血清抗百日咳毒素中和抗体检测方法的建立及应用  

Development and application of a method for determination of serum neutralizing antibody against pertussis toxin

作　　者：张雨笑 谌志筠[1] 孙琳 何秋水[1,3] ZHANG Yu-xiao;CHEN Zhi-yun;SUN Lin;HE Qiu-shui(Department of Medical Microbiology,School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学基础医学院病原生物学系,北京100069 [2]首都医科大学附属北京儿童医院呼吸疾病研究室,北京100045 [3]芬兰图尔库大学医学微生物和免疫学系,图尔库20520

出　　处：《中国生物制品学杂志》2021年第1期93-96,共4页Chinese Journal of Biologicals

基　　金：国家重大传染病防治科技重大专项(2017ZX10202101-004-001);北京市自然科学基金-海淀原始创新联合基金前沿项目(19L2043)。

摘　　要：目的建立血清抗百日咳毒素(pertussis toxin,PT)中和抗体的检测方法,并进行应用。方法将不同浓度的PT蛋白(0.1~105.5 ng/mL)和105个/mL的CHO细胞悬浮液加入96孔板,均100μL/孔,37℃孵育24 h,显微镜下观察细胞簇集程度,确定中和试验中PT的最适终浓度。将待检血清(1∶4~1∶4 096)及最适终浓度的PT加入96孔板,均50μL/孔,37℃中和3 h;加入105个/mL的CHO细胞悬浮液,100μL/孔,继续培养24 h,镜下观察细胞簇集程度。用建立的方法检测8份健康人血清样品的中和抗体含量,重复检测4次,计算变异系数(CV)。采用PT-IgG抗体ELISA试剂盒及建立的方法分别检测109份20~39岁健康人群血清样本的PT-IgG抗体及PT中和抗体水平,并分析两者的分布情况及相关性。结果确定PT最适终浓度为0.84 ng/m L。8份血清样品重复检测4次的CV为0.00%~12.28%。109份健康人血清中和抗体效价分布范围为<4~256,中位数为32,几何平均效价(GMT)为27.12(95%CI:20.78~35.39);PT-IgG抗体分布范围为0.03~184.80 IU/mL,中位数为21.17 IU/mL,GMT为13.33 IU/mL(95%CI:9.95~17.87 IU/mL)。PT-IgG IU/mL抗体浓度与中和抗体效价呈明显相关性(r=0.77,P <0.01),部分血清存在抗体浓度高,中和抗体效价低的现象。结论成功建立了血清抗PT中和抗体的检测方法,该方法具有良好的重复性,可用于评价抗PT中和抗体的功能。Objective To develop and apply a method for determination of neutralizing antibody against pertussis toxin(PT).Methods PT at various concentrations(0.1 ~ 105.5 ng/mL)and CHO cells suspension at a concentration of 105 cells/m L were added onto a 96-well microplate,100 μL per well,incubated at 37 ℃ for 24 h and observed for cell cluster by microscopy to optimize the final PT concentration in neutralization test.The serum samples at dilutions of 1∶4~1∶4 096 and PT at the optimal final concentration were added onto a 96-well microplate,50 μL per well,and incubated at 37 ℃ for 3 h for neutralization,then added with CHO cell suspension at a concentration of 105 cells/mL,100 μL per well,further incubated for 24 h and observed for cell cluster by microscopy.The neutralizing antibody contents in eight healthy human serum samples were determined by the developed method for 4 times,and the CVs of determination results were calculated.The IgG and neutralizing antibody levels against PT in 109 serum samples from healthy individuals at ages of 20~39 years were determined by ELISA kit for PT-IgG and the developed method respectively,and the distribution of antibodies and relationship between the determination results was analyzed.Results The optimal final concentration of PT was 0.84 ng/mL.The CVs of determination results of eight serum samples in 4 repeat tests were 0 ~12.28%.The neutralizing antibody titers in 109 healthy human serum samples ranged from less than 4 to 256,with a median of 32 and a geometric mean of 27.12(95% CI: 20.78~35.39).However,the PT-IgG titers ranged from 0.03 to184.80 IU/mL,with a median of 21.17 IU/mL and a geometric mean of 13.33 IU/mL(95% CI: 9.95~17.87 IU/mL).The PT-IgG concentration was significantly related to the neutralizing antibody titer(r = 0.77,P < 0.01),while high PT-IgG and low neutralizing antibody titer were observed in partial serum samples.Conclusion A method for determination of serum neutralizing antibody against PT was successfully developed,which showed good reproducib

关 键 词：百日咳 百日咳毒素 CHO细胞 IGG抗体 中和抗体 

分 类 号：R378.42[医药卫生—病原生物学]