A、C、Y、W135群脑膜炎奈瑟菌抗体血清体外杀菌试验方法优化  

Optimization of serum bactericidal assay for serogroups A C Y W135 from Neisseria meningitidis

作　　者：段静留 温晔 彭少丹 朱涛[1] DUAN Jing-liu;WEN Ye;PENG Shao-dan;ZHU Tao(College of Bioengineering of Tianjin University of Science ＆ Technology, Tianjin 300457, Chin)

机构地区：[1]天津科技大学生物工程学院,天津300457

出　　处：《微生物学免疫学进展》2018年第3期18-26,共9页Progress In Microbiology and Immunology

基　　金：国家"重大新药创制"科技重大专项资助项目(2015ZX09102009)

摘　　要：目的进一步优化脑膜炎奈瑟菌的血清抗体特异性体外杀菌试验,建立更加标准的A、C、W135、Y群脑膜炎奈瑟菌血清杀菌试验(serum bactericidal assay,SBA)方法,并验证其可行性及稳定性。方法 (1)用经典溶血方法测定补体活性,并根据非特异性杀菌率(non-specific killing rates,NSK)筛选出最佳试验补体;(2)对影响最终菌落数的关键因素,如靶菌起始浓度、培养基、染色剂浓度等进行优化;(3)根据质控血清的测定结果,确定补体最佳稀释比例和孵育时间;(4)对优化后的方法进行准确性、线性和精密度验证。结果补体选用Pel-freez-11536,溶血活性约为400 U/m L,除C血清群靶菌孵育时间需在45 min以内,其余血清群靶菌NSK均≤25%;对数期A、C、W135、Y血清群脑膜炎奈瑟菌做靶菌的最佳起始浓度为A600 nm的菌液分别稀释10~5、10~5×2、10~5×4、10~5×4倍;4种血清群脑膜炎奈瑟菌在BHI+0.5%血平板培养基上生长正常,且培养基颜色较浅,染色效果较明显;4种血清群靶菌的染色剂TTC最佳添加量均为0.001%;补体最佳用量为1∶3稀释,活性单位约133 U/m L;最佳孵育时间除C血清群为45 min外,其余血清群均以60 min为宜;优化后的方法测定质控血清的稀释倍数与对应的SBA滴度呈显著负相关,斜率在-1.1^-0.9之间,Pearson相关系数在-1.0^-0.9之间,P<0.001,多次试验的CV均<15%。结论对传统的SBA方法进行了优化,建立了更具有可比性、重复性较好的简单、可行、稳定的SBA方法。Objective To optimize the serum bactericidal assay（ SBA）,and development a more standardized SBA on serogroups A,C,Y and W135 from Neisseria meningitidis,and to validate its feasibility and stability. Methods（1） Determination of complement level by the classical hemolysis method and calculation of the non-specific killing rates of the target bacteria,to select the best tested complement.（2） Optimization of the critical factors,such as initial concentration of target bacteria,medium and colorant concentration.（3） According to the SBA titer in the quality control serum CDC1992,the best usage of complement and incubation time were selected.（4） The optimized method was verified for accuracy,linearity and precision. Results The optimal complement was Pel-freez-11536 with an activity about 400 U/m L,while it＇s nonspecific killing rates was ≤25% except the target bacteria of group C,the non-specific killing rates of group C could also be accepted when incubated in 45 minutes; The optimal dilution of Neisseria meningitidis of A,C,W135,Y in logarithmic phase by A600 nmmeasurement were as follows： 10^5,2×10^5,4×10^5 and 4×10^5,respectively. Four serogroups Neisseria meningitidis grew well on BHI ＋ 0. 5% blood medium with a light color. The optimal concentration of colorant,triphenyltetrazolium chloride was 0.001%. The best dilution of complement was 1 ∶3,about 133 U/m L. The best incubation time for the serogroups A,W135 and Y were 60 minutes,except for group C 45 minutes. The determined dilution for the quality control serum was significantly negatively correlated with the corresponding SBA titer by the optimized method,and the slope was between1.1 - 0.9,Pearson＇s r was between 1.0 - 0.9,P0.001; and multiple test CV results were 15%. Conclusion The simple,feasible and stable SBA was developed with advantageous on more comparative and reproducible by the optimized conventional SBA.

关 键 词：抗体血清体外杀菌试验 脑膜炎奈瑟菌 补体 质控血清 

分 类 号：R378.15[医药卫生—病原生物学] R392-33[医药卫生—基础医学]