血清抗百日咳毒素中和抗体检测方法的建立及应用

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100 μL/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:...

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

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 reproducibility and might be used for the evalution of function of the neutralizing antibody.