使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法
1Baker Heart and Diabetes Institute, 2Department of Physiology, Anatomy and Microbiology,La Trobe University, 3Florey Institute of Neuroscience and Mental Health,University of Melbourne, 4Department of Physiology, Centre for Muscle Research (CMR),The University of Melbourne, 5Department of Diabetes, Central Clinical School,Monash University, 6Baker Department of Cardiometabolic Health,The University of Melbourne, 7Department of Physiology and Department of Medicine Alfred Hospital,Monash University
Summary
介绍了全面的实验室实验方案和分析工作流程，用于快速、经济高效且直接的基于细胞的比色测定，以检测针对 AAV6 的中和元素。
Abstract
重组腺相关病毒（rAAV）已被证明是转移遗传物质以治疗实验室和临床各种健康状况的安全和成功的载体。然而，预先存在的针对AAV衣壳的中和抗体（NAbs）对在大型动物实验模型和人类群体中成功施用基因疗法构成了持续的挑战。对宿主对AAV的免疫力进行初步筛查是必要的，以确保基于AAV的基因疗法作为研究工具和临床上可行的治疗剂的有效性。该协议描述了一种比色 体外 测定，以检测针对AAV血清型6（AAV6）的中和因子。该测定利用编码碱性磷酸酶（AP）报告基因的AAV与其底物NBT / BCIP之间的反应，其组合产生不溶性可量化的紫色染色剂。
在该协议中，将血清样品与表达AAV的AP结合并孵育以允许潜在的中和活性发生。随后将病毒血清混合物添加到细胞中，以允许任何未中和的AAV的病毒转导。加入NBT / BCIP底物并进行显色反应，对应于病毒转导和中和活性。使用免费软件工具定量着色区域的比例，以产生中和滴度。该测定显示着色与病毒浓度之间具有很强的正相关关系。在给予重组AAV6之前和之后对绵羊血清样本的评估导致中和活性急剧增加（增加125至>10，000倍）。该测定显示出足够的灵敏度，可以检测>1：32，000血清稀释液中的中和活性。该测定提供了一种简单、快速且经济高效的方法来检测针对 AAV 的 NAb。
Introduction
腺相关病毒（AAV）越来越多地被用作将基因疗法递送到影响心血管，肺，循环，眼部和中枢神经系统的各种健康状况的试验治疗的载体1，2，3，4，5。AAV载体作为领先的基因治疗平台的受欢迎程度源于其积极的安全性，长期转基因表达和广泛的组织特异性倾向1，6。动物研究的成功结果为五十多项AAV基因治疗临床试验铺平了道路，这些临床试验已成功达到其疗效终点7，以及美国食品和药物管理局批准的首个市售AAV基因治疗药物的发布8。继最初的成功之后，AAV作为首选载体继续在基础和临床研究领域获得牵引力，并且是目前美国和欧洲唯一批准用于临床的体内基因疗法9。尽管如此，预先存在的针对AAV载体衣壳的中和抗体（NAbs）的存在仍然是临床前研究和临床试验疗效的障碍。NAbs存在于幼稚的人类和动物种群中，并在体内施用AAV载体1后抑制基因转导。AAV血清阳性是大多数基因治疗试验的排除标准，因此宿主免疫的初步筛查在实验室和临床中都至关重要。建立一种可以检测NAbs对AAV存在的测定是任何基于AAV基因治疗的研究项目管道中必不可少的一步。本报告重点介绍AAV6，由于其在横纹肌（心脏和骨骼肌）中的高效和选择性转导，研究人员一直对AAV6感兴趣1，10，11，12。基因治疗被认为是一种针对心脏的有前途的策略，因为如果没有侵入性的心脏直视手术，很难专门针对心脏。
中和活性通常使用基于细胞的体外或体内转导抑制测定法测定。体内 NAb测定通常涉及将来自测试对象（例如，人类或大型动物）的血清施用于小鼠体内，然后使用具有报告基因的AAV，然后测试报告基因或相应抗原的表达。体外检测通过将来自人类或大型动物的血清或血浆与表达报告基因的重组 AAV （rAAV） 以连续稀释液培养来确定 NAb 滴度。细胞被血清/病毒混合物感染，并与对照组相比，评估报告基因表达被抑制的程度。体外检测广泛用于NAb筛选，因为与体内测定相比，其成本相对较低，测试速度快，标准化和验证能力更强13，14。据报道，体内测定通常具有更高的灵敏度15，16，但对体外测定也提出了相同的要求14，17。
迄今为止， 体外 NAb测定主要使用发光（荧光素酶）作为报告基因来检测中和。尽管基于光的方法在许多情况下都有其优点，但在某些情况下，比色/显色NAb测定可能是有利的。用于评估中和的比色法已成功用于其他病毒，如流感和腺病毒18，19。它们的吸引力源于其简单性，低成本以及对日常实验室仪器和工具的要求20。使用基于发光的报告基因的NAb检测需要昂贵的底物试剂盒、光度计和相应的软件进行分析21。这种比色测定的优点是只需要光学显微镜和非常便宜的底物。比色法与发光法的灵敏度报告产生了相互矛盾的结果。一项研究表明，基于发光的 ELISA 检测方法与比色法相比具有更高的灵敏度和可比的再现性22，而另一项研究表明，基于比色法的 ELISA 检测可赋予更高的灵敏度23。这里，提供了针对AAV 的体外 NAb测定的详细方案，该方案利用编码碱性磷酸酶（AP）报告基因的AAV与硝基蓝色四唑/5-溴-4-氯-3-吲哚基磷酸（NBT / BCIP）底物之间的显色反应。该分步方案是根据先前的一份报告开发的，该报告利用hPLAP（人胎盘碱性磷酸酶）报告基因（AAV6-hPLAP）来检测针对AAV24的中和活性。该测定具有成本效益，具有时效性，易于设置，并且需要最少的技术技能，实验室设备和试剂。此外，这种测定的简单性使其有可能针对不同类型的细胞，组织或病毒血清型的广泛应用进行优化。
Protocol
动物护理和实验的各个方面都是按照弗洛里神经科学和心理健康研究所的指导方针和澳大利亚科学目的动物护理和使用守则进行的，遵循参考文献25。1.5-3岁的美丽诺母羊用于研究。 图1提供了测定方案的示意图。 图1：N…
Representative Results
转导测定，以确定板覆盖的最佳病毒剂量HT1080细胞是一种成熟的纤维肉瘤细胞系，被选择用于该测定。浓度为1×104 HT1080细胞/孔，在96孔板的每个孔中提供〜50%的细胞汇合度。为了确定测定的最佳病毒浓度，在每个细胞的含vg颗粒浓度范围内（MOI：0，150 ，500，1500，1500，1500，500，1500，5000，1500，15000，15000，5000，5000和150000）的范围内…
Discussion
该报告描述了一种比色测定法，该测定法通过评估与 体外 病毒转导程度相对应的显色反应来评估给定血清样品中AAV中和的程度。该方案的开发基于碱性磷酸酶与NBT / BCIP之间的已知显色反应，NBT / BCIP已被广泛用作免疫组织化学等应用中检测蛋白质靶标的染色工具，并作为评估病毒转导的报告工具24，33，34，<sup c…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究由国家卫生和医学研究委员会对JRM和CJT（ID 1163732）的项目赠款资助，部分由维多利亚州政府的运营基础设施支持计划资助。SB由贝克心脏和糖尿病研究所 – 拉筹伯大学联合博士奖学金支持。KLW得到了The Shine On Foundation和澳大利亚国家心脏基金会（ID 102539）的未来领袖奖学金的支持。JRM由国家卫生和医学研究委员会高级研究奖学金（ID 1078985）提供支持。
Materials
| 0.05% Trypsin/EDTA | Gibco | 25300-054 | |
| 50 mL conical centrifuge tube | Falcon | 14-432-22 | Or equivalent |
| 75 cm2 square flasks | Falcon | 353136 | Or equivalent |
| 96 well flat bottomed plate | Falcon | 353072 | |
| AAV6-CMV-hPLAP Vector | Muscle Research & Therapeutics Lab (University of Melbourne, Australia) AAV6-CMV-hPLAP can be provided upon request. | ||
| Aluminium foil | |||
| Anti-AAV6 (intact particle) mouse monoclonal antibody, (ADK6) | PROGEN | 610159 | Positive control monoclonal antibody |
| BCIP/NBT | SIGMAFAST | B5655 | |
| Cell and tissue culture safety cabinet | |||
| Electronic Pipette | 5 & 10 mL stripette inserts | ||
| Fetal Bovine Serum | Gibco | 10099-141 | |
| Haemocytometer | |||
| High glucose Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11965118 | |
| HT1080 cells | ATCC | ||
| ImageJ Software | Freely available: https://imagej.nih.gov/ij/download.html | ||
| Incubator | 37 °C, 5% CO2 | ||
| Light microscope with camera | Capable of taking photos with a 4x objective lens | ||
| Oven | For a 65 °C incubation | ||
| Paraformaldehyde | MERCK | 30525-89-4 | |
| Penicillin Streptomycin | Gibco | 15140-122 | |
| Phosphate buffered saline | |||
| Pipettes and tips | 20 μL, 200 μL & 1 mL single pipettes and tips & 200 μL multichannel pipette | ||
| Stericup quick release filter | Millipore | S2GPU10RE | Used for combining media reagents |
| Trypan blue solution | Sigma-Aldrich | T8154 | |
| VACUETTE TUBE 8 ml CAT Serum Separator Clot Activator | Greiner BIO-ONE | 455071 | Used for serum collection & processing from sheep |
| Water bath |
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Citar este artigo
Bass-Stringer, S., Thomas, C. J., May, C. N., Gregorevic, P., Weeks, K. L., McMullen, J. R. A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay. J. Vis. Exp. (178), e63419, doi:10.3791/63419 (2021).