人肺炎球菌参考血清09CS的制备及其中13个肺炎球菌结合疫苗血清型抗体的IgG含量和调理吞噬滴度

Preparation of human pneumococcal reference serum 09CS and calibration of Ig G content and opsonophagocytic titer of 13-valent pneumococcal conjugate vaccine serotypes

目的制备人肺炎球菌参考血清,对其中13价肺炎球菌结合疫苗(pneumococcal conjugate vaccine,PCV)血清型(1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F型)的抗荚膜多糖抗体Ig G含量和调理吞噬滴度进行定值。方法用23价肺炎荚膜多糖疫苗免疫20名健康成人,采集免疫后血浆,进行混合、去纤维蛋白原、过滤、分装、冻干,制备人肺炎球菌参考血清09CS。在WHO细菌性呼吸道病原参考实验室(University of Alabama at Birmingham,UAB)和兰州生物制品研究所有限责任公司(Lanzhou Institute of Biological Products,LIBP)实验室,采用WHO推荐的Pn PS ELISA法,以国际标准血清89SF为标准,对其中13价PCV血清型抗荚膜多糖抗体Ig G含量定值;以临时定值的09CS为标准,检测12份WHO校正血清、16份LIBP质控血清和89SF,对定值的准确性进一步验证。同时以调理吞噬杀菌试验(opsonophagocytic killing assay,OPA)测定针对13个血清型的调理吞噬滴度。结果制备了5 000支(0.5 ml/支)冻干人肺炎球菌参考血清09CS,残留水分含量2.3%。确定了09CS中13价PCV血清型抗荚膜多糖抗体Ig G含量的临时定值;以09CS为标准检测的12份WHO校正血清和16份LIBP质控血清的13价PCV血清型抗荚膜多糖抗体结果,与以89SF为标准检测的结果,均具有良好的直线相关关系(r≈1.00,P<0.05);以09CS为标准检测的89SF的13价PCV血清型抗荚膜多糖抗体的新值与其原定值比较,各血清型的误差百分率的绝对值均<20%。确立了09CS中13价PCV血清型调理吞噬滴度。结论 LIBP成功制备了人肺炎球菌参考血清09CS,完成了对其中的13价PCV血清型抗荚膜多糖抗体Ig G含量的准确定值,并确定了调理吞噬滴度。

Objective To prepare human pneumoccocal reference serum and calibrate its Ig G content and opsoniphagocytic titer of 13-valent pneumococcal conjugate vaccine（PCV）serotypes（1,3,4,5,6A,6B,7F,9V,14,18 C,19A,19 F and 23F）. Methods Twenty healthy adults were immunized with one dose of 23-valent pneumococcal polysaccharide vaccine,of whom the plasma were collected,pooled,defibrinated,filtered,aliquoted and lyophilized to p repare human pneumococcal reference serum 09 CS. The Ig G contents of 09 CS against capsular polysaccharide of 13-valent PCV serotypes were calibrated by WHO recommended standardized Pn PS ELISA using international reference serum 89 SF as a standard in WHO Reference Laboratory of Bacterial Respiratory Pathogens（University of Alabama at Birmingham,UAB）and a laboratory in Lanzhou Institute of Biological Products Co.,Ltd.（LIBP）. Provisional Ig G contents of 09 CS were further validated by testing a panel of 12 WHO calibration serum samples and a panel of 16 LIBP quality controls using the temporarily calibrated 09 CS as a standard. Meanwhile,opsoniphagocytic titers against 13-valent PCV serotypes were measured by opsonophagocytic killing assay（OPA）. Results A total of 5 000 containers（0. 5 ml for each） of freezedried human pnemoccocal reference serum 09 CS were prepared,of which the residual moisture content was 2. 3%. The Ig G contents of 09 CS against capsular polysaccharide of 13-valent PCV serotypes were calibrated temporarily. The determination results of antibody against capsular polysaccharide of 13-valent PCV serotypes in 12 WHO calibration serum samples and a panel of 16 LIBP quality controls using 09 CS as a standard were linearily correlated with those using 89 SF as a standard（r ≈ 1. 00,P〈 0. 05）. However,the antibody contents against capsular polysaccharide of 13-valent PCV serotypes determined using 89 SF as a standard were matched well with their previously assigned values,with difference of less than 20%. The opsoniphagocytic titers of 13-valent PCV serotypes in 09 CS were established. Conclusion Human pneumococcal reference serum 09 CS was successfully prepared by LIBP by adequate calibration of Ig G content of capsular polysaccharide of 13-valent PCV serotypes as well as opsoniphagocytic titers.