一种优化的血凝抑制 (HI) 测定流感特异抗体滴
Summary
本协议描述了如何进行血凝抑制试验, 以量化流感疫苗接受者血清样本中的流感特异性抗体滴。第一种方法是通过血凝法确定最佳病毒抗原浓度。第二种方法通过血凝抑制来量化流感特异抗体滴。
Abstract
抗体滴是常用的替代标志, 用于血清保护流感和其他病原体。对抗体产生前和后接种的详细知识需要了解疫苗诱导免疫。本文介绍了一个可靠的点对点协议来确定流感特异抗体滴。第一个协议描述了一种方法来指定血凝所需的抗原量, 它标准化了第二个协议中随后使用的浓度 (血凝试验, HA 测定)。第二个协议描述了对不同病毒株的流感特异性抗体滴的定量化, 使用人血清或细胞培养清 (血凝抑制法, HI 测定) 的序列稀释。
作为一个应用实例, 我们展示了一个健康的队列的抗体应答, 它接受了三价灭活流感疫苗。此外, 还说明了不同流感病毒之间的交叉反应性, 并解释了使用不同类型的动物红细胞 (红细胞) 减少交叉反应性的方法。讨论突出了所提出的化验的优缺点, 以及如何确定流感特异抗体滴可以提高对疫苗相关免疫的认识。
Introduction
感染流感病毒与相当大的发病率, 死亡率和高医疗费用相关的1,2,3,4。特别是, 老年人、新生儿、孕妇和慢性病患者面临更严重的临床后果。因此, 预防循环流感病毒株的接种是降低高危人群疾病负担的主要措施。疫苗接种后个体免疫应答的增加,例如, 高于保护性阈值的流感特异性抗体, 降低了个体感染的风险, 并在总体上减少了病毒在人群中传播的可能性5. 详细了解不同人群及各年龄组的疫苗诱导体液免疫应答是回答重要临床问题的关键因素6、7、8,9, 例如: 为什么有些老年患者尽管曾接种过疫苗, 但仍有感染？什么是 “好” 和 “足够” 疫苗诱导的保护？疫苗应多久免疫患者才能达到保护性滴？什么是最有效的剂量？新佐剂对后接种抗体滴的影响是什么？对疫苗特异抗体的测定可以帮助回答这些重要问题, 提高疫苗接种效果。
病毒特异性抗体滴的定量可以用各种免疫学方法进行。这包括固相10或基于磁珠的 ELISA11化验、HI 化验12和中和分析13。以 ELISA 为基础的方法可以筛选相对大量的血清标本对各种抗原。另外, 可以分别探讨病原体特异免疫球蛋白 (Ig) 和 IgG。虽然抗原的特征,如例如, 线性氨基酸序列或类似病毒的微粒可能影响抗体的结合, 潜在的表位的光谱是非常宽广的并且不提供信息关于是否抗体响应具有功能相关性。
与此相反, 中和试验确定了抗体在功能上抑制细胞感染的潜能, 从而反映了中和电位。然而, 这种方法是非常劳动密集型, 需要培养特定的细胞系和活病毒, 因此, 它是费时, 昂贵, 并要求特殊设备。
本文介绍了一个循序渐进的世界卫生组织 (WHO) 为基础的 HI 协议12 , 以量化流感特异性抗体滴。血凝是某些病毒导致红细胞凝集的一种特征性效应。这种效果与患者血清的抑制, 使抑制抗体浓度的测量, 这反映了中和效应。
我们修改了 WHO 协议的工作流程, 以便在同一时间更有效地处理多个样本, 从而减少所需的时间。第一个协议描述了特定流感抗原的凝集电位的测定。这样做, 确定了正确的流感抗原浓度的第二个协议。这部分应重复每一个新的病毒抗原, 以及每一批血液。
第二个协议描述了流感特异性抗体滴的测定。针对流感病毒和人血清样本的研究, 提出的协议进行了优化但是, 它也可以用于小鼠血清样本或细胞培养清的刺激免疫细胞,例如, 特定病毒 B 细胞。结果可以确定为绝对测量滴。在许多疫苗研究中, 每个特定种群都显示了几何平均滴和95% 置信区间。对于解释, seroprotection 或血清经常被用来描述人口对某种病毒的易感性。Seroprotection 被定义为≥1:40 的效价, 血清为超过4倍的效价增加与 seroprotective 滴在两个时间点之间的成就 (最常见的预接种和30天后接种使用)。
这两种协议都很容易使用, 而且可以适应范围广泛的研究问题。特别是, 他们可以用来确定可靠和快速的抗体滴对其他各种病毒的血凝能力, 如麻疹, polyomaviruses, 腮腺炎, 或风疹14,15,16.
Protocol
Representative Results
Discussion
对疫苗接种前后流感病毒特异性抗体滴的定量是一种重要的研究手段。基于保护病毒感染的替代措施, 如 seroprotection (> 1:40) 或血清 (4 倍效价增加), 疫苗接种策略可以优化9。使用所提供的协议可以确定: (i) 特定病毒的血凝潜能, 和 (ii) 抗体滴的病毒的利益。
修改和故障排除:
此协议基于 WHO 标准12。我们用 PCR…
Disclosures
The authors have nothing to disclose.
Acknowledgements
没有.
Materials
| 25 ml Disposable Multichannel Pipette Reservoirs | Integra | 4312 | |
| 8-well PCR tubes | Brand GMBH | 781332 | For serum aliquots |
| 96-well microtiter plate, U-shaped | TPP | 92097 | For HI assay when using mammalian RBCs |
| 96-well microtiter plate, V-shaped | Corning Costar | 3897 | For HI assay when using avian RBCs |
| Aqua ad iniect. Steril | Bichsel AG | 1000004 | For preparing influenza antigen and cholera filtrate solutions |
| Chicken RBC (10%) | Cedarlane | CLC8800 | 10% suspension of chicken red blood cells in Alsever's solution |
| Cholera filtrate | Sigma-Aldrich | C8772 | Used as receptor destroying enzyme (RDE) |
| Dulbecco's PBS | Sigma-Aldrich | D8537 | For diluting the serum samples, RBCs and antigens |
| Eppendorf Multichannel pipette, 12-channel, 10-100 µl | Sigma-Aldrich | Z683949 | |
| Eppendorf Multichannel pipette, 8-channel, 10-100 µl | Sigma-Aldrich | Z683930 | |
| Guinea Pig RBC (10%) | Cedarlane | CLC1800 | 10% suspension of guinea pig red blood cells in Alsever's solution |
| Influenza Anti-A/California/7/09 HA serum | NIBSC | 14/134 | Used as positive control at the HI assay |
| Influenza Anti-A/Switzerland/9715293/2013-like HA serum | NIBSC | 14/272 | Used as positive control at the HI assay |
| Influenza Anti-A/Texas/50/2012-Like HA Serum | NIBSC | 13/178 | Used as positive control at the HI assay |
| Influenza Anti-B/Brisbane/60/2008-HA serum | NIBSC | 13/254 | Used as positive control at the HI assay |
| Influenza Anti-B/Massachusetts/02/2012 HA serum | NIBSC | 13/182 | Used as positive control at the HI assay |
| Influenza antigen A/California/7/09 (H1N1)(NYMC-X181) | NIBSC | 12/168 | Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181) virus (ca. 46µgHA/ml) |
| Influenza antigen A/Switzerland/9715293/2013 (NIB88) | NIBSC | 14/254 | Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55µgHA/ml) |
| Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223) | NIBSC | 13/112 | Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74µgHA/ml) |
| Influenza antigen B/Brisbane/60/2008 | NIBSC | 13/234 | Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42µgHA/ml) |
| Influenza antigen B/Massachusetts/02/2012 | NIBSC | 13/134 | Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35µgHA/ml) |
| Serum-Tubes | S-Monovette, Sardstedt | 01.1601.100 | For serum extraction with clotting activator |
| Single Donor Human RBC, Type 0 | Innovative Research | IPLA-WB3 | Suspension of single donor human red blood cells in Alsever's solution (ca. 26%) |
| Turkey RBC (10%) | Cedarlane | CLC1180 | 10% suspension of turkey red blood cells in Alsever's solution |
| Phosphate Buffered Saline (PBS) | Gibco |
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