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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 establish a method for the detection of hepatitis B virus surface antigen (HBsAg) in nasal spray recombinant hepatitis B vaccine. Methods The detection method of HBsAg in nasal spray recombinant hepatitis B vaccine was established by optimizing the treatment conditions of chitosanase (reaction temperature, enzyme concentration and enzymolysis time), and the specificity, linear range, accuracy , Precision (repeatability, intermediate precision) and durability verification. The results showed that the conditions of chitosanase treatment were as follows: 2 U / ml chitosanase was incubated in water bath at 37 ℃ for 60 min, then HBsAg content was quantitatively detected by a chemiluminescence-based kit. The addition of chitosanase did not interfere with the detection of HBsAg in the vaccine. The HBsAg concentration was linear with the luminescence value (r2≥0.95) at the concentration of 1.25-160 ng / ml. The mean of high, middle and low concentration vaccines The recoveries were 100.18%, 103.54% and 113.88%, respectively. The coefficients of variation (CV) of repeatability and intermediate precision were all less than 20%. The concentration of chitosan was between 1.5-2.5 U / ml and the enzymolysis time For 60 min, the measured value of CV are in line with the verification standard of ≤ 20%. Conclusion The detection method of HBsAg in nasal spray recombinant hepatitis B vaccine is established. The method has good specificity, accuracy, precision and durability and can be used for the quality control of the vaccine.