抗类鼻疽伯克霍尔德菌多抗血清的制备及评价

摘要

目的制备抗类鼻疽伯克霍尔德菌(Burkholderia pseudoamllei,其抗原简写为BP)多抗血清,评价不同处理方式对抗原免疫原性的影响.方法 采用超声破碎、甲醛灭活、热灭活3种方式处理细菌抗原接种新西兰兔和BALB/C小鼠,检测不同抗原免疫动物抗血清的ELISA效价及凝集试验效价.结果 (1)ELISA方案最佳条件:二抗稀释度为1/8 000,抗原包被浓度为4 μg/mL或8 μg/mL;凝集反应最佳条件:细菌浓度为6×109 CFU/mL、凝集温度为37 ℃、观察时刻为4 h.(2)超声破碎抗原(U-BP)免疫新西兰兔和小鼠的抗血清ELISA效价高达1/64 000,最高血清凝集效价分别为1/32和1/64,甲醛灭活抗原(F-BP)抗血清ELISA效价高达1/16 000,凝集效价为1/128和1/64;热灭活抗原(H-BP)抗血清ELISA效价达1/8 000,凝集效价为1/16和1/64.(3)F-BP和H-BP具有较强的免疫原性,U-BP在小鼠中的免疫原性较弱(P<0.05).结论 建立了抗类鼻疽伯克霍尔德杆菌多克隆抗体的检测方法,制备了高效价的抗类鼻疽伯克霍尔德杆菌多抗血清;甲醛灭活和热灭活细菌具有较好的免疫原性,为下一步类鼻疽伯克霍尔德杆菌的致病机制研究和疫苗研发打下了基础.%Objective To prepare multi-antibody of Burkholderia pseudomallei(B. Pseudomallei) and evaluate the immunogenici-ty of antigens under different inactivation treatments. Methods Bacterial antigen was inactivated by three different methods, including ultrasound cracking, formaldehyde inactivation and heat inactivation, and inoculated into New-Zealand rabbits and BALB/C mice, followed by quality and specificity evaluation by ELISA and agglutination test. Results 1. The optimized condition of ELISA was 1:8000 of the dilution of secondary antibodies, 4 or 8fig/mL of the concentration of coating antigen. The optimized condition of agglutination test was 6×109 cfu/mL of bacteria density, 37 ℃ of reaction temperature, and 4 h for observation. 2. ELISA titer of serum from rabbits and mice inoculated by ultrasound-cracked bacteria antigen (U-BP) was up to 1/64 000, with top agglutination test titers of 1/32 and 1/64 respectively. ELISA titer of serum from rabbits and mice, inoculated by formaldehyde-inactivated bacteria antigen (F-BP) was 1/16 000, with top agglutination test titers of 1/128 and 1/64 respectively. For heat-inactivated bacteria antigen (H-BP) ,ELISA titer was up to 1/8 000, with agglutination test titers of 1/16 and 1/64 respectively. 3. The immunoge-nicity of H-BP and F-BP antigen was strong, but that of U-BP antigen was weak in mice (P<0. 05). Conclusion Detection method of polyclone antibody against B. Pseudomallei was established and polyclonal antiserum of B. Pseudomallei with high titer was prepared. H-BP and F-BP antigen might have stronger immunogenicity, which set substantial basis for future study on B. Pseudomallei.