制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体
1National Exposure Research Laboratory,U.S. Environmental Protection Agency, 2National Risk Management Research Laboratory,U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education, 4Department of Biological Sciences, McMicken College of Arts and Sciences,University of Cincinnati
Summary
In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.
Abstract
其病因与人体暴露于环境中的病原体的影响是全球主要的关注，因此，评估接触和感染的使用成本效益，高通量方法的能力是不可缺少的。该原稿描述了能够同时测量抗体在人唾液的存在下，以多种病原体的基于珠子的多重免疫测定的开发和分析。唾液是在本申请中特别有吸引力的，因为它是无创，更便宜和更容易收集比血清。从环境中的病原体的抗原偶联到羧基化微球（珠）和用于测量使用基于珠子的，溶液相测定法在人唾液样品的非常小体积的抗体。珠再加上从空肠弯曲菌，幽门螺旋杆菌，弓形虫，诺如病毒（G I.1和G II.4）抗原，甲型肝炎病毒。以保证抗原充分耦合到珠，耦合用物种特异性的，动物来源的初级捕获抗体证实，随后用生物素化抗物种二次检测抗体和链霉抗R-藻红蛋白记者（SAPE）温育。作为衡量的非特异性结合的对照，一个小珠组被同样处理的其他不同的是它不连接到任何抗原。抗原耦合和控制珠子然后用前瞻性收集的人唾液样品孵育，基于流式细胞术的原理的高通量分析仪测量，和抗体的每个抗原的存在下，在中位数荧光强度单位测定（MFI） 。这个多重免疫测定具有许多优点，包括具有较少样本更多的数据;降低了成本和劳动;并能够自定义检测到许多感兴趣的目标。结果表明，该唾液多重免疫测定可以是能够识别先前接触和感染的，它可以是especiaLLY有用的涉及大型人群监测研究。
Introduction
腹泻相关的疾病的百分之八十八世界范围内与人体接触到受污染的水，不安全的食品和卫生条件差/卫生有关，造成约1.5万人死亡，其中大多数是儿童1。这是公共卫生官员和决策者关注的一个重要原因。在努力，调查与水性和其他环境中的病原体相关风险和疾病，我们开发了多重免疫测定来衡量人类样本2-4的抗体。这种方法可以适用于流行病学研究，以确定人体暴露于这些病原体和更好地确定immunoprevalence和事件感染。
唾液持有相当大的希望作为替代血清人类生物标志物研究。间使用唾液的优点是，非侵入性和易于样品收集，成本低的，并且样品可以容易地从儿童5-7 <收集/ SUP>。血清和唾液样本已经被反对H.抗体广泛的研究幽门螺杆菌 2,3,8， 恶性疟原虫 9，阿米巴 10，隐孢子虫 3,11， 肺炎链球菌 12，肝炎病毒A和C 13-14， 诺如病毒 2-4,15，T。弓形虫 2-4，登革热病毒16，人免疫缺陷病毒（HIV）17，和大肠杆菌 O157：H7 18。
一个多重免疫测定允许在单个样本量同时和一个周期或运行中的多个分析物的分析。从C抗原复空肠弯曲菌 ，T。弓形虫，幽门螺旋杆菌，A型肝炎病毒，和两个诺如病毒被用来测量人体唾液的IgG 2-4和IgA 3,4-和血浆的IgG 2,3抗体反应使用这些病原体珠为基础的复用免疫。当在暴露于微生物的水，土壤和食品的流行病学研究结合使用，在此研究中所描述测定的类型可以提供有价值的信息，以提高所造成的环境中的病原体感染的理解。而且，从这些研究获得的唾液抗体的数据可以被用来改善风险评估模型19-22。
Protocol
从机构审查委员会获得批准（IRB＃08-1844，北卡罗莱纳州教堂山分校，北卡罗来纳大学，美国），用于刺激沟唾液样本从博克龙海滩，波多黎各，前往沙滩的集合作为美国的一部分环保局（USEPA）国家流行病学和娱乐（NEEAR）水研究23环境评估，评估中游泳暴露和疾病。研究对象签署知情同意书，并指导对训练的USEPA承包商使用唾液收集装置。唾液样品运送在冰上，在接收，它们被离心，并?…
Representative Results
一个唯一的珠集被用来作为测量非特异性结合对照和样品采样变性。这些珠粒相同处理，以与异常的抗原偶联珠，他们不与在偶合步骤中的任何抗原温育。从所有的唾液样本培养控制珠获得MFI值> 500被拆除，由于从进一步分析，从血清中怀疑被污染，其余的答复日志分配。唾液可以与血清污染如果牙龈与附连到收集装置，或者如果在研究参与者都有牙周病海绵擦太剧烈?…
Discussion
这些结果表明，在多重免疫测定方法是用于唾液样品是免疫阳性或免疫阴性区分是有用的。为了确定免疫阳性，单个切点通过计算平均值加上的与所有的唾液样品的测试的控制耦合珠粒的日志变换MFI响应的三个标准偏差的发展。截止点，得到以评估曝光和immunoprevalence到单个或多种病原体的能力。这种辨别力可能是调查与接触环境和其他病原体相关的健康风险在人口研究非常有用。
<p class="jove_c…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Clarissa Curioso was supported through an appointment to the Research Participation Program at the U.S. Environmental Protection Agency administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA.
Materials
| Equipment and Software | |||
| Microcentrifuge | Thermo Electron Corporation | 75002446 | Used to centrifuge samples |
| Vortex Mixer | VWR | G560 | Used to mix samples |
| Sonicator (mini) | Fisher Scientific | 15-337-22 | Used to separate beads |
| Pipettors P10, P20, P100, P1000, 8 ch. | Capp | Various | |
| Hemacytometer (Bright Line) | Housser Scientific | 3200 | Used to count coupled beads |
| Multiscreen Vacuum Manifold | Millipore | MSVMHTS00 | Used in washing steps to remove supernatant |
| MicroShaker | VWR | 12620-926 | Used to agitate beads during incubations |
| Tube rack (1.5mL and 0.5mL) (assorted) | VWR | 30128-346 | |
| Weighing Scale | Mettler or other | Used to measure wash reagents for making buffers | |
| Dynabead Sample Mixer | Invitrogen | 947-01 | Used during coupling incubation step |
| MatLab (R2014b) | The MathWorks, Inc. | Used to analyze antibody response data | |
| Microsoft Excel 2014 | Microsoft Corporation | Used to analyze antibody response data | |
| Luminex Analyzer with xPonent 3.1 software | Luminex Corporation | LX200-XPON3.1 | Instrument and software used to run assay |
| Antigens | |||
| GI.1 Norwalk Virus : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| GII.4 Norovirus VA387 : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| Hepatitis A Virus : grade II concentrate from cell culture | Meridian Life Sciences | 8505 | Antigen coupled at 100 µg |
| Helicobacter pylori : lysate | Meridian Life Sciences | R14101 | Antigen coupled at 25 µg |
| Toxoplasma gondii : recombinant p30 (SAG1) | Meridian Life Sciences | R18426 | Antigen coupled at 25 µg |
| Campylobacter jejuni : heat killed whole cells | KPL | 50-92-93 | Antigen coupled at 50 µg |
| Primary Antibodies | |||
| Guinea pig anti-Norovirus | (CCHMC)* | NA | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| BacTraceAffinity Purified Antibody to Helicobacter pylori | KPL | 01-93-94 | Used for coupling confirmation |
| Goat pAb to Toxoplasma gondii | Abcam | Ab23507 | Used for coupling confirmation |
| BacTrace Goat anti-Campylobacter species | KPL | 01-92-93 | Used for coupling confirmation |
| Secondary Antibodies | |||
| Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L) | Jackson | 705-065-149 | Used for coupling confirmation |
| Biotinylated Rabbit anti-Goat IgG (H+L) | KPL | 16-13-06 | Used for coupling confirmation |
| Biotinylated Goat anti-Mouse IgG (H+L) | KPL | 16-18-06 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG(H+L) | KPL | 176-1506 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(ᵞ) | KPL | 16-10-02 | Used for Salivary Immunoassay |
| Consumables | |||
| 1.5 mL copolymer microcentrifuge tubes | USA Scientific | 1415-2500 | Used as low binding microcentrifuge tubes |
| 10 µL pipette tip refills | BioVentures | 5030050C | |
| 200 µL pipette tip refills | BioVentures | 5030080C | |
| 1000 µL pipette tip refills | BioVentures | 5130140C | |
| Aluminum foil | Various Vendors | Used keep beads in the dark during incubations | |
| Deep Well plates | VWR | 40002-009 | Used for diluting saliva samples |
| Multiscreen Filter Plates | Millipore | MABVN1250 | Used to run assays |
| Oracol saliva collection system | Malvern Medical Developments Limited | Used for saliva collection | |
| Reagents | |||
| Carboxylated microspheres (beads) | Luminex Corporation | Dependent on bead set | Antigens are coupled to the microspheres |
| EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) | Pierce | 77149 or 22980 | Used in bead activation |
| Sulfo-NHS (N-hydroxysulfosuccinimide) | Pierce | 24510 | Used in bead activation |
| Steptavidin-R-phycoerythrin (1mg/mL) | Molecular Probes | S-866 | Used as reporter |
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | M-2933 | Used for coupling |
| Tween-20 (Polyoxyethylenesorbitan monolaurate) | Sigma | P-9416 | Used in wash buffer to remove non-specific binding |
| Protein Buffers | |||
| PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)** | Filter Sterilize and store at 4°C | ||
| PBS, pH 7.4 | Sigma | P-3813 | 138 mM NaCl, 2.7 mM KCl |
| BSA | Sigma | A-7888 | 0.1% (w/v) |
| Tween-20 | Sigma | P-9416 | 0.2% (v/v) |
| Sodium Azide (0.05% azide)** | Sigma | S-8032 | **Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE's. Dispose of according to applicable laws. |
| MES/ Coupling Buffer (0.05 M MES, pH 5.0) | |||
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | S-3139 | |
| 5 N NaOH | Fisher | SS256-500 | |
| Assay Buffer (PBS, 1% BSA, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 1% BSA, pH 7.4 | Sigma | P-3688 | 138 mM NaCl, 2.7 mM KCl, 1% BSA |
| Activation Buffer (0.1 M NaH2PO4, pH 6.2) | Filter Sterilize and store at 4°C | ||
| NaH2PO4 (Sodium phosphate, monobasic anhydrous) | Sigma | S-3139 | 0.1M NaH2PO4 |
| 5 N NaOH | Fisher | SS256-500 | |
| Wash Buffer (PBS, 0.05% Tween-20, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 0.05% Tween-20, pH 7.4 | Sigma | P-3563 | 138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN |
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Citar este artículo
Augustine, S. A. J., Eason, T. N., Simmons, K. J., Curioso, C. L., Griffin, S. M., Ramudit, M. K. D., Plunkett, T. R. Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens. J. Vis. Exp. (115), e54415, doi:10.3791/54415 (2016).