鼻腔喷雾型重组乙型肝炎疫苗中乙肝病毒表面抗原含量检测方法的建立及验证

Development and verification of a method for determination of hepatitis B surface antigen in recombinant hepatitis B vaccine for nasal spray

目的建立鼻腔喷雾型重组乙型肝炎疫苗中乙肝病毒表面抗原(HBsAg)含量检测方法,并进行验证。方法通过对壳聚糖酶的处理条件(反应温度、酶浓度和酶解时间)进行优化,建立鼻腔喷雾型重组乙肝疫苗中HBsAg含量检测方法,并对该方法进行特异性、线性范围、准确度、精密度(重复性、中间精密度)及耐用性验证。结果确定壳聚糖酶处理条件为:2 U/ml的壳聚糖酶于37℃水浴中孵育60 min,然后采用基于化学发光法的试剂盒定量检测HBsAg含量。壳聚糖酶的加入不会对疫苗中HBsAg含量检测产生干扰;HBsAg浓度在1.25~160 ng/ml之间与发光值具有良好的线性(r2≥0.95);高、中、低浓度疫苗的平均回收率分别为100.18%、103.54%和113.88%;重复性及中间精密度验证结果的变异系数(CV)均≤20%;壳聚糖酶浓度在1.5~2.5 U/ml之间以及酶解时间为60 min,测定值CV均符合≤20%的验证审定标准。结论建立了鼻腔喷雾型重组乙型肝炎疫苗中HBsAg含量检测方法,该方法具有良好的特异性、准确度、精密度及耐用性,可用于该疫苗的质量控制。

Objective To develop and verify a method for determination of HBs Ag content in recombinant hepatitis B vaccine for nasal spray. Methods The condition for treatment of chitosanase, including reaction temperature, enzyme concentration and enzymolysis time, were optimized, based on which a chemiluminescent immunoassay（CLIA）method for determination of HBs Ag in recombinant hepatitis B vaccine was developed and verified for specificity, linear range,accuracy and precision（reproducibility and intermediate precision） and robustness. Results The optimal condition for treatment of chitosanase was as follows： the chitosanase at a concentration of 2 U/ml was incubated in a 37 ℃ water bath for 60 min, and determined for HBs Ag content by CLIA kit. The addition of chitosanase showed no interference to the determination of HBs Ag content. The HBs Ag content at a range of 1. 25 ~ 160 ng/ml showed good linear relationship to the chemiluminescence（r2 ≥ 0. 95）. The mean recovery rates of test samples at high, moderate and low concentrations were 100. 18%, 103. 54% and 113. 88% respectively. The CVs in verification for reproducibility and intermediate precision were not more than 20%. When the concentration of chitosanase was 1. 5 ~ 2. 5 U/ml, and the enzyme treatment time was 60 min, the CVs of determinat results were not more than 20%. Conclusion The CLIA method for determination of HBs Ag content in recombinant hepatitis B vaccine was developed, which showed good specificity,accuracy, precision and robustness, and might be used for quality control of the vaccine.