目的: 对mRNA疫苗dsRNA残留量的常用检测方法均相时间分辨荧光法(homogeneous time resolved fluorescence,HTRF法)、酶联免疫法(enzyme linked immunosorbent assay,ELISA法)和斑点杂交法(Dot Blot法)进行比较研究,为mRNA疫苗质量控制提供技术支撑。方法: 分别根据HTRF法、ELISA法和Dot Blot法特点,配制相应浓度标准品,通过建立标准曲线,计算样品dsRNA浓度,考察3种方法的适用性、准确度、精密度、定量限和曲线拟合关系。结果: HTRF法检测dsRNA残留量的回收率为110.7%,重复性的RSD为5.6%,中间精密度的RSD≤19.0%,在1.56~100.00 ng·mL-1的质量浓度范围内,拟合关系良好(r=0.999 3);ELISA法检测dsRNA残留量的回收率为103.2%,重复性的RSD为19.5%,中间精密度的RSD≤11.7%,在7.81~500.00 ng·mL-1的质量浓度范围内,拟合关系良好(r=0.999 8);Dot Blot法检测dsRNA残留量的回收率为101.5%,重复性的RSD为18.5%,中间精密度的RSD≤42.2%,在800.00~80 000.00 ng·mL-1的质量浓度范围内,拟合关系良好(r=0.999 2)。HTRF法和ELISA法的准确度和精密度良好,HTRF法可检测较低浓度的dsRNA残留量。结论: 3种方法均可有效检测mRNA疫苗中的dsRNA残留量。
Objective: To provide technical support for mRNA vaccine quality control by compareing with 3 methods of homogeneous time resolved fluorescence(HTRF),enzyme linked immunosorbent assay(ELISA) and Dot Blot for detecting dsRNA residues. Methods: According to the characteristics of HTRF,ELISA and Dot Blot assay respectively, the standards with different concentration were prepared and the dsRNA residues of samples were calculated by the standard curve. And tests were investigated in applicability, accuracy, precision, limit of quantitation and non-linear response. Results: The HTRF method showed that the recovery was 110.7% with the RSD of repeatability and intermediate precision were 5.6% and ≤19.0% respectively, the non-linear range of dsRNA fell into 1.56-100.00 ng·mL-1(r=0.999 3). The ELISA method showed that the recovery was 103.2% with the RSD of repeatability and intermediate precision were 19.5% and ≤11.7% respectively, the non-linear range of dsRNA fell into 7.81-500.00 ng·mL-1(r=0.999 8). The Dot Blot method showed that the recovery was 101.5% with the RSD of repeatability and intermediate precision were 18.5% and ≤42.2% respectively, the non-linear range of dsRNA fell into 800.00-80 000.00 ng·mL-1(r=0.999 2). The accuracy and precision of the HTRF and ELISA method are effective, and the HTRF method could detect lower concentrations of dsRNA residues. Conclusion: The three methods all could determine the dsRNA residues in mRNA vaccines effectively.
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