Summary

体外ELISA测试评估狂犬病疫苗效力

Published: May 11, 2020
doi:

Summary

在这里,我们描述了一个间接的ELISA三明治免疫捕获,以确定狂犬病疫苗中的免疫原性糖蛋白含量。本测试使用中和单克隆抗体(mAb-D1)识别糖蛋白修剪器。它是体内NIH测试的替代方案,用于在生产过程中遵循疫苗效力的一致性。

Abstract

全球对动物福利的日益关注鼓励制造商和国家控制实验室 (OMCL) 遵循实验室动物检测的替换、减少和改进的 3R 战略。建议在世卫组织和欧洲各级制定体外方法,作为NIH检测的替代品,以评估狂犬病疫苗的效力。在狂犬病病毒 (RABV) 颗粒的表面,糖蛋白的修剪器构成诱导病毒中和抗体 (VNAbs) 的主要免疫原。ELISA测试,其中中和单克隆抗体(mAb-D1)识别糖蛋白的三氯体形式,已经开发,以确定原生折叠三氯糖蛋白的内容以及疫苗批次的生产。这种体外效力测试与NIH测试良好一致,并发现适合RABV疫苗制造商和OMCL的合作试验。在不久的将来,避免使用动物是一个可以实现的目标。

所介绍的方法基于使用mAb-D1的间接ELISA三明治免疫捕获,该mAb-D1可识别三角RABV糖蛋白的抗原位点III(aa 330至338),即免疫原性RABV抗原。mAb-D1 用于疫苗批次中糖蛋白修剪器的涂层和检测。由于表位因其构象特性而得到识别,因此 mAb-D1 无法捕获和检测潜在的变性糖蛋白(免疫原性较低)。要测试的疫苗在mAb-D1的培养板中孵育。结合三聚三叶胶质的拉巴糖蛋白通过再次添加mAb-D1来识别,用过氧化物酶标记,然后在基质和色蛋白的存在下显示。对经测试的疫苗和参考疫苗的吸收度进行比较,可以确定免疫原糖蛋白含量。

Introduction

50多年来,NIH测试1被用作一种黄金标准方法,在批次发布前评估狂犬病疫苗的功效。该测试包括对带有疫苗的小鼠组进行腹内免疫,然后14天后与狂犬病病毒挑战病毒标准(CVS)菌株(RABV)进行脑内(IC)挑战。从在IC挑战中幸存下来的小鼠比例来评估效力。虽然世卫组织2和欧洲药典3仍然需要NIH测试来评估疫苗的功效,但该测试面临几个障碍:结果高度可变4;传染性RABV在挑战期间使用,这既需要技术技能和严格的生物安全措施;大量动物被使用,挑战的严重性引起了严重的伦理问题这种测试的一个不太严重的变异已经形成:在腹管内免疫两周后,小鼠不受IC的挑战,而是通过体外中和测试,在血清中存在特定的RABV中和抗体(VNAbs)进行流血和测试。然而,这项试验仍然需要牺牲大量的实验鼠,虽然它已经用于兽医疫苗66,7,7并已考虑为人类疫苗8。.

截至目前,国际9国和欧洲10项建议都鼓励制造商和国家控制实验室(官方药物控制实验室 -OMCL)实施实验室动物检测的更换、减少和改进,即3R战略。欧洲第2010/63/EU号指令(自2013/01/01月起生效)也加强了对疫苗制造商和实验室参与狂犬病疫苗质量控制以及狂犬病研究的限制。因此,开发、验证和使用替代体外方法现已成为优先事项。这些不仅在道德上是健全的,而且还可以减少批次测试成本,并将结果的时间缩短到数小时,而不是第3周。

在RABV粒子的表面,糖蛋白采用12、13、14、15、1613,14,15,16的三角状。12在狂犬病疫苗中,这种原生三分体构成诱导VNAbs17的主要免疫原,而单体、可溶性或变性糖蛋白的免疫原性差18、19。,19因此,沿疫苗生产过程中保存糖蛋白的修剪器是保存最佳免疫原性潜力的良好指标。几种免疫化学方法,如抗体结合测试20、21、,21单径向免疫扩散(SRD)测试22和ELISA测试23、24、25、26、27,均被世卫组织技术报告系列23,24,25,26,272和欧洲专著3推荐,以量化狂犬病疫苗中的抗原含量。制造商使用这些来监测疫苗生产的一致性,并由OMCL评估人类疫苗的批次28的一致性配方即使NIH测试仍考虑其效力。

然而,所有这些免疫化学方法并不等效。SRD测试需要预先处理,可能会改变膜锚定修剪器,并导致糖蛋白22,29,29的可溶性或变性形式。因此,SRD在区分免疫原性和非免疫原性糖蛋白方面效率不高,导致对疫苗批次的免疫原性评价不完善。相比之下,ELISA测试更敏感22,保留了糖蛋白的原生结构,更适合确定糖蛋白的原生折叠修剪器的含量。ELISA 测试可以使用兔子多克隆或小鼠单克隆抗糖蛋白抗体纯化和浓缩与硫酸铵。研究表明,NIH测试与ELISA在疫苗中评估的抗原含量良好一致,并得出结论,ELISA方法适用于体外效力测试。这主张ELISA测试至少可以补充甚至取代NIH测试44,26,27,30,31,32,33。,26,27,30,31,32,33今天,欧洲药典建议使用经过验证的血清学或免疫化学测定作为NIH测试3的替代品。完全避免动物使用疫苗效力已成为一个现实的观点。

下面介绍的方法是基于一个间接的ELISA三明治免疫捕获使用小鼠单克隆抗体克隆(mAb-D1),识别的抗原位点III(a330至338)的三角RABV糖蛋白15,34。15,该方法最初是在巴斯德研究所26、30,30中开发,然后由国家国家麻醉研究所(ANSM)实验室(即法国OMCL4,4,33)进行优化验证。mAb-D1 既用于使板敏感,又用于检测捕获的抗原。这允许对糖蛋白修剪器进行特定定量,即免疫原性RABV抗原。用于检测的 mAb-D1 标有过氧化物酶,在基材和色蛋白存在时会显示此特性。对经测试的疫苗和参考疫苗的吸收度进行比较,可以确定免疫原糖蛋白含量。值得注意的是,同一类型的测定可以适用于不同的mAbs识别不同的抗原位点的RABV糖蛋白35。与硫酸铵抗糖蛋白多克隆兔免疫球蛋白G(IgG)或单克隆小鼠球蛋白一起获得和纯化或浓缩的方法,在36之前与过氧化物酶37结合抗体的方法中得到了广泛的描述

Protocol

1. 安全防范措施 注:此方法适用于活式 RABV 和灭活疫苗。 使用良好的实验室实践和安全程序。 佩戴足够的个人防护设备 (PPE),包括一次性外套、手套、面罩、眼镜等。 当活病毒被定化时,使用II类生物安全柜。 将任何与样品接触的材料(试剂、洗涤溶液等)视为传染性材料。 通过浸入漂白液(次氯酸钠的2.5%)进行30分钟的净化,处?…

Representative Results

在下面的示例中,使用了参考疫苗09,包括纯化灭活狂犬病病毒颗粒(PV疫苗毒株)。在测定病毒蛋白总量(BCA测试)和通过SDS-聚丙烯酰胺凝胶电泳评估糖蛋白百分比后,确定了其中的糖蛋白修剪器(10 μg/mL)含量。或者,可以使用经过校准的参考疫苗,例如,世卫组织第六届狂犬病疫苗国际标准(NIBSC代码:07/162)。 <p class="jove_content" fo:keep-together.within-page="1"…

Discussion

mAb-D1识别的表位位于RABV糖蛋白的抗原部位III,它不仅是VNAbs诱导的免疫主导物,而且还涉及神经毒性、致病性40、41,41和受体识别42。沿糖蛋白的另一个重要的抗原位点,站点II43,有几个MAbs已被隔离,如mAb-WI-111235。这些也可用于类似类型的实验。

ELISA的这种体外方法的一个局限?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

必须承认西尔维·莫尔格博士和让-米歇尔·查萨尔博士的主要参与,以建立疫苗批次的ELISA测定赛,并组织国际讲习班和合作研究。我们感谢萨布丽娜·卡利对手稿的批判性解读。皮埃尔·佩林博士负责mAb-D1的隔离和表征。这项工作主要由巴斯德研究所资助。

Materials

Class II Biological Safety Cabinet ThermoFisher Scientific 10445753 if titrating live virus
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 µL, MAXISORP ThermoFisher Scientific 439454 good for binding to the loaded antibody
Equip Labo Polypropylene Laboratory Fume Hood ThermoFisher Scientific 12576606 for the preparation of sulfuric acid
Immunology Plate Strong
Adsorption MAXISORP Flat Bottom
Well F96
Dutscher 55303 good for binding to the loaded antibody
Microplate Sealing Tape(100 sheets) ThermoFisher Scientific 15036
Microplate  single mode reader Sunrise TECAN
Microplate shaker-incubator Dutscher 441504
Microplate washer Wellwash ThermoFisher Scientific 5165000
Multichannel pipette (30-300 µL) 12 channels ThermoFisher Scientific 4661180N
Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 μL micro, 2-20 μL, 20-200 μL & 100-1000 μL) ThermoFisher Scientific 4700880

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Jallet, C., Tordo, N. In Vitro ELISA Test to Evaluate Rabies Vaccine Potency. J. Vis. Exp. (159), e59641, doi:10.3791/59641 (2020).

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