Chapter 30 : Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT)

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT), Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818722/9781555818715_CH30-1.gif /docserver/preview/fulltext/10.1128/9781555818722/9781555818715_CH30-2.gif


CD3CD8TcR αβ thymus-derived T cells form the major effector cell component of the immune response against intracellular pathogens. Naive CD8 T cells encounter antigen, expand, and divide, and effector cells mediate a range of functions. These include major histocompatibility complex (MHC) class I restricted killing of infected host cells, noncytolytic suppression, and release of soluble cytokines and chemokines. After the acute infection is contained the number of active effector cells declines, but antigen-specific memory CD8 T cells persist, ready to expand rapidly upon antigen re-exposure. Most CD8 antigen-specific T cells recognize viral antigen as a peptide presented by an MHC class I molecule. The T-cell receptor of the antigen-specific CD8 cell interacts with the MHC class I antigenic peptide complex on the surface of the antigen-presenting cell (1).

Citation: Shacklett B, Nixon D. 2016. Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT), p 290-295. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch30
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Murphy K, . 2012. T cell-mediated immunity, p 335 352. In Murphy K (ed), Janeway's Immunology, 8th ed. Garland Science, New York, NY.
2. Picker LJ, Singh MK, Zdraveski Z, Treer JR, Waldrop SL, Bergstresser PR, Maino VC . 1995. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86 : 1408 1419.[PubMed]
3. Lamoreaux L, Roederer M, and, Koup R . 2006. Intracellular cytokine optimization and standard operating procedure. Nature Protocols 1 : 1507 1516.[CrossRef].[PubMed]
4. Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM . 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274 : 94 96.[PubMed].[CrossRef]
5. Doherty PC . 1998. The new numerology of immunity mediated by virus-specific CD8 + T cells. Curr Opin Microbiol 1 : 419 422.[PubMed].[CrossRef]
6. McMichael AJ, O'Callaghan CA . 1998. A new look at T cells. J Exp Med 187 : 1367 1371.[PubMed].[CrossRef]
7. Russell ND, Hudgens MG, Ha R, Havenar-Daughton C, McElrath MJ . 2003. Moving to human immunodeficiency virus type 1 vaccine efficacy trials: defining T cell responses as potential correlates of immunity. J Infect Dis 187 : 226 242.[CrossRef].[PubMed]
8. Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A . 1983. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65 : 109 121.[PubMed].[CrossRef]
9. Czerkinsky C, Andersson G, Ekre HP, Nilsson LA, Klareskog L, Ouchterlony O . 1988. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 110 : 29 36.[PubMed].[CrossRef]
10. Weiskopf D, Angelo MA, de Azeredo EL, Sidney J, Greenbaum JA, Fernando AN, Broadwater A, Kolla RV, De Silva AD, de Silva AM, Mattia KA, Doranz BJ, Grey HM, Shresta S, Peters B, Sette A . 2013. Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8 + T cells. Proc Natl Acad Sci USA 110 : E2046 2053.[CrossRef].[PubMed]
11. Ledgerwood JE, Hu Z, Gordon IJ, Yamshchikov G, Enama ME, Plummer S, Bailer R, Pearce MB, Tumpey TM, Koup RA, Mascola JR, Nabel GJ, Graham BS . 2012. Influenza virus h5 DNA vaccination is immunogenic by intramuscular and intradermal routes in humans. Clin Vaccine Immunol 19 : 1792 1797.[CrossRef].[PubMed]
12. Lindestam Arlehamn CS, Gerasimova A, Mele F, Henderson R, Swann J, Greenbaum JA, Kim Y, Sidney J, James EA, Taplitz R, McKinney DM, Kwok WW, Grey H, Sallusto F, Peters B, Sette A . 2013. Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3 +CCR6 + Th1 subset. PLoS Pathog 9 : e1003130.[CrossRef].[PubMed]
13. Danta M, Semmo N, Fabris P, Brown D, Pybus OG, Sabin CA, Bhagani S, Emery VC, Dusheiko GM, Klenerman P . 2008. Impact of HIV on host-virus interactions during early hepatitis C virus infection. J Infect Dis 197 : 1558 1566.[CrossRef].[PubMed]
14. Ferraro B, Talbott KT, Balakrishnan A, Cisper N, Morrow MP, Hutnick NA, Myles DJ, Shedlock DJ, Obeng-Adjei N, Yan J, Kayatani AK, Richie N, Cabrera W, Shiver R, Khan AS, Brown AS, Yang M, Wille-Reece U, Birkett AJ, Sardesai NY, Weiner DB . 2013. Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA. Infect Immun 81 : 3709 3720.[CrossRef].[PubMed]
15. Kim KH, Greenfield W, Shotts E, Nakagawa M . 2007. Detection of human papillomavirus type 16-specific T lymphocytes by a recombinant vaccinia virus-based enzyme-linked immunospot assay. Clin Vaccine Immunol 14 : 362 368.[CrossRef].[PubMed]
16. Ewer K, Deeks J, Alvarez L, Bryant G, Waller S, Andersen P, Monk P, Lalvani A . 2003. Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 361 : 1168 1173.[CrossRef].[PubMed]
17. Asai T, Storkus WJ, Whiteside TL . 2000. Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol 7 : 145 154.[PubMed]
18. Maier R, Miller S, Kurrer M, Krebs P, de Giuli R, Kremer M, Scandella E, Ludewig B . 2005. Quantification and characterization of myosin peptide-specific CD4 + T cells in autoimmune myocarditis. J Immunol Methods 304 : 117 125.[CrossRef].[PubMed]
19. Anthony DD, Lehmann PV . 2003. T-cell epitope mapping using the ELISPOT approach. Methods 29 : 260 269.[PubMed].[CrossRef]
20. Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, Johnston MN, Corcoran C, Wurcel AG, Fitzpatrick CA, Feeney ME, Rodriguez WR, Basgoz N, Draenert R, Stone DR, Brander C, Goulder PJ, Rosenberg ES, Altfeld M, Walker BD . 2003. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 77 : 2081 2092.[PubMed].[CrossRef]
21. de Souza MS, Ratto-Kim S, Chuenarom W, Schuetz A, Chantakulkij S, Nuntapinit B, Valencia-Micolta A, Thelian D, Nitayaphan S, Pitisuttithum P, Paris RM, Kaewkungwal J, Michael NL, Rerks-Ngarm S, Mathieson B, Marovich M, Currier JR, Kim JH . 2012. The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope. J Immunol 188 : 5166 5176.[CrossRef].[PubMed]
22. Long HM, Leese AM, Chagoury OL, Connerty SR, Quarcoopome J, Quinn LL, Shannon-Lowe C, Rickinson AB . 2011. Cytotoxic CD4 +T cell responses to EBV contrast with CD8 responses in breadth of lytic cycle antigen choice and in lytic cycle recognition. J Immunol 187 : 92 101.[CrossRef].[PubMed]
23. Larsson M, Jin X, Ramratnam B, Ogg GS, Engelmayer J, Demoitie MA, McMichael AJ, Cox WI, Steinman RM, Nixon D, Bhardwaj N . 1999. A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS 13 : 767 777.[PubMed].[CrossRef]
24. Streeck H, Frahm N, Walker BD . 2009. The role of IFN-gamma Elispot assay in HIV vaccine research. Nat Protoc 4 : 461 469.[CrossRef].[PubMed]
25. Sun Y, Iglesias E, Samri A, Kamkamidze G, Decoville T, Carcelain G, Autran B . 2003. A systematic comparison of methods to measure HIV-1 specific CD8 T cells. J Immunol Methods 272 : 23 34.[PubMed].[CrossRef]
26. Xu J, Lori F, Lisziewicz J . 2003. Vaccinia assay for the rapid detection of functional HIV-specific CD8 + cytotoxic T lymphocytes. J Immunol Methods 276 : 45 57.[PubMed].[CrossRef]
27. Mwau M, McMichael AJ, Hanke T . 2002. Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res Hum Retroviruses 18 : 611 618.[CrossRef].[PubMed]
28. Hudgens MG, Self SG, Chiu YL, Russell ND, Horton H, McElrath MJ . 2004. Statistical considerations for the design and analysis of the ELISpot assay in HIV-1 vaccine trials. J Immunol Methods 288 : 19 34.[CrossRef].[PubMed]
29. Janetzki S, Schaed S, Blachere NE, Ben-Porat L, Houghton AN, Panageas KS . 2004. Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291 : 175 183.[CrossRef].[PubMed]
30. Cox JH, Ferrari G, Janetzki S . 2006. Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods 38 : 274 282.[CrossRef].[PubMed]
31. Janetzki S, Cox JH, Oden N, Ferrari G . 2005. Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302 : 51 86.[CrossRef].[PubMed]
32. Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, Cox JH . 2002. A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 260 : 157 172.[PubMed].[CrossRef]
33. Murphy KM, Stockinger B . 2010. Effector T cell plasticity: flexibility in the face of changing circumstances. Nat Immunol 11 : 674 680.[CrossRef].[PubMed]
34. Mucida D, Husain MM, Muroi S, van Wijk F, Shinnakasu R, Naoe Y, Reis BS, Huang Y, Lambolez F, Docherty M, Attinger A, Shui JW, Kim G, Lena CJ, Sakaguchi S, Miyamoto C, Wang P, Atarashi K, Park Y, Nakayama T, Honda K, Ellmeier W, Kronenberg M, Taniuchi I, Cheroutre H . 2013. Transcriptional reprogramming of mature CD4( +) helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes. Nat Immunol 14 : 281 289.
35. Akondy RS, Monson ND, Miller JD, Edupuganti S, Teuwen D, Wu H, Quyyumi F, Garg S, Altman JD, Del Rio C, Keyserling HL, Ploss A, Rice CM, Orenstein WA, Mulligan MJ, Ahmed R . 2009. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8 + T cell response. J Immunol 183 : 7919 7930.[CrossRef].[PubMed]
36. Boulet S, Ndongala ML, Bernard NF . 2010. Dual-color ELISPOT assay for the simultaneous detection of IL-2 and/or IFN-gamma secreting T cells. Cold Spring Harb Protoc 2010 :pdb prot5369.
37. Ahlborg N, Axelsson B . 2012. Dual- and triple-color fluorospot. Methods Mol Biol 792 : 77 85.[CrossRef].[PubMed]
38. Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A . 2008. Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57 : 303 315.[CrossRef].[PubMed]
39. Ashoor I, Najafian N, Korin Y, Reed EF, Mohanakumar T, Ikle D, Heeger PS, Lin M . 2013. Standardization and cross validation of alloreactive IFNgamma ELISPOT assays within the clinical trials in organ transplantation consortium. Am J Transplant 13 : 1871 1879.[CrossRef].[PubMed]
40. Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, D'Souza PM . 2005. Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retrovir 21 : 68 81.[CrossRef].[PubMed]
41. Ott PA, Berner BR, Herzog BA, Guerkov R, Yonkers NL, Durinovic-Bello I, Tary-Lehmann M, Lehmann PV, Anthony DD . 2004. CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases. J Immunol Methods 285 : 223 235.[CrossRef].[PubMed]
42. Larsson M, Wilkens DT, Fonteneau JF, Beadle TJ, Merritt MJ, Kost RG, Haslett PA, Cu-Uvin S, Bhardwaj N, Nixon DF, Shacklett BL . 2002. Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells. AIDS 16 : 171 180.[PubMed].[CrossRef]
43. Altfeld MA, Trocha A, Eldridge RL, Rosenberg ES, Phillips MN, Addo MM, Sekaly RP, Kalams SA, Burchett SA, McIntosh K, Walker BD, Goulder PJ . 2000. Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay. J Virol 74 : 8541 8549.[PubMed].[CrossRef]
44. Jennes W, Kestens L, Nixon D, Shacklett B . 2002. Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15—the Amplispot assay. J Immunol Methods 270 : 99.[PubMed].[CrossRef]
45. Calarota SA, Otero M, Hermanstayne K, Lewis M, Rosati M, Felber BK, Pavlakis GN, Boyer JD, Weiner DB . 2003. Use of interleukin 15 to enhance interferon-gamma production by antigen-specific stimulated lymphocytes from rhesus macaques. J Immunol Methods 279 : 55 67.[PubMed].[CrossRef]
46. Chitnis V, Pahwa R, Pahwa S . 2003. Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15. Clin Immunol 107 : 36 45.[PubMed].[CrossRef]
47. Britten CM, Meyer RG, Kreer T, Drexler I, Wolfel T, Herr W . 2002. The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8( +) T lymphocytes in IFN-gamma ELISPOT assays. J Immunol Methods 259 : 95 110.[PubMed].[CrossRef]
48. Herr W, Linn B, Leister N, Wandel E, Meyer zum Buschenfelde KH, Wolfel T . 1997. The use of computer-assisted video image analysis for the quantification of CD8 + T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens. J Immunol Methods 203 : 141 152.[PubMed].[CrossRef]
49. Karlsson RK, Jennes W, Page-Shafer K, Nixon DF, Shacklett BL . 2004. Poorly soluble peptides can mimic authentic ELISPOT responses. J Immunol Methods 285 : 89 92.[CrossRef].[PubMed]
50. Alexander N, Fox A, Lien VT, Dong T, Lee LY, Hang Nle K, Mai le Q, Horby P . 2013. Defining ELISpot cut-offs from unreplicated test and control wells. J Immunol Methods 392 : 57 62.[CrossRef].[PubMed]


Generic image for table

Methods of enhancing ELISPOT sensitivity

Citation: Shacklett B, Nixon D. 2016. Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT), p 290-295. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch30
Generic image for table

Parameters influencing ELISPOT assay variability

Citation: Shacklett B, Nixon D. 2016. Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT), p 290-295. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch30
Generic image for table

Methods of analyzing ELISPOT data

Citation: Shacklett B, Nixon D. 2016. Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT), p 290-295. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch30

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error