人血清中福氏2a及宋内志贺菌O-特异性多糖抗体IgG含量间接ELISA定量检测方法的验证及应用  

Verification and application of indirect ELISA for quantitative detection of content of antibody IgG against Shigella flexneri 2a and Shigella sonnei 0-specific polysaccharide in human serum

作　　者：李红[1] 石刚 郭丽娜 陈琼[1] 付宏斌 王国东 胡小华 朱卫华 王斌[1] 叶强[1] LI Hong;SHI Gang;GUO Lina;CHEN Qiong;FU Hongbin;WANG Guodong;HU Xiaohua;ZHU Weihua;WANG Bin;YE Qiang(Key Laboratory of Method and Standardization for Quality Control of Biotechnical Products,National Institutes for Food and Drug Control,Beijing 102629,China;不详)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京102629 [2]北京智飞绿竹生物制药有限公司,北京100176

出　　处：《中国生物制品学杂志》2023年第6期714-718,723,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(民口)重大新药创制(2018ZX09101-001)。

摘　　要：目的验证人血清中福氏2a及宋内志贺菌O-特异性多糖(分别简称福氏2a多糖及宋内多糖)抗体IgG含量的ELISA定量检测方法,并用于福氏宋内痢疾双价疫苗免疫前后人血清样本的检测。方法将参考血清及质控血清按一定比例混合,制备成10份不同浓度的血清样本(编号为S1~S10),采用企业提供的间接ELISA定量检测方法检测样本,连续测定6 d,计算多糖抗体IgG回收率和变异系数(coefficient of variation,CV),并以回收率CV低于20%的相应参考血清含量作为定量限(limit of quantitation,LOQ)。采用相同检测方法连续6 d测定血清样本S10及质控血清,每天重复检测3次,计算CV;采用参考血清进行多糖竞争抑制ELISA试验,计算血清对多糖的抑制率。采用企业提供的方法检测参考血清,确定线性范围,获得曲线方程,计算相关系数(R^(2))。采用验证的方法检测1842份福氏宋内痢疾双价疫苗免疫前后的人血清样本。结果10份血清样本中福氏2a和宋内多糖抗体IgG回收率分别为73.21%~222.68%和83.67%~123.56%;试验间及试验内精密性CV均<30%;参考血清对100μg/mL福氏2a和宋内多糖的抑制率分别为85.95%和88.42%;福氏2a和宋内多糖抗体IgG检测的线性范围分别为0.05~0.125和0.025~0.125 EU/mL,R^(2)均≥0.99,LOQ分别为0.050和0.025 EU/mL。福氏宋内痢疾双价疫苗免疫后血清IgG含量大幅高于免疫前。结论企业提供的ELISA定量检测方法具有良好的准确性、精密性及特异性,可用于痢疾疫苗免疫血清的检测。Objective To verify an ELISA method for quantitative detection of antibody IgG against Shigella flexneri 2a and Shigella sonnei O-specific polysaccharide in human serum and use it to detect human serum samples before and after immunization with flexneri and sonnei dysentery bivalent vaccine.Methods The reference serum and quality control serum were mixed in a certain proportion to prepare 10 serum samples of different concentrations(numbered as S1~S10),which was determined continuously for 6 d by the indirect ELISA quantitative detection method provided by the enterprise,and the recovery rate and coefficient of variation(CV)were calculated.The reference serum content with a recovery rate of CV less than 20%was used as the limit of quantitation(LOQ).The serum sample S10 and reference serum were measured by the same method for 6 d,3 times of repeated test a day,and the CV was calculated;The inhibition rate of serum to polysaccharide was calculated by using the competitive inhibition ELISA with reference serum.The reference serum was detected by the method provided by the enterprise,the linear range was determined,the curve equation was obtained,and the correlation coefficient(R^(2))was calculated.The verified method was used to detect 1842 human serum samples before and after immunization with flexneri and sonnei dysentery bivalent vaccine.Results The recovery rates of Shigella flexneri 2a and Shigella sonnei polysaccharide antibody IgG in 10 serum samples were 73.21%~222.68%and 83.67%~123.56%,respectively;CV of precision verification between and within tests were all less than 30%;The inhibition rates of reference serum on 100μg/mL Shigella flexneri 2a and Shigella sonnei polysaccharide were 85.95%and 88.42%,respectively;The linear ranges of Shigella flexneri 2a and Shigella sonnei polysaccharide antibody IgG were 0.05~0.125 and 0.025~0.125 EU/mL,with the R^(2)not less than 0.99,and the LOQ were 0.050 and 0.025 EU/mL,respectively.The content of antibody IgG in serum after immunization with flexneri and sonnei dys

关 键 词：多糖 抗体IGG 酶联免疫吸附试验 志贺菌 痢疾疫苗 

分 类 号：R446.6[医药卫生—诊断学]