C群脑膜炎球菌家兔免疫血清的制备及其群特异性和相关方法专属性分析  

Prearation and group specificity of rabbit antisera against group C Neisseria meningococcus and their speciality in immunological methods

作　　者：刘方蕾[1] 吴兵[1] 侯亚莉[1] 杨英英[1] 王晶[1] 于旭博 范锋锋[1] 孔素娟[1] 袁菲[1] 赵志强[1] 谢贵林[1] 

机构地区：[1]兰州生物制品研究所有限责任公司,甘肃兰州730046

出　　处：《中国生物制品学杂志》2014年第11期1421-1425,1432,共6页Chinese Journal of Biologicals

基　　金：国家"重大新药创制"专项(2013ZX09402-302-215);甘肃省科技重大专项(0801NKDA003);甘肃省"十二五陇药产业发展"专项

摘　　要：目的制备C群脑膜炎球菌家兔免疫血清,并进行群特异性和相关方法专属性分析。方法用制备的C群脑膜炎球菌菌液免疫青紫兰家兔,经耳缘静脉注射8×109个/ml菌液,0.5~1.0 ml/只,每周1、3、5免疫,共免疫4周,于第6周开始加强免疫,每周2次,连续3周,于末次免疫1周后,经颈动脉采血,分离血清,采用免疫双扩散法检测血清效价。采用ELISA、Western blot及火箭免疫电泳法分析其群特异性和相应方法专属性。结果建立了家兔免疫血清制备方法,制备的3份（1、2、3）免疫血清相应多糖抗体效价分别为1︰8、1︰8和1︰16。自制免疫血清1、3在使用工作浓度下,符合免疫学质量控制ELISA方法的特异性和专属性要求。自制免疫血清3可作为C群脑膜炎球菌结合物Western blot检测的Anti-PS血清,在该检测灵敏度下可完全达到与破伤风类毒素（TT）无交叉反应;血清1、2的轻微交叉反应可通过相应抗原预吸收血清消除,从而达到结合物Western blot检测的Anti-PS使用要求。自制3份免疫血清均可用于火箭免疫电泳分析。结论自制的家兔免疫血清可替代商品诊断血清,并达到了ELISA、Western blot及火箭免疫电泳法群特异性和相应方法专属性要求,可用于对C群脑膜炎球菌结合物制备过程的多糖抗原性分析及结合疫苗质量控制。Objective To prepare rabbit antisera against group C Neisseria meningococcus and analyze their group specificity as well as the speciality of relevant methods. Methods The Chinchilla rabbits were immunized through ear marginal vein with prepared group C N. meningococcus liquid at a concentration of 8 × 109 bacteria / ml,0. 5 ~ 1. 0 ml for each,on Monday,Wednesday and Friday every week for 4 weeks,while boosted starting from week 6 for 3 weeks,twice a week. Serum samples were collected one week after the last immunization and determined for titer by double immunodiffusion test. The group specificity and the speciality of relevant methods were analyzed by ELISA,Western blot and rocket immunoelectrophoresis（RIE）. Results The method for preparation of rabbit antisera was developed. The polysaccharide antibody titers of prepared samples 1,2 and 3 were 1 ∶ 8,1 ∶ 8 and 1 ∶ 16 respectively. The prepared serum samples 1 and 3,at working concentrations,met the requirement for specificity of ELISA. However,the serum sample 3 could be used as the Anti-PS serum for determination of group C meningococcal conjugate by Western blot,while no cross reaction with tetanus toxoid（TT） was observed at the detection sensitivity. The mild cross reactions of serum samples 1 and 2 were eliminated by pre-absorption with the corresponding antigen so as to meet the requirements for Anti-PS for determination of group C meningococcal conjugate by Western blot. All the three prepared serum samples were suitable for rocket immunoelectrophoresis（IE）. Conclusion Self-produced antisera,with comparable titer with commercial one,met the requirements for group specificity of ELISA,Western blot and RIE as well as the speciality of the relevant methods,which could be used for analysis of antigenicity of polysaccharide in quality control of vaccine and during production of group C meningococcal conjugate.

关 键 词：脑膜炎球菌 免疫血清 特异性 专属性 

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