Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Isfahan R. Chambers; Tiffany R. Cone; Kyra Oswald-Richter; Wonder P. Drake

doi:10.3791/2221

JoVE Journal > Immunology and Infection

Please note that all translations are automatically generated. Click here for the English version.

Imunologia e Infecção

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Published: November 23, 2010

doi:

10.3791/2221

Isfahan R. Chambers*¹, Tiffany R. Cone*¹, Kyra Oswald-Richter, Wonder P. Drake²

¹Department of Medicine,Vanderbilt University School of Medicine, ²Department of Microbiology and Immunology,Vanderbilt University School of Medicine

Summary

可以通过使用的酶联免疫检测鉴定的微生物指标在特发性疾病的适应性免疫。

Abstract

适应性免疫是清除细胞内病原体的一个重要组成部分。检测和量化这些反应在人类的能力是一个重要的诊断工具。酶联免疫斑点法（ELISPOT）获得其对微生物的抗原，包括人群，如那些与艾滋病毒感染，移植免疫抑制，并使用类固醇的细胞免疫反应的能力，以确定的普及。此实验的能力量化针对特定微生物抗原的免疫反应，以及区分这些对策Th1或Th2型性格。酶联免疫斑点法不限于炎症部位。它是多才多艺的，在其评估的外周血，以及支气管肺泡灌洗，脑脊髓液，腹水，如积极参与网站内的免疫反应的能力。对一个或多个抗原免疫反应的检测是可能的，以及微生物蛋白质内的特定抗原决定簇。此实验有利于检测的免疫反应，随着时间的推移，以及在宿主T细胞识别抗原的区别。双色酶联免疫斑点法检测可用于检测两种细胞因子的同时的表达。该技术的最新应用，包括诊断为肺外结核，以及调查传染病抗原的自身免疫性疾病的贡献。

Protocol

对于下面的协议，我们用外周血单个核细胞（PBMC）的细胞的利息。然而，该协议可用于其他类型的细胞。使用无菌技术在组织培养罩进行检测。第1天：板和细胞的制备取出ELISPOT板，并在引擎盖打开它。 150μL，使用多通道移液器的1X PBS洗板3次，如果有的话，是可以接受的，否则单以及吹打。拿出一包水库，打开引擎盖。新增2微克/…

Discussion

酶联免疫斑点法检测已成为最重要和最广泛使用的检测，监测人体的免疫反应和各种其他物种。酶联免疫斑点法检测，免疫细胞的频率可以测量在单细胞水平上没有膨胀或操纵细胞群。酶联免疫斑点法检测已被广泛应用于各种疾病，包括感染，癌症，过敏症和自身免疫性疾病的抗原特异性免疫反应调查。这个实验中的应用已基本适用于量化是非常重要的抗原特异性细胞或特异性抗原的免疫反应，?…

Declarações

The authors have nothing to disclose.

Acknowledgements

范德比尔特CTSA的成员授予1 UL1 RR024975的一部分，由国家研究资源，国立卫生研究院中心的支持。这项工作是由美国国立卫生研究院R01 HL 83839; R01人工智能65744。

Materials

A. Preparation of Media

B. Preparation of Tris Buffer

Add 800 ml of ddH₂O to 1 L beaker.
Measure 12 grams of Tris and add to the beaker.
Add 0.12 grams of magnesium chloride hexahydrate (MgCl₂:6H₂O) into the beaker.
Stir with magnetic stirring rod on automatic stirring plate.
Once dissolved, pH the solution to 9.5.
Add 200 mL of ddH₂O to the beaker, bringing it up to 1000mL.
Filter the solution with a 0.22 μm filter.

C. Materials necessary for ELISPOT analysis:

Referências

Dosanjh, D. P., Hinks, T. S., Innes, J. A., Deeks, J. J., Pasvol, G., Hackforth, S., Varia, H., Millington, K. A., Gunatheesan, R., Guyot-Revol, V., Lalvani, A. Improved diagnostic evaluation of suspected tuberculosis. Ann Intern Med. 148, 325-336 (2008).
Sung, H. K. E. Diagnosis of abdominal tuberculosis by T-cell based assays on peripheral blood and peritoneal fluid mononuclear cells. Journal of Infection. 59, 409-415 (2009).
Kim, S. -. H. Diagnosis of Central Nervous System Tuberculosis by T-Cell-Based Assays on Peripheral Blood and Cerebrospinal Fluid Mononuclear Cells. Clinical and Vaccine Immunology. , 1356-1362 (2008).
Drake, W. P., Dhason, M. S., Nadaf, M., Shepherd, B. E., Vadivelu, S., Hajizadeh, R., Newman, L. S., Kalams, S. A. Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis. Infect Immun. 75, 527-530 (2007).
Allen, S. S., Evans, W., Carlisle, J., Hajizadeh, R., Nadaf, M., Shepherd, B. E., Pride, D. T., Johnson, J. E., Drake, W. P. Superoxide dismutase A antigens derived from molecular analysis of sarcoidosis granulomas elicit systemic Th-1 immune responses. Respir Res. 25, 36-36 (2008).
Mutsvangwa, J., Millington, K. A., Chaka, K., Mavhudzi, T., Cheung, Y. B., Mason, P. R., Butterworth, A. E., Corbett, E. L., Lalvani, A. Identifying recent Mycobacterium tuberculosis transmission in the setting of high HIV and TB burden. Thorax. 65, 315-320 (2010).
Streeck, H., Frahm, N., Walker, B. D. The role of IFN-gamma Elispot assay in HIV vaccine research. Nat Protoc. 4, 461-469 (2009).
Chaudhary, N., Staab, J. F., Marr, K. A. Healthy human T-Cell Responses to Aspergillus fumigatus antigens. PLoS One. 5, e9036-e9036 (2010).
Manigold, T., Mori, A., Graumann, R., Llop, E., Simon, V., Ferr, S. M., Valdivieso, F., Castillo, C., Hjelle, B., Vial, P. Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus. PLoS Pathog. 6, e1000779-e1000779 (2010).
Ndongala, M. L., Kamya, P., Boulet, S., Peretz, Y., Rouleau, D., Tremblay, C., Leblanc, R., Côté, P., Baril, J. G., Thomas, R., Vézina, S., Boulassel, M. R., Routy, J. P., Sékaly, R. P., Bernard, N. F. Changes in function of HIV-specific T-cell responses with increasing time from infection. Viral Immunol. 2, 159-168 (2010).

Play Video

PDF

DOI

DOWNLOAD MATERIALS LIST

Citar este artigo

Chambers, I. R., Cone, T. R., Oswald-Richter, K., Drake, W. P. Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens. J. Vis. Exp. (45), e2221, doi:10.3791/2221 (2010).