title: Improved efficacy by using the pTnT-rhtTG plasmid for the detection of celiac disease specific tissue transglutaminase autoantibodies in radioligand binding assays.
abstract: BACKGROUND
  Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays.
  
  
  METHODS
  Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³⁵S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively.
  
  
  RESULTS
  Median incorporation of [³⁵S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively.
  
  
  CONCLUSIONS
  The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.
authors.affiliation: Unit of Diabetes and Celiac Disease, Department of Clinical Sciences, University Hospital MAS, Lund University, Malmö, Sweden. jennifer.kjelleras@med.lu.se
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kjellerås
authors.forename: Fariba
authors.initials: F
authors.lastname: Vaziri-Sani
authors.forename: Daniel
authors.initials: D
authors.lastname: Agardh
mesh_terms: D000293:Adolescent; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000097:blood* / Q000276:immunology; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D015415:Biomarkers / Q000097:blood*; D016022:Case-Control Studies; D002446:Celiac Disease / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D002474:Cell-Free System / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D012372:ROC Curve; D011869:Radioligand Assay; D012156:Reticulocytes / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000032:analysis / Q000378:metabolism; D013548:Sweden; D011503:Transglutaminases / Q000097:blood* / Q000276:immunology; D055815:Young Adult; D013205:Staphylococcal Protein A
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000951:Antigens, Neoplasm; D001323:Autoantibodies; D015415:Biomarkers; D008715:Methionine; D012685:Sepharose; D013462:Sulfur Radioisotopes; D011503:Transglutaminases; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A; C049937:anti-IgA