基于蛋黄抗体的细胞术微球测定的标准化
1Programa de Pós-Graduação em Biotecnologia da Universidade Federal do Amazonas – UFAM, 2Instituto Leônidas e Maria Deane (ILMD),Fundação Oswaldo Cruz (FIOCRUZ), 3Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro do Instituto Leônidas e Maria Deane (ILMD),Fundação Oswaldo Cruz (FIOCRUZ), 4Programa de Pós-graduação em Ciências Aplicadas à Hematologia – Universidade Estadual do Amazonas/Fundação Hospitalar de Hematologia e Hemoterapia do Amazonas, 5Programa de Pós-Graduação em Imunologia Básica e Aplicada, Instituto de Ciências Biológicas,Universidade Federal do Amazonas (UFAM)
Summary
该协议描述了一种使用IgY抗体进行抗原检测的乳胶珠制备乳胶珠的方法。
Abstract
免疫测定是检测多种分子靶标的重要测试。在目前可用的方法中，细胞仪微球测定在近几十年来越来越突出。设备读取的每个微球代表被测分子之间相互作用能力的分析事件。在一次检测中读取数千个此类事件，从而确保高检测准确性和重现性。该方法还可用于验证用于诊断疾病的新输入，例如IgY抗体。这些抗体是通过用目标抗原对鸡进行免疫，然后从动物鸡蛋的蛋黄中提取免疫球蛋白而获得的;因此，这是一种获得抗体的无痛且高效的方法。除了高精度验证该测定的抗体识别能力的方法外，本文还提出了一种提取这些抗体的方法，确定抗体和乳胶珠的最佳偶联条件，并确定测试的灵敏度。
Introduction
在旨在诊断疾病的免疫测定技术中，细胞术微球测定已成为一种高度灵敏和可靠的方法，因为它允许在一次测定中分析数千个颗粒1。该技术除了具有高生产率并允许使用较小体积的样品外，还具有极大的灵活性，因为它允许检测多种分子，例如细胞因子，粘附分子，抗体同种型和蛋白质2,3。
不同的颗粒用于开发这些测定，其中包括乳胶珠，这是一种有效且低成本的输入。这些可以在其表面呈现修饰，例如存在允许某些分子共价或非共价偶联的官能团或蛋白质3,4,5。
这些免疫测定使用抗原和抗体等成分来检测疾病标志物，通常需要来自哺乳动物（如小鼠、兔子和山羊）的抗体。这会产生与伦理问题相关的问题，因为哺乳动物的免疫通常需要许多动物才能获得良好的产量，以及导致动物痛苦的程序的频繁执行6,7。另一种方法是使用从免疫鸡的蛋黄中分离的 IgY 抗体，因为在蛋黄中可以找到高浓度的针对接种抗原的特异性抗体;一只鸡的产量相当于在一年内生产 4.3 只兔子 6,7。
因此，该协议的目的是提供一种使用乳胶珠流式细胞术评估从鸡蛋黄中获得的IgY抗体的方法。为此，我们提出了一种使用乳胶珠进行三明治形式的细胞术微球免疫测定的标准化方法。作为模型，我们使用针对恶性疟原虫富含组氨酸的蛋白II抗原（IgY-Pf HRP2）的IgY抗体。我们描述了一种提取抗体的方法，讨论了确定这些抗体与乳胶珠偶联浓度的关键步骤，并评估了抗原的检测限。流式细胞术的高精度，加上乳胶珠的低成本，使该技术适用于免疫测定工具的分析，例如抗体和抗原。该方法可用于检测各种目标。
Protocol
注意：有关本协议中使用的所有材料、试剂和仪器的详细信息，请参阅 材料表 。 1. 从蛋黄中提取IgY。 鸡蛋的卫生化将鸡蛋（新鲜产下或产卵后 4 天，来自 Gallus gallus Dekalb White 谱系）浸入 0.2% 稀释的次氯酸钠溶液中，在流水下快速冲洗，然后轻轻擦拭以备后用或立即使用。注意：如果不立即使用，请在4°C下保持长达15天。…
Representative Results
图2提供了 通过酸化 提取IgY抗体的过程，然后使用辛酸进行分离的图形表示（图2）。 图 2：IgY 抗体的提取步骤和使用辛酸分离的示意图 。 （A）分离蛋黄，在锥形管中回收，冷冻?…
Discussion
通过降低pH值然后使用辛酸进行脂质分离来沉淀IgY抗体的方法可有效地分离总抗体而不会损失任何功能。这里提出的方法比Redwan等人报告的方法更简单，更便宜11，后者也使用酸化和辛酸沉淀，但方案更复杂和费力。与常用的从蛋黄中分离IgY的方法相比，该方法也具有优势，例如使用PEG沉淀12,13，硫酸钠14,15和氯仿<sup class="xref"…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢FIOCRUZ（“Leônidas e Maria Deane – ILMD/Fiocruz Amazônia-PROEP-LABS/ILMD FIOCRUZ AMAZÔNIA”）、生物技术研究生课程（亚马逊联邦大学的PPGBIOTEC – UFAM）、尼加拉瓜佩索阿尔协会（CAPES）和亚马逊国家宪法权利保护基金会（FAPEAM）提供奖学金。图 2 和 图 4 是使用 biorender.com 创建的。
Materials
| Anti-Chicken IgY (H+L), highly cross-adsorbed, CF 488A antibody produced in donkey | Sigma-Aldrich | SAB4600031 | |
| Anti-mouse IgG (H+L), F(ab′)2 | Sigma-Aldrich | SAB4600388 | |
| BD FACSCanto II | BD Biosciences | BF-FACSC2 | |
| BD FACSDiva CS&T research beads (CS&T research beads) | BD Biosciences | 655050 | |
| BD FACSDiva software 7.0 | BD Biosciences | 655677 | |
| Bio-Rad Protein Assay Dye Reagent Concentrate | Bio-Rad | #5000006 | |
| Bovine serum albumin | Sigma-Aldrich | A4503 | |
| Caprilic acid | Sigma-Aldrich | O3907 | |
| Centrifuge 5702 R | Eppendorf | Z606936 | |
| Chloride 37% acid molecular grade | NEON | 02618 NEON | |
| CML latex, 4% w/v | Invitrogen | C37253 | |
| Megafuge 8R | Thermo Scientific | TS-HM8R | |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Hydrochloride Powder (EDC) | Sigma-Aldrich | E7750-1G | |
| N-Hydroxysuccinimide (NHS) | Sigma-Aldrich | 130672-25G | |
| Phosphate buffered saline | Sigma-Aldrich | 1003335620 | |
| Sodium hydroxide | Acs Cientifica | P.10.0594.024.00.27 | |
| Sodium hypochlorite | Acs Cientifica | R09211000 | |
| Thermo Mixer Heat/Cool | KASVI | K80-120R |
Referências
- Salzer, U., Sack, U., Fuchs, I. Flow cytometry in the diagnosis and follow up of human primary immunodeficiencies. Electronic Journal of the International Federation of Clinical Chemistry and Laboratory. 30 (4), 407 (2019).
- de Figueiredo, A. M., Glória, J. C., Chaves, Y. O., Neves, W. L. L., Mariúba, L. A. M. Diagnostic applications of microsphere-based flow cytometry: A review. Experimental Biology and Medicine. 247 (20), 1852-1861 (2022).
- Morgan, E., et al. Cytometric bead array: A multiplexed assay platform with applications in various areas of biology. Clinical Immunology. 110 (3), 252-266 (2004).
- Graham, H., Chandler, D. J., Dunbar, S. A. The genesis and evolution of bead-based multiplexing. Methods. 158, 2-11 (2019).
- Kellar, K. Multiplexed microsphere-based flow cytometric assays. Experimental Hematology. 30 (11), 1227-1237 (2002).
- Schade, R., et al. Chicken egg yolk antibodies (IgY-technology): A review of progress in production and use in research and human and veterinary medicine. ATLA Alternatives to Laboratory Animals. 33 (2), 129-154 (2005).
- Xu, Y., et al. Application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: A review. Biotechnology Advances. 29 (6), 860-868 (2011).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1-2), 248-254 (1976).
- Sousa, L. P., et al. A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments. BMC Infectious Diseases. 14 (1), 49 (2014).
- Klimentzou, P., et al. Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha. Peptides. 27 (1), 183-193 (2006).
- Redwan, E. M., Aljadawi, A. A., Uversky, V. N. Simple and efficient protocol for immunoglobulin Y purification from chicken egg yolk. Poultry Science. 100 (3), 100956 (2021).
- Lee, H. Y., Abeyrathne, E. D. N. S., Choi, I., Suh, J. W., Ahn, D. U. K. Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents. Poultry Science. 93 (10), 2668-2677 (2014).
- Pauly, D., Chacana, P., Calzado, E., Brembs, B., Schade, R. IgY Technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. Journal of Visualized Experiments. (51), e3084 (2011).
- Chang, H. M., Lu, T. C., Chen, C. C., Tu, Y. Y., Hwang, J. Y. Isolation of immunoglobulin from egg yolk by anionic polysaccharides. Journal of Agricultural and Food Chemistry. 48 (4), 995-999 (2000).
- Ko, K. Y., Ahn, D. U. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. Poultry Science. 86 (2), 400-407 (2007).
- Polson, A. Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunological Communications. 19 (3), 253-258 (1990).
- Simonova, M. A., et al. xMAP-based analysis of three most prevalent staphylococcal toxins in Staphylococcus aureus cultures. Analytical and Bioanalytical Chemistry. 406 (25), 6447-6452 (2014).
- Sharma, P., et al. A multiplex assay for detection of staphylococcal and streptococcal exotoxins. PLoS One. 10 (8), e0135986 (2015).
- Merbah, M., et al. Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-subtypes. Journal of Virological Methods. 230, 45-52 (2016).
Citar este artigo
Corrêa Glória, J., Oliveira Chaves, Y., Marques de Figueiredo, A., Coutinho de Souza, C., Duarte da Silva, E. R., Lopes Batista, J. C., Nogueira, P. A., Morais Mariúba, L. A. Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies. J. Vis. Exp. (195), e65123, doi:10.3791/65123 (2023).