Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD, USA.
Influenza Other Respir Viruses. 2009 Sep;3(5):233-40. doi: 10.1111/j.1750-2659.2009.00094.x.
Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA-specific antibodies only.
Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA-mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA.
Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live-attenuated vaccines increased NI titers against a given subtype at approximately the same rate.
The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA.
神经氨酸酶（NA）抗体有助于在流感病毒感染期间提供保护，但在疫苗试验中通常不分析 NA 抑制（NI）滴度。原因之一是传统的硫代巴比妥酸（TBA）NI 测定法繁琐，该方法使用化学方法在存在血清的情况下，定量分析 NA 与底物孵育后释放的游离唾液酸。此外，由于需要使用宿主新型血凝素（HA）亚型的病毒来仅检测针对 NA 的特异性抗体，因此该测定法很复杂。
我们的主要目标是将比色 NI 测定法小型化，以适合对大量样品进行定量分析，并使用雪貂和人血清验证小型化格式的特异性和敏感性。另一个目的是使用反向遗传学构建具有最近流感 A 疫苗株和 H6 HA 的 NA 的 HA 错配病毒试剂。
通过小型化测定法分析雪貂抗血清表明，其敏感性和特异性与传统测定法相当。在小型化和常规测定法中，均测量到接种疫苗的人类志愿者血清中 NI 滴度的相似增加。灭活和减毒疫苗对特定亚型的 NI 滴度增加率大致相同。
这里描述的 TBA 方法的试剂和小型化格式为针对 NA 的功能性抗体的实用血清学监测提供了平台。
