犬种布鲁氏菌标准阳性血清的研制与应用

Preparation and application of standard positive serum of Brucella canis

摘要:

【背景】犬种布鲁氏菌(Brucella canis)是布鲁氏菌6个经典种之一，是严重危害犬只健康的人畜共患病原。【目的】制备犬种布鲁氏菌阳性血清，为犬布病诊断试剂的开发与应用提供标准物质。【方法】用犬种布鲁氏菌灭活菌液(约1.5×10¹⁰ CFU/mL)多次免疫实验用比格犬，制备获得犬种布鲁氏菌病阳性血清，采用试管凝集试验方法，以欧盟犬种布鲁氏菌标准阳性血清溯源并标定血清效价。再以本研究制备的标准阳性血清为标准物质，质控本实验室研制的犬种布鲁氏菌虎红平板凝集试验抗原和犬种布鲁氏菌间接ELISA抗体检测试剂盒的检测灵敏度，并用上述2种方法同时检测北京地区宠物犬血清中犬种布鲁氏菌抗体，比较各方法与补体结合试验结果的符合率。【结果】经试管凝集试验测定，制备获得的犬种布鲁氏菌阳性血清效价为2 400 IU/mL，采用犬阴性血清将阳性血清效价稀释至1 000 IU/mL，分装冻干，即为本研究制备的犬种布鲁氏菌标准阳性血清。将该标准阳性血清作为质控品，检测本实验室研制的犬种布鲁氏菌虎红平板凝集试验抗原和犬种布鲁氏菌间接ELISA抗体检测试剂盒的检测灵敏度均为20 IU/mL。用犬种布鲁氏菌虎红平板凝集试验抗原与犬种布鲁氏菌间接ELISA抗体检测试剂盒对北京地区宠物犬血清临床样品进行检测，与金标准的补体结合试验进行符合率比对，虎红平板凝集试验和补体结合试验的阳性率均为6.25%，间接ELISA抗体检测试剂盒阳性率为5.97%，虎红平板凝集试验、间接ELISA抗体检测试剂盒与补体结合试验的符合率分别为94.31%和94.60%。【结论】本研究制备的犬种布鲁氏菌标准阳性血清为犬种布鲁氏菌诊断试剂的研发提供了必要的质控样品。

关键词:犬种布鲁氏菌;阳性血清;效价标定;血清凝集试验;间接ELISA

Abstract:

[Background] Brucella canis, one of the six representative species of Brucella, is a zoonotic pathogen that poses a serious threat to the health of dogs. [Objective] To prepare the B. canis positive serum, providing the reference material for the development and application of diagnostic reagents for canine brucellosis. [Methods] Beagle dogs were immunized multiple times with the inactivated bacterial suspension (approximately 1.5×10¹⁰ CFU/mL) to obtain the positive serum of B. canis. By conducting the serum agglutination test, we calibrated the serum titer with the international standard serum provided by the European Union. We then used the standard positive serum prepared in this study as the reference material for quality control of the detection sensitivity of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory. Furthermore, we used the above two methods to detect the B. canis antibody in the serum samples of pet dogs in Beijing and analyzed the coincidence rates of results between the two methods and the complement fixation test. [Results] The results of the serum agglutination test showed that the titer of B. canis positive serum was 2 400 IU/mL. The serum was diluted with the negative dog serum to reach a titer of 1 000 IU/mL, subpackaged, and freeze-dried to give the standard positive serum of B. canis. With the standard positive serum for quality control, the detection sensitivities of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory were both 20 IU/mL. The positive rates obtained by both the B. canis antigen for rose bengal test and the complement fixation test was 6.25% and that obtained by the B. canis indirect ELISA antibody detection kit was 5.97%. The coincidence rates of results between the two detection methods and the complement fixation test were 94.31% and 94.60%, respectively. [Conclusion] The standard positive serum of B. canis that we prepared in this study provides a quality control sample for the development of diagnostic reagents for B. canis.

Key words:Brucella canis; positive serum;calibration of titer;serum agglutination test;indirect ELISA