optimal dilution degree of serum is 1:160. Also, the developed assay was proved to be quite repetitive, specific and sensitive. Conclusion] An indirect ELISA method for detecting the infection of multi-serotypes of Riemerella anatipestifer was established in this study and could be used for evaluating the immune effects of RA vaccine and for diagnosing RA infections with different serotypes of RA.