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Immunology and Infection

השימוש באינטרפרון-γ Assay האנזים המקושר Immunospot לאפיין רומן T-cell אפיטופים של וירוס הפפילומה האנושי

doi: 10.3791/3657 Published: March 8, 2012

Materials

Name Company Catalog Number Comments
CD8 T cell isolation kit Miltenyi Biotec 130-094-156
Phytohemagglutinin Remel, Thermo Fisher Scientific R-30852801 2mg/vial
Yssel’s medium Gemini Bio Products 400-102 1% pooled human serum
96-well round bottom plate BD Biosciences 08-772-17
Recombinant human IL-2 R&D Systems 202-IL-050 50μg
24-well plate Corning 07-200-84
Phosphate-buffered saline Cellgro MT-21-031-CV
Primary anti-IFN-γ monoclonal antibody (D1K) Mabtech 3420-3-1000
Biotin-conjugated anti-IFN-γ monoclonal antibody (7B-6) Mabtech 3420-6-1000
Multiscreen HA ELISPOT plate EMD Millipore MAHA S45 10
Dimethyl Sulphoxide Sigma-Aldrich D2650 100 ml bottle
Pooled Serum, human Atlanta Biologicals S40510H Heat inactivate
RPMI 1640 Cellgro MT-10-040-CV
Tween-20 Sigma-Aldrich P2287-100ml
Vectastain Elite ABC Kit Vector Laboratories NC9313719
Stable diaminobenzidine Open Biosystems MBI 1241
AID EliSpot Reader Classic Autoimmun Diagnostika GmbH ELR06

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References

  1. Nakagawa, M. A favorable clinical trend is associated with CD8 T-cell immune responses to the human papillomavirus type 16 e6 antigens in women being studied for abnormal pap smear results. J. Low. Genit. Tract Dis. 14, 124-129 (2010).
  2. Walls, E., Crawford, L. Lymphocytes: A practical approach. Klaus, G. G. B. IRL Press. 149-149 (1987).
  3. Nakagawa, M., Kim, K. H., Moscicki, A. B. Patterns of CD8 T-cell epitopes within the human papillomavirus type 16 (HPV 16) E6 protein among young women whose HPV 16 infection has become undetectable. Clin. Diagn. Lab Immunol. 12, 1003-1005 (2005).
  4. Wang, X., Santin, A. D., Bellone, S., Gupta, S., Nakagawa, M. A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells. Cancer Immunol. Immunother. 58, 301-308 (2009).
  5. Larsson, M. 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 (1999).
  6. Nakagawa, M. Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia. J. Infect. Dis. 175, 927-931 (1997).
  7. Wang, X., Moscicki, A. B., Tsang, L., Brockman, A., Nakagawa, M. Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable. Clin. Vaccine Immunol. 15, 937-945 (2008).
  8. Evans, M. Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6. J. Immunol. 167, 5420-5428 (2001).
  9. Shi, Y., Smith, K. D., Kurilla, M. G., Lutz, C. T. Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro. J. Immunol. 159, 1844-1852 (1997).
  10. Varadarajan, N. A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis. The Journal of Clinical Investigation. (2011).
  11. Nakagawa, M., Kim, K. H., Moscicki, A. B. Different methods of identifying new antigenic epitopes of human papillomavirus type 16 E6 and E7 proteins. Clin. Diagn. Lab Immunol. 11, 889-896 (2004).
  12. Nakagawa, M., Kim, K. H., Gillam, T. M., Moscicki, A. B. HLA class I binding promiscuity of the CD8 T-cell epitopes of human papillomavirus type 16 E6 protein. J. Virol. 81, 1412-1423 (2007).
  13. Brown, S. A. T cell epitope "hotspots" on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry. AIDS Res. Hum. Retroviruses. 21, 165-170 (2005).
  14. Masemola, A. M. Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa. J. Immunol. 173, 4607-4617 (2004).
  15. Walker, B. D., Korber, B. T. Immune control of HIV: the obstacles of HLA and viral diversity. Nat. Immunol. 2, 473-475 (2001).
השימוש באינטרפרון-γ Assay האנזים המקושר Immunospot לאפיין רומן T-cell אפיטופים של וירוס הפפילומה האנושי
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Cite this Article

Wang, X., Greenfield, W. W., Coleman, H. N., James, L. E., Nakagawa, M. Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus. J. Vis. Exp. (61), e3657, doi:10.3791/3657 (2012).More

Wang, X., Greenfield, W. W., Coleman, H. N., James, L. E., Nakagawa, M. Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus. J. Vis. Exp. (61), e3657, doi:10.3791/3657 (2012).

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