几种戊型肝炎试剂的比较及IgG抗体定量方法的建立

目的 利用多种抗戊型肝炎病毒(HEV)IgM、IgG检测试剂对戊型肝炎恢复期血清进行确认,建立戊型肝炎病毒ISG抗体的定量检测方法,初步应用于戊型肝炎疫苗接种后抗体效价检测.方法 应用多种抗-HEV IgM、IgG试剂对江苏省确诊的戊型肝炎病人发病后6个月以上的血清样品进行检测;抗-HEV IgG阳性样本应用ORF2 C端抗原和ORF3抗原Western blot进行确认,用WHO抗-HEV Ig标准品标定确认阳性的混合血清抗-HEV IgG的含量,制备抗-HEV IgG定量检测的线性标准品,建立戊型肝炎疫茼免疫后抗体定量检测方法.结果 42份戊型肝炎恢复期血清采用G、K、MP、万泰4种抗-HEV IgG试剂检测的阳性率分别为71.4%、78.6%、92.9%、100%,G试剂阳性率低于MP(X2=5.19,P<0.05)和万泰试剂(χ~2=11.76,P<0.01),K试剂阳性率明显低于万泰试剂(χ~2=7.96,P<0.01);MP、G、X、万泰、K试剂抗-HEV IgM试剂的阳性率分别为21.4%、7.1%、21.4%、64.3%、78.6%;万泰和K试剂的阳性率均明显高于MP(χ~2=15.75,P<0.01;X2=27.43,P<0.01).Western blot确认试验分别有30和18份血清与ORF2、ORF3抗原有阳性反应.13份血清混合为HEV-D01,其抗-HEV IgG浓度经标定为57.94 U/ml.制备了浓度范围为0.077～0.877 U/ml的7个1.5倍系列稀释的定最线性标准品,用于戊肝疫苗临床试验,试验过程中对高、中、低浓度质控品的定量值均在均数±2s范围内,变异系数(cv)分别为16%、16%、12%.结论 不同抗-HEV IgM和IgG试剂之间的质量存在较大差别,建立了抗-HEV IsG定量检测标准品,适用于戊肝疫苗的临床血清抗体IgG定量检测.

Evaluation of the diagnostic kits for hepatitis E and establishment of a quantification method for de-tecting anti-HEY IgG

Objective To evaluate anti-HEV IgG and IgM diagnostic kits with sera from convalescent hepatitis E patients and to establish the quantification method of detecting anti-HEV lgG.Methods Detect 42 convalescent serum samples of over 6 months after onset of hepatitis E patients from Jiangsu province with anti-HEV IgM and IgG diagnostic kits. Select and mix the anti-HEV IgG positive sera which were confirmed by Western blot with ORF2 and ORF3 antigen. The mixed serum was calibrated with a WHO anti-HEV Ig standard. A series quantitative linear standard was made for quantitative detection of anti-HEV IgG in hepatitis E vaccine clinical trials phase Ⅲ. Results The positive rates of the anti-HEV IgG di-agnose kits of G, K, MP, Wantai were 71.4%, 78.6%, 92.9% and 100% respectively. The positive rates of G was lower than that of MP (χ~2 = 5.19, P<0.05) and obviously lower than Wantai (χ~2 = 11.76,P<0.01). The positive rates of K was also obviously lower than that of Wantai (χ~2 =7.96, P <0.01).The positive rates of the anti-HEV IgM diagnose kits of MP, G, X, Wantai, K were 21.4%, 7.1%,21.4%, 64.3%, 78.6% respectively. The positive rate of both K and Wantai were obviously higher than that of MP(χ~2 = 15.75 ,P<0.01 ; X2 = 27.43 ,P< 0.01). With the Western blot confirmation test, 30 and 18 sera were reactive to ORF2 and ORF3 antigen separately. The anti-HEV IgG concentration of HEV-D01 mixed by 13 samples was 57.94 U/ml by the calibration. Prepare seven 1.5-fold dilution series of quantita-tive linear standard for HEV vaccine clinical trials phase Ⅲ, concentration range from 0.077 to 0.877 U/ml. The quantitive values of high, medium and low concentrations quality control samples lay in the range of average ± 2s, and the CV of quantitative values were 16%, 16%, 12% respectively. Conclusion The quality of different anti-HEY IgM and IgG diagnose kits were different. This study had set up a set of anti-HEV IgG linear quantitative standard, which fit for detecting anti-HEV IgG antibodies quantitatively in HEVvaccine clinical trial phase Ⅲ.