title: An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.
abstract: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.
  
  
  
  Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.
  
  
  
  Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104and 2 × 105PBMC per well upon stimulation with T-activated® IE-1 (R2= 0.97) and pp65 (R2= 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+(Th), CD3+CD8+(CTL), CD3-CD56+(NK) and CD3+CD56+(NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.
  
  
  
  The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Barabas
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Theresa
authors.initials: T
authors.lastname: Spindler
authors.affiliation: Institute of Medical Microbiology and Hygiene, University Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Kiener
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Tonar
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Lugner
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Batzilla
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Hanna
authors.initials: H
authors.lastname: Bendfeldt
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Rascle
authors.affiliation: Institute of Medical Microbiology and Hygiene, University Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Asbach
authors.affiliation: Institute of Medical Microbiology and Hygiene, University Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Wagner
authors.affiliation: Lophius Biosciences GmbH, Am BioPark 13, 93053, Regensburg, Germany. Ludwig.Deml@lophius.com.|Institute of Medical Microbiology and Hygiene, University Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany. Ludwig.Deml@lophius.com.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Deml
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D005260:Female; D006801:Humans; D017874:Immediate-Early Proteins / Q000276:immunology; D007111:Immunity, Cellular; D007371:Interferon-gamma / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology* / Q000821:virology; D008297:Male; D008875:Middle Aged; D015166:Monitoring, Immunologic; D055611:Natural Killer T-Cells / Q000276:immunology* / Q000821:virology; D015588:Observer Variation; D010750:Phosphoproteins / Q000276:immunology; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000276:immunology; D055815:Young Adult
chemical_list: C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma
keywords: CD4 +; CD8 +; CMV; Cell-mediated immunity; Cytomegalovirus; Cytotoxic T lymphocyte (CTL); ELISpot; IE-1; NKT-like; Natural killer (NK); T helper (Th); pp65