Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, United States.
Vaccine. 2012 Mar 23;30(14):2475-82. doi: 10.1016/j.vaccine.2011.12.056. Epub 2011 Dec 22.
Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1 μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.
流感疫苗接种是预防流感及其严重并发症的主要方法。已获得许可的季节性或大流行性流感灭活疫苗的配方中含有预定量的血凝素 (HA)，这是引发保护作用的关键抗原。目前尚无定量神经氨酸酶 (NA) 的监管方法，神经氨酸酶是另一种被认为具有保护能力的主要膜结合蛋白。这主要是由于目前定量这些抗原的方法在灵敏度和选择性方面均存在局限性。目前用于建立疫苗中 HA 浓度的方法依赖于间接测量，而这些方法容易受到相当大的实验变异性的影响。我们提出了一种液相色谱-串联质谱 (LC/MS/MS) 方法，用于在复杂混合物中绝对定量病毒蛋白。通过使用同位素稀释方法，可以直接且快速地测定 H1N1、H3N2 和 B 亚型的 HA 和 NA。每种亚型的 HA 分析都使用了三个肽，以确保蛋白质的完全消化和测量的准确性。该方法已应用于纯化的病毒制剂、单价批量浓缩物、三价灭活流感疫苗，甚至是改良的粗鸡胚尿囊液，具有更高的速度、灵敏度、精密度和准确性。使用该方法很容易检测到 1 μg/mL 的蛋白。该方法的灵敏度涵盖了疫苗制剂中预期的范围，包括基于佐剂的疫苗。这种 LC/MS/MS 方法大大提高了定量季节性和大流行性流感疫苗中病毒蛋白含量的选择性、准确性和精密度，并减少了在下次流感大流行期间为公共卫生用途提供流感疫苗所需的时间和精力。
