乙型肝炎病毒表面抗体中和验证方法的建立与初步应用

Establishment and preliminary application of neutralization verification method for antibody to hepatitis B virus surface antigen

目的建立一种乙型肝炎病毒表面抗体(抗-HBs)中和验证方法并初步应用。方法采用双抗原夹心时间分辨荧光免疫分析法(TRFIA)原理建立了抗-HBs中和验证方法,固相包被乙型肝炎病毒表面抗原[HBsAg(ad/ay)]作为捕获抗原,N1-(P-异硫氰基苄基)-二乙三胺四乙酸铕钠标记HBsAg(ad/ay)作为检测抗原,采用HBsAg阳性混合样本作为中和HBsAg。自建的抗-HBs中和验证方法采用检测国家参考品、国家标准品和血清盘样本进行性能评估。丰华公司抗-HBs TRFIA试剂与Abbott公司抗-HBs化学发光微粒子免疫法(CMIA)试剂平行对比检测了171例临床样本,对2种试剂抗-HBs不符合样本以及同时阳性样本,采用自建抗-HBs中和验证方法进行中和实验。结果自建抗-HBs中和验证方法的固相包被HBsAg(ad/ay)的工作浓度为5.0μg/mL,标记物母液工作稀释比例为1∶1500,中和HBsAg的工作稀释比例为1∶1000。自建中和验证方法检测20份国家阴性参考品均为无反应性;检测国家精密度参考品的CV为4.10%(n=10);最低检出量不高于10.00 IU/L;检测国家标准品9.40~160.00 IU/L,实测值与理论值的相对偏倚在-4.08%~4.36%内,线性相关系数能达0.99以上。检测20份血清盘阴性样本均为无反应性;检测20份血清盘阳性样本均有反应性;检测血清盘灵敏度样本L1~L5,L1为有反应性;检测血清盘精密度样本的CV为5.56%(n=10)。抗-HBs TRFIA试剂与CMIA试剂对比检测了171例临床样本,其中9份样本CMIA试剂阴性而TRFIA试剂阳性的样本,有5份CMIA试剂抗-HBs在8.00~10.00 IU/L间,中和验证实验为有反应性;3份CMIA试剂阳性而TRFIA试剂阴性的样本中,中和验证实验为有反应性。69份TRFIA试剂与CMIA试剂抗-HBs同时阳性样本,中和验证实验均为有反应性。结论自建抗-HBs中和验证方法性能良好。建议各临床实验室尽量不要采用不同的检测系统同时进行抗-HBs定量检测并进行直接比较。

Objective To establish a neutralization verification method for antibody to hepatitis B virus surface antigen(anti-HBs),and evaluate its preliminary application.Methods The anti-HBs neutralization verification method was based on the double antigen sandwich method of time-resolved fluorescence immunoassay(TRFIA).Hepatitis B virus surface antigen[HBsAg(ad/ay)]was coated on the solid phase as capture antigen,HBsAg(ad/ay)was labeled N1-(P-isothiocyanatobenzyl)-diethylenetriamine tetraacetate sodium as a detection antigen.The performance evaluation of the self-developed anti-HBs neutralization verification method was evaluated by testing national reference specimens,national standards,and serum disk samples.171 clinical samples were detected by TRFIA reagent and chemiluminescent microparticle immunoassay(CMIA)reagent in parallel.The non-compliant samples and positive samples of two reagents were tested by the self-developed anti-HBs neutralization verification method.Results The self-developed anti-HBs neutralization verification method for the solid phase coated HBsAg(ad/ay)was 5.0μg/mL,the working ratio of the mark mother liquor was 1:1500,and the working dilution ratio of neutralizing HBsAg was 1∶1000.The national precision reference specimens and the national standards were tested by the self-developed neutralization verification method,there were non-reactivity of 20 national negative reference specimens,the coefficient of variation of the national precision reference specimens was 4.10%(n=10),the lower limit of detection was better than 10.00 IU/L,the relative biases between the measured values and the theoretical values were within-4.08%-4.36%for the national standards between 9.40-160.00 IU/L,the linear correlation coefficient could reach 0.99 or above.The serum disk samples were tested by the self-developed neutralization verification method,there were non-reactivity of 20 serum-negative samples,there were reactivity of 20 serum-positive samples,there was reactivity of L1 in serum-sensitivity samples L1 to L5,the coefficient of variation of serum-precision samples was 5.56%(n=10).171 clinical samples were tested with anti-HBs TRFIA reagent of Fenghua company and CMIA reagent of Abbott company.Among them,9 samples were anti-HBs negative for CMIA reagent and anti-HBs positive for TRFIA reagent,and the anti-HBs values of 5 samples were between 8.00 and 10.00 IU/L were tested by CMIA reagent tested,which were reactive by the neutralization verification experiment tested.3 samples were anti-HBs positive by the CMIA reagent detected,while the TRFIA reagent detected anti-HBs as negative,and the neutralization verification experiment was reactive.Conclusion The performance of the self-developed anti-HBs neutralization verification method are well.It is recommended that clinical laboratories should not use different detection systems to perform quantitative anti-HBs tests simultaneously and make direct comparisons.