鸭疫里默氏杆菌血清2型ELISA抗体检测方法的建立和应用

Establishment and application of an ELISA method for detecting antibodies of Riemerella anatipestifer serotype 2

摘要:

【背景】 鸭疫里默氏杆菌(Riemerella anatipestifer, RA)感染对家禽养殖业造成了巨大的经济损失。RA血清型众多且相互之间没有交叉保护，因此，建立一种快速检测方法鉴定流行菌株的血清型，并制备血清型特异性的疫苗进行免疫对于防治该病具有重要的意义。【目的】 建立一种血清2型特异性的鸭疫里默氏杆菌ELISA抗体检测方法，能够有效地检测2型RA感染和免疫鸭血清中的特异性抗体。【方法】 通过热酚水法(hot phenol-water method)提取鸭疫里默氏杆菌血清2型菌株脂多糖(lipopolysaccharide, LPS)，包被ELISA板。通过优化抗原包被条件、待检血清/兔抗鸭酶标抗体的稀释倍数和孵育条件，以及底物的显色时间等反应条件，建立一种检测血清2型RA抗体的间接ELISA方法。【结果】 优化反应条件后确定的最佳方案为：以5 μg/mL LPS作为包被抗原、100 μL/孔PBS作为包被液加入酶标板，4 ℃包被12 h；以5%脱脂奶粉作为封闭液、200 μL/孔，37 ℃封闭2 h；待测血清稀释度为1:400、孵育时间为1 h；兔抗鸭酶标二抗稀释度为1:10 000，孵育时间为1 h；显色时间为5 min。该反应条件下的P/N平均值最大。通过检测80份健康非免疫鸭血清，按平均值+3个标准差(standard deviation, SD)确立阳性判断标准为OD₄₅₀≥0.151。特异性试验结果表明：该方法检测RA血清2型阳性血清为阳性结果；检测RA血清1型、6型、7型和10型鸭阳性血清、禽致病性大肠杆菌阳性鸭血清、禽巴氏杆菌阳性鸭血清、禽沙门菌阳性鸭血清、呼肠孤病毒阳性鸭血清和雏鸭肝炎阳性鸭血清均呈阴性结果。敏感性试验结果表明：对阳性鸭血清的检测限度为 > 50 000倍稀释。重复性试验结果表明：该检测方法的批间和批内变异率均小于10%。该检测方法对血清2型RA感染7 d后鸭血清中的抗体检测结果为100%阳性；对血清2型RA灭活疫苗免疫后的抗体检测结果表明，免疫后14 d的血清抗体阳性率为80%、免疫后28 d的血清抗体阳性率为100%。【结论】 该方法的建立填补了鸭疫里默氏杆菌血清2型特异性抗体检测方法的空白，为RA的流行病学调查和疫苗研发提供了有效的数据支持与科学依据，具有重要的理论和应用价值。

Abstract:

[Background] Riemerella anatipestifer (RA) causes enormous losses to the poultry industry due to its multiple serotypes and lack of effective cross protection. Therefore, establishing a rapid detection method to identify the serotypes of prevalent RA strains and preparing serotype-specific vaccines for immunization are of great significance for preventing RA infection. [Objective] To establish an ELISA method for detecting antibodies in the sera of ducks infected with RA serotype 2 strains or vaccinated with the serotype 2 vaccine. [Methods] The lipopolysaccharide (LPS) of RA serotype 2 strains was extracted by the hot phenol-water method and then used for coating the ELISA plate. By optimizing the antigen coating conditions, dilution ratio and incubation conditions of the serum sample and the HRP-conjugated anti-duck secondary antibodies, and substrate development time, we established an indirect ELISA method for detecting antibodies of RA serotype 2. [Results] The optimal reaction conditions were determined as follows: 5 μg/mL LPS as the coating antigen, PBS as the coating solution, 100 μL/well, coating ELISA plates at 4 ℃ for 12 h; 5% skimmed milk powder as the blocking solution, 200 μL/well, blocking at 37 ℃ for 2 h; serum sample dilution of 1:400 and incubation for 1 h; Rabbit Anti-Duck IgG (H & L)-HRP (secondary antibody) dilution of 1:10 000 and incubation for 1 h; color development time of 5 min. The highest P/N value was obtained under the optimal reaction conditions. By testing 80 serum samples from healthy non-immunized ducks, we established the positive judgment criterion as OD₄₅₀≥0.151 based on the mean (X)+3 times of standard deviation (SD). The specificity test of the established method showed positive results for the RA serotype 2-positive serum but negative results for the RA serotype 1, 6, 7, and 10-positive duck sera, avian pathogenic Escherichia coli-positive duck sera, avian Pasteurella-positive duck sera, avian Salmonella-positive duck sera, reovirus-positive duck sera, and duck hepatitis-positive duck sera. The sensitivity test showed that the limit of detection for positive duck sera was dilution factor > 50 000. The repeatability test showed that the inter- and intra-batch coefficients of variation of the method were less than 10%. The established ELISA method showed a 100% positive result for the serum samples from RA serotype 2-infected ducks on day seven post infection. It generated a 80% positive result for antibodies on day 14 and a 100% positive result on day 28 post vaccination with the inactivated vaccine against RA serotype 2. [Conclusion] The established method fills the gap in detection of RA serotype 2 antibodies and provides effective data support and a scientific basis for the epidemiological investigations and vaccine development of RA, showing both theoretical and practical values.