title: Lymphocyte proliferation specific for recall, CMV and HIV antigens in miniaturized and automated format.
abstract: Lymphoproliferation assay (LPA) is used to test specific T-cell responses. LPA is performed in 96-well plates with 2-5×10⁵ PBMC/well. In order to test numerous antigens, as in the case of epitope mapping or screening of antigenic panels from relevant pathogens, PBMC numbers may not be sufficient. We developed a miniaturized and automated procedure to perform LPA in 384- and 1536-well plates with one fourth to one twentieth of PBMC numbers used for standard assays. Here, we demonstrate that the procedure is reliable and robust using recall antigens and protein and peptide antigens from CMV and HIV. By using HIV specific T-cell lines, we also demonstrate that sensitivity ranges overlap with those of standard LPA and that as few as 3 specific cells/well provide a positive signal. This procedure is consistent with our policy to miniaturize assays for specific T-cell immunity, as we have already established for cytokine secretion assays.
authors.affiliation: Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children Hospital, Rome, Italy. lipira@email.it
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Li Pira
authors.forename: Nadia
authors.initials: N
authors.lastname: Starc
authors.forename: Antonella
authors.initials: A
authors.lastname: Conforti
authors.forename: Alice
authors.initials: A
authors.lastname: Bertaina
authors.forename: Sergio
authors.initials: S
authors.lastname: Rutella
authors.forename: Franco
authors.initials: F
authors.lastname: Locatelli
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Manca
mesh_terms: D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002460:Cell Line; D049109:Cell Proliferation; D003587:Cytomegalovirus / Q000276:immunology; D006678:HIV / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000276:immunology; D054303:HIV Reverse Transcriptase / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques / Q000295:instrumentation / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D055616:Microtechnology / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D015203:Reproducibility of Results
chemical_list: D000956:Antigens, Viral; D015699:HIV Envelope Protein gp120; D054303:HIV Reverse Transcriptase