JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Imunologia e Infecção

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Published: September 07, 2018
doi:
10.3791/57843
, ,
1Department of Cell Biology and Neuroscience and Graduate Program in Cellular and Molecular Pharmacology,Rutgers University, 2Department of Cell Biology and Neuroscience,Rutgers University, 3Rutgers Cancer Institute of New Jersey

Summary

本文描述了一种用 ELISA 方法表征小鼠 t 依赖性和 t 无关免疫球蛋白 (同种) 反应的协议。这种方法单独使用或结合流式细胞术将允许研究人员确定在 B 细胞介导的小鼠同种反应在 t 依赖性和 t 独立抗原免疫的差异。

Abstract

抗体, 也称为免疫球蛋白 (Ig), 分泌的分化 B 淋巴细胞, plasmablasts/血浆细胞, 在体液免疫提供了强大的防御入侵病原体通过不同的机制。疫苗接种的一个主要目标是诱导保护性抗原特异抗体, 以防止危及生命的感染。胸腺依赖 (TD) 和胸腺独立 (TI) 抗原可以引起强健的抗原特异性 IgM 反应, 也可以诱导生产同种交换抗体 (IgG, IgA 和 IgE), 以及产生的记忆 B 细胞的帮助由抗原呈现细胞 (apc) 提供。在这里, 我们描述了一个协议, 以表征 TD 和 TI 同种反应的小鼠使用酶联免疫吸附试验 (ELISA)。在本议定书中, 抗原-共轭模型抗原 TNP-KLH (明矾) 和 TNP 多糖 (PBS) 对小鼠进行了腹腔 (ip) 免疫, 从而诱发了 TD 和 TI 的反应。为诱发 TD 记忆反应, TNP-KLH 在明矾中的助推免疫接种是在第一次免疫后3周使用相同的抗原/佐剂。小鼠血清在免疫前后的不同时间点收获。血清总免疫球蛋白水平和 TNP 特异抗体随后分别使用同种特异性三明治和间接 ELISA 进行量化。为了正确量化每个同种的血清浓度, 样品需要适当稀释, 以适应标准曲线的线性范围。使用该协议, 我们一直得到可靠的结果, 具有较高的特异性和灵敏度。当与其他补充方法, 如流式细胞术,在体外培养脾脏 B 细胞和免疫组化染色 (IHC), 这项协议将允许研究人员获得全面了解抗体给定实验设置中的响应。

Introduction

B 淋巴细胞是体液免疫的主要参与者, 是哺乳动物中唯一能够产生抗体的细胞类型, 也称为免疫球蛋白 (Ig)1,2。B 细胞分泌的抗体通过多种机制, 包括中和、opsonization 和补充活化, 为入侵病原体提供了强大的防御, 导致保护性免疫3。b 细胞分泌抗体仅在特定 B 细胞完全活化后才能实现, 通常需要两个不同的信号3。信号1由抗原 (Ag) 的直接捆绑中继到 b 细胞受体 (BCR) 在特定天真 b 细胞的表面表达3。根据信号来源 2, B 细胞活化可分为胸腺依赖性 (TD) 或胸腺独立 (TI)3,4。在 TD 抗原反应中, 信号2由活化同源 CD4 t (tH) 细胞提供, 表达 CD154, 在 B 细胞1,2,3表达的共刺激受体 CD40 的配体。在 TI 抗原反应中, 信号2来自于与收费类似的受体的接触 (TLRs 在1型 ti ag 的情况下) 或 BCRs 的广泛交联 (在2钛 ag 的情况下) 在 B 细胞3,4。1型钛 (TI-1) 抗原是 TLRs 的微生物配体, 包括细菌脂多糖 (LPS)、病毒 rna 和微生物 CpG DNA4、5。2型钛 (TI-2) 抗原具有高度重复性的结构, 并且能够通过 BCRs4,6的多重交联, 向 B 细胞传递长期持久的信号。TI-2 抗原的典型例子包括肺炎球菌多糖和抗原共轭多糖6,7。TD 和 TI 抗原均可诱发强健的抗原特异性 IgM 反应, 也可诱发同种交换抗体 (IgG、IgA 和 IgE) 的产生, 并借助抗原呈现细胞 (apc) 提供的帮助, 如树突状细胞 (DCs)1 ,2,3。此外, td 和 TI 抗原都能在 apc 的帮助下诱发记忆反应, 但 td 抗原在诱导记忆 B 细胞生成3、8时更有效。

在本议定书中, 通过腹腔 (ip) 免疫与抗原共轭模型抗原 24, 6-trinitrophenyl 锁孔帽贝血蓝蛋白 (TNP KLH) 和 TNP 多糖 (中性, 高度分枝) 诱发小鼠的 TD 和 TI 的反应。和高质量), 分别为9,10,11。TD 抗原通常与佐剂一起使用, 以提高抗体的产生12。在我们的协议中, TNP KLH 注射了明矾, 这是免疫研究中常用的佐剂12。其他可以使用的佐剂的例子包括完整或不完整的弗氏的辅助 (CFA 或 IFA), 单磷酰脂 A/海藻糖 dicorynomycolate (“Ribi” 佐剂), 和中央核苷酸等13, 14. 免疫后, 小鼠血清在不同的时间点收获, 血清中的 TNP 特异抗体使用同种特异的酶联免疫吸附试验 (ELISA)9、10进行量化,11。

ELISA 是一种以板材为基础的检测方法, 广泛应用于医学诊断工具, 也是生物医学研究15、16的分析工具。它用于检测和量化包括抗体、激素、细胞因子、趋化因子和各种抗原等的物质。elisa 可以用几种不同的格式进行, 包括直接、间接、三明治和竞争性 elisa15、16。一般而言, 它涉及将抗原固定在固体表面, 通常是一个96井微量滴定板, 它是用一个主要抗体孵化的。孵化后, 未绑定抗体被冲走。在直接 ELISA 中, 主要抗体直接与一种酶 (典型的辣根过氧化物酶或碱性磷酸酶) 结合, 它可以将显色基底切割成一个由信号检测仪器检测到的可见颜色变化, 如分光光度计15,16。相比之下, 如果用酶链二次抗体来结合原抗体, 那么这被认为是间接 ELISA15,16。直接 elisa 更快, 而间接 elisa 更敏感15,16。在三明治 ELISA 中, 这些盘子上涂有一种 “捕获” 抗体, 用来固定样本中感兴趣的抗原, 然后用另一种 “检测” 抗体直接或间接地检测到捕获的抗原15,16. 三明治 ELISA 具有很高的特异性, 因为抗原是由两种不同的抗原抗体检测出来的。在竞争性 ELISA 中, 在样本抗原和板块绑定抗原之间建立了与原抗体结合的竞争, 然后通过测量基片中信号的减少来量化样本中的抗原浓度。15,16. 竞争性 ELISA 可使用上述直接或间接格式进行, 对仅有一个表位15、16的小抗原的检测非常有用。

抗体测量的替代技术包括无线电免疫分析 (RIA), 电化学发光 (ECL) 检测和表面等离子共振 (SPR) 检测17。RIA 是第一个利用核素试剂18,19测量抗原 (或抗体) 具有高度特异性和灵敏度的免疫分析法。然而, 由于对放射性毒性、处置成本、保质期以及使用放射性材料的特殊许可证等问题的关注, ELISA 是一种比较方便的常用20、21技术。ECL 是一种高度敏感的检测方法, 其中化学发光反应是利用电产生高活性物种从电极表面的稳定前体, 并可用于测量的数量的分析 (如抗原或抗体)22。但是, ECL 需要一种特殊的仪器, 因此不像 ELISA23那样广泛使用。SPR 是一种直接的检测方法, 可用于测量传感器芯片表面上固定化分子 (e.、抗原) 的配体 (e.、抗体) 的结合情况24。SPR 非常明确地检测了交互作用, 不需要使用标记试剂作为 ELISA。然而, SPR 还需要一个特殊的设备, 灵敏度低于 ELISA17。鉴于替代方法的局限性, ELISA 是本协议中最适合我们使用的技术。在这里, 我们描述了使用三明治 elisa 分析总的同种水平和间接 ELISA 的程序, 以分析抗原特异性的 isotypes。

Protocol

本议定书遵循罗格斯大学机构动物研究伦理学委员会的指导方针。所有的老鼠都是按照 NIH 的指导方针和动物保育和使用委员会批准的一项牲畜协议使用的。 1. 小鼠的制备和单纯小鼠血清的收集 保持所有老鼠在一个特定的无病原动物设施的免疫实验。 使用与性别匹配的, 年轻的成人 (8–12周老) 淘汰赛和 littermate 控制小鼠, 共享相同的父母和笼子进行免疫研究。…

Representative Results

我们使用这个协议来调查免疫系统的一个关键调节器, TRAF3, 在 TI 和 TD 同种反应9,10,11的作用。TRAF3 直接或间接地调节了一些先天和适应性免疫受体的信号传导, 包括 TNF 受体超家族、类似收费受体和 T 细胞 receptor/CD28, 其中27,28。我们推测 TRAF3 在不同的免疫细?…

Discussion

在这里, 我们描述的协议, 以表征的 TD 和 TI 同种反应的小鼠使用 ELISA。成功实施本议定书需要使用表 1中规定的材料, 包括 ELISA 化验板、免疫 Ags、小鼠同种特异抗体和标准。应注意避免使用组织培养治疗板的 ELISA。稀释的标准和血清样品应在单独未经处理的板 (圆底), 然后添加到 ELISA 板。使用该协议, 我们一直得到可靠的结果, 具有较高的特异性和灵敏度。

此协?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国国立卫生研究院资助的 R01 CA158402 (p 谢) 和 R21 AI128264 (p 谢), 国防部赠款 W81XWH-13-1-0242 (p 谢), 一个试点奖从新泽西州癌症研究所通过批准号码 P30CA072720来自国家癌症研究所 (p 谢), 一个布希生物医学补助金 (p 谢), 一个胜利者 Stollar 奖学金 (a Lalani), 和安妮 b. 和詹姆斯 b. Leathem 奖学金 (朱)。

Materials

VersaMax Tunable Microplate Reader MDS Analytical Technologies VERSAMAX Equipment to read the plates
SOFTmax PRO 5.3 MDS Analytical Technologies SOFTmax PRO 5.3 Software for the plate reader
GraphPad Prism GraphPad Prism Software for graphing and statistics
TNP-AECM-polysaccharide (FICOLL) Biosearch Technologies F-1300-10 A TI Ag for immunization
TNP-KLH Biosearch Technologies T-5060-5 A TD Ag for immunization
TNP(38)-BSA Biosearch Technologies T-5050-10 (conjugation ratio: 38) Coating Ag for TNP-specific ELISA
TNP(3)-BSA Biosearch Technologies T-5050-10 (conjugation ratio: 3) Coating Ag for high affinity TNP-specific Ig
Imject Alum Fisher Scientific  PI-77161 Alum adjuvant for immunization
Falcon Polypropylene tubes Fisher Scientific  14-959-11A For incubation of TNP-KLH/alum
BD Insulin Syringe Fisher Scientific  14-829-1B For i.p. injection of mice
Immuno 96-Well Plates, Flat-Bottom Fisher Scientific  14-245-61 For ELISA
Untreated 96-Well Microplates, Round-Bottom VWR 82050-622 For serial dilutions of standards and samples
Phosphatase substrate, 5 mg Tablets Sigma S0942-200TAB AP substrate
Diethanolamine VWR IC15251690 A component of AP substrate buffer
Goat anti-mouse IgM SouthernBiotech 1020-01 Capture Ab for mouse IgM
Goat anti-mouse IgG1 SouthernBiotech 1070-01 Capture Ab for mouse IgG1
Goat anti-mouse IgG2a SouthernBiotech 1080-01 Capture Ab for mouse IgG2a
Goat anti-mouse IgG2b SouthernBiotech 1090-01 Capture Ab for mouse IgG2b
Goat anti-mouse IgG3 SouthernBiotech 1100-01 Capture Ab for mouse IgG3
Goat anti-mouse IgA SouthernBiotech 1040-01 Capture Ab for mouse IgA
Goat anti-mouse IgE SouthernBiotech 1110-01 Capture Ab for mouse IgE
AP-Goat anti-mouse IgM SouthernBiotech 1020-04 Detection Ab for mouse IgM
AP-Goat anti-mouse IgG1 SouthernBiotech 1070-04 Detection Ab for mouse IgG1
AP-Goat anti-mouse IgG2a SouthernBiotech 1080-04 Detection Ab for mouse IgG2a
AP-Goat anti-mouse IgG2b SouthernBiotech 1090-04 Detection Ab for mouse IgG2b
AP-Goat anti-mouse IgG3 SouthernBiotech 1100-04 Detection Ab for mouse IgG3
AP-Goat anti-mouse IgA SouthernBiotech 1040-04 Detection Ab for mouse IgA
AP-Goat anti-mouse IgE SouthernBiotech 1110-04 Detection Ab for mouse IgE
Mouse IgM standard BD Biosciences 553472 TNP-specific IgM, Clone  G155-228
Mouse IgG1 standard BD Biosciences 554054 TNP-specific IgG1, Clone  107.3
Mouse IgG2a standard BD Biosciences 556651 TNP-specific IgG2a, Clone  G155-178
Mouse IgG2b standard BD Biosciences 554055 TNP-specific IgG2b, Clone  49.2
Mouse IgG3 standard BD Biosciences 553486 KLH-specific IgG3, Clone  A112-3
Mouse IgA standard BD Biosciences 550924 Mineral oil-induced IgA, Clone  MOPC-320
Mouse IgE standard BD Biosciences 557079 TNP-specific IgE, Clone  C38-2
DOWNLOAD MATERIALS LIST

Referências

  1. Moise, A., Nedelcu, F. D., Toader, M. A., Sora, S. M., Tica, A., Ferastraoaru, D. E., Constantinescu, I. Primary immunodeficiencies of the B lymphocyte. Journal of Medicine and Life. 3, 60-63 (2010).
  2. Bishop, G. A., Haxhinasto, S. A., Stunz, L. L., Hostager, B. S. Antigen-specific B-lymphocyte activation. Critical Reviews in Immunology. 23, 149-197 (2003).
  3. Murphy, K. . Janeway’s Immunobiology. 8th Edition. 1, (2012).
  4. Vinuesa, C. G., Chang, P. P. Innate B cell helpers reveal novel types of antibody responses. Nature Immunology. 14, 119-126 (2013).
  5. Bekeredjian-Ding, I., Jego, G. Toll-like receptors–sentries in the B-cell response. Immunology. 128, 311-323 (2009).
  6. Mond, J. J., Lees, A., Snapper, C. M. T cell-independent antigens type 2. Annual Review of Immunology. 13, 655-692 (1995).
  7. Garcia de Vinuesa, C., O’Leary, P., Sze, D. M., Toellner, K. M., MacLennan, I. C. T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci. European Journal of Immunology. 29, 1314-1323 (1999).
  8. Kurosaki, T., Kometani, K., Ise, W. Memory B cells. Nature Reviews Immunology. 15, 149-159 (2015).
  9. Xie, P., Stunz, L. L., Larison, K. D., Yang, B., Bishop, G. A. Tumor necrosis factor receptor-associated factor 3 is a critical regulator of B cell homeostasis in secondary lymphoid organs. Immunity. 27, 253-267 (2007).
  10. Xie, P., Kraus, Z. J., Stunz, L. L., Liu, Y., Bishop, G. A. TNF Receptor-Associated Factor 3 Is Required for T Cell-Mediated Immunity and TCR/CD28 Signaling. The Journal of Immunology. 186, 143-155 (2011).
  11. Lalani, A. I., Moore, C. R., Luo, C., Kreider, B. Z., Liu, Y., Morse, H. C., Xie, P. Myeloid Cell TRAF3 Regulates Immune Responses and Inhibits Inflammation and Tumor Development in Mice. The Journal of Immunology. 194, 334-348 (2015).
  12. Lee, S., Nguyen, M. T. Recent advances of vaccine adjuvants for infectious diseases. Immune Network. 15, 51-57 (2015).
  13. Gavin, A. L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B., Nemazee, D. Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science. 314, 1936-1938 (2006).
  14. Klinman, D. M., Currie, D., Gursel, I., Verthelyi, D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunological Reviews. 199, 201-216 (2004).
  15. Gan, S. D., Patel, K. R. Enzyme immunoassay and enzyme-linked immunosorbent assay. Journal of Investigative Dermatology. 133, 12 (2013).
  16. Shah, K., Maghsoudlou, P. Enzyme-linked immunosorbent assay (ELISA): the basics. British Journal of Hospital Medicine. 77, 98-101 (2016).
  17. Nencini, F., Pratesi, S., Petroni, G., Matucci, A., Maggi, E., Vultaggio, A. Assays and strategies for immunogenicity assessment of biological agents. Drug Development Research. 75, 4-6 (2014).
  18. Haber, E., Page, L. B., Richards, F. F. Radio immunoassay employing gel filtration. Analytical Biochemistry. 12, 163-172 (1965).
  19. Yalow, R. S., Berson, S. A. Immunoassay of endogenous plasma insulin in man. 1960. Obesity Research. 4, 583-600 (1996).
  20. Lequin, R. M. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry. 51, 2415-2418 (2005).
  21. Wreghitt, T. G., Tedder, R. S., Nagington, J., Ferns, R. B. Antibody assays for varicella-zoster virus: comparison of competitive enzyme-linked immunosorbent assay (ELISA), competitive radioimmunoassay (RIA), complement fixation, and indirect immunofluorescence assays. Journal of Medical Virology. 13, 361-370 (1984).
  22. Mathew, B. C., Biju, R. S., Thapalia, N. An overview of electrochemiluminescent (ECL) technology in laboratory investigations. Kathmandu University Medical Journal. 3, 91-93 (2005).
  23. Mikulskis, A., Yeung, D., Subramanyam, M., Amaravadi, L. Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development. The Journal of Immunological Methods. 365, 38-49 (2011).
  24. Wadhwa, M., Bird, C., Dilger, P., Gaines-Das, R., Thorpe, R. Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals. The Journal of Immunological Methods. 278, 1-17 (2003).
  25. Wolforth, J. B. Methods of Blood Collection in the Mouse. Laboratory Animals. 29, 47-53 (2000).
  26. Stunz, L. L., Busch, L. K., Munroe, M. E., Sigmund, C. D., Tygrett, L. T., Waldschmidt, T. J., Bishop, G. A. Expression of the Cytoplasmic Tail of LMP1 in Mice Induces Hyperactivation of B Lymphocytes and Disordered Lymphoid Architecture. Immunity. 21, 255-266 (2004).
  27. Xie, P. TRAF molecules in cell signaling and in human diseases. Journal of Molecular Signaling. 8, 7 (2013).
  28. Lalani, A. I., Zhu, S., Gokhale, S., Jin, J., Xie, P. TRAF molecules in inflammation and inflammatory diseases. Current Pharmacology Reports. 4, 64-90 (2018).
  29. Tate, J., Ward, G. Interferences in immunoassay. The Clinical Biochemist Reviews. 25, 105-120 (2004).
  30. Specter, S., Friedman, H. Age- and sex-related differences in antibody formation and blastogenic responsiveness of splenocytes from RIII mice developing virus-induced mammary adenocarcinoma. The Journal of the National Cancer Institute. 67, 1347-1351 (1981).
  31. Giefing-Kroll, C., Berger, P., Lepperdinger, G., Grubeck-Loebenstein, B. How sex and age affect immune responses, susceptibility to infections, and response to vaccination. Aging Cell. 14, 309-321 (2015).
  32. Kaminski, D. A., Stavnezer, J. Antibody class switching differs among SJL, C57BL/6 and 129 mice. International Immunology. 19, 545-556 (2007).
  33. Sellers, R. S., Clifford, C. B., Treuting, P. M., Brayton, C. Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice. Veterinary Pathology. 49, 32-43 (2012).
  34. Conour, L. A., Murray, K. A., Brown, M. J. Preparation of animals for research–issues to consider for rodents and rabbits. Institute of Laboratory Animal Resources Journal. 47, 283-293 (2006).
  35. Vlkova, M., Rohousova, I., Hostomska, J., Pohankova, L., Zidkova, L., Drahota, J., Valenzuela, J. G., Volf, P. Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi. PLOS Neglected Tropical Diseases. 6, 1719 (2012).
  36. Mestas, J., Hughes, C. C. Of mice and not men: differences between mouse and human immunology. The Journal of Immunology. 172, 2731-2738 (2004).
  37. Ruane, D., Chorny, A., Lee, H., Faith, J., Pandey, G., Shan, M., Simchoni, N., Rahman, A., Garg, A., Weinstein, E. G., et al. Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses. The Journal of Experimental Medicine. 213, 53-73 (2016).
  38. Chorny, A., Puga, I., Cerutti, A. Innate signaling networks in mucosal IgA class switching. Advances in Immunology. 107, 31-69 (2010).
  39. Van Praet, J. T., Donovan, E., Vanassche, I., Drennan, M. B., Windels, F., Dendooven, A., Allais, L., Cuvelier, C. A., van de Loo, F., Norris, P. S., et al. Commensal microbiota influence systemic autoimmune responses. The EMBO Journal. 34, 466-474 (2015).
  40. Nguyen, Q. N., Himes, J. E., Martinez, D. R., Permar, S. R. The Impact of the Gut Microbiota on Humoral Immunity to Pathogens and Vaccination in Early Infancy. PLOS Pathogens. 12, 1005997 (2016).
  41. Jeevan-Raj, B. P., Robert, I., Heyer, V., Page, A., Wang, J. H., Cammas, F., Alt, F. W., Losson, R., Reina-San-Martin, B. Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination. The Journal of Experimental Medicine. 208, 1649-1660 (2011).
  42. Chen, Z., Getahun, A., Chen, X., Dollin, Y., Cambier, J. C., Wang, J. H. Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination. The Journal of Immunology. 195, 5461-5471 (2015).
  43. Boboila, C., Yan, C., Wesemann, D. R., Jankovic, M., Wang, J. H., Manis, J., Nussenzweig, A., Nussenzweig, M., Alt, F. W. Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4. The Journal of Experimental Medicine. 207, 417-427 (2010).
  44. Shah, H. B., Koelsch, K. A. B-Cell ELISPOT: For the Identification of Antigen-Specific Antibody-Secreting Cells. Methods in Molecular Biology. 1312, 419-426 (2015).
  45. Bonsignori, M., Moody, M. A. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay. Cells. 1, 15-26 (2012).
  46. Sasaki, Y., Derudder, E., Hobeika, E., Pelanda, R., Reth, M., Rajewsky, K., Schmidt-Supprian, M. Canonical NF-kappaB activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation. Immunity. 24, 729-739 (2006).
  47. Goodlad, J. R., Macartney, J. C. Germinal-center cell proliferation in response to T-independent antigens: a stathmokinetic, morphometric and immunohistochemical study in vivo. European Journal of Immunology. 25, 1918-1926 (1995).
  48. Xie, P., Poovassery, J., Stunz, L. L., Smith, S. M., Schultz, M. L., Carlin, L. E., Bishop, G. A. Enhanced Toll-like receptor (TLR) responses of TNFR-associated factor 3 (TRAF3)-deficient B lymphocytes. Journal of Leukocyte Biology. 90, 1149-1157 (2011).
  49. Kaku, H., Horikawa, K., Obata, Y., Kato, I., Okamoto, H., Sakaguchi, N., Gerondakis, S., Takatsu, K. NF-kappaB is required for CD38-mediated induction of C(gamma)1 germline transcripts in murine B lymphocytes. International Immunology. 14, 1055-1064 (2002).
  50. Dudley, D. D., Chaudhuri, J., Bassing, C. H., Alt, F. W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. Advances in Immunology. 86, 43-112 (2005).
  51. Lange, H., Hecht, O., Zemlin, M., Trad, A., Tanasa, R. I., Schroeder, H. W., Lemke, H. Immunoglobulin class switching appears to be regulated by B-cell antigen receptor-specific T-cell action. European Journal of Immunology. 42, 1016-1029 (2012).
  52. Moore, C. R., Liu, Y., Shao, C. S., Covey, L. R., Morse, H. C., Xie, P. Specific deletion of TRAF3 in B lymphocytes leads to B lymphoma development in mice. Leukemia. 26, 1122-1127 (2012).
  53. Bergmann, B., Grimsholm, O., Thorarinsdottir, K., Ren, W., Jirholt, P., Gjertsson, I., Martensson, I. L. Memory B cells in mouse models. Scandinavian Journal of Immunology. 78, 149-156 (2013).
  54. McHeyzer-Williams, L. J., Milpied, P. J., Okitsu, S. L., McHeyzer-Williams, M. G. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nature Immunology. 16, 296-305 (2015).
  55. Elgueta, R., Marks, E., Nowak, E., Menezes, S., Benson, M., Raman, V. S., Ortiz, C., O’Connell, S., Hess, H., Lord, G. M., et al. CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen. The Journal of Immunology. 194, 505-513 (2015).

Tags

– Enzyme-linked Immunosorbent Assay (ELISA)- Immunoglobulin (Ig) Isotype- Thymus-dependent- Thymus-independent- B Cell Activation- Germinal Center- TNP-polysaccharide- Retro-orbital Bleeding- Ig Isotype-specific Capturing Antibodies- TNP-38BSA Antigen- TNP-3BSA Antigen- Blocking Buffer- Ig Isotype Standard
check_url/pt/57843?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Lalani, A. I., Zhu, S., Xie, P. Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay. J. Vis. Exp. (139), e57843, doi:10.3791/57843 (2018).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image