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Clinical and Vaccine Immunology, December 2008, p. 1811-1818, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00322-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method{triangledown}

Giuseppina Li Pira,1* Federico Ivaldi,1 Chiara Dentone,2 Elda Righi,2 Valerio Del Bono,2 Claudio Viscoli,2 Gerrit Koopman,3 and Fabrizio Manca4

Laboratory of Cellular Immunology, Advanced Biotechnology Center, Genoa, Italy,1 Department of Infectious Diseases, San Martino Hospital-University of Genoa, Genoa, Italy,2 Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands,3 Laboratory of Clinical and Experimental Immunology, G. Gaslini Institute, Genoa, Italy4

Received 4 September 2008/ Returned for modification 11 October 2008/ Accepted 15 October 2008

The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-µl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-{gamma}) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 104 PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.

* Corresponding author. Mailing address: Laboratory of Cellular Immunology, Advanced Biotechnology Center, Largo Benzi 10, 16132 Genoa, Italy. Phone and fax: 39 010 5737370. E-mail: lipira{at}email.it

{triangledown} Published ahead of print on 22 October 2008.

Clinical and Vaccine Immunology, December 2008, p. 1811-1818, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00322-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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