title: A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes.
abstract: OBJECTIVE
  Autoantibodies against the zinc transporter 8 (ZnT8A) are common in type 1 diabetes (T1D). ZnT8A analyses are complicated by the fact that there are three variants of the autoantigen at amino acid position 325 representing ZnT8-R (Arginine), ZnT8-W (Tryptophan) and ZnT8-Q (Glutamin). The aims of the study were: 1) to develop an autoantigen triple mix Radio-Binding Assay (RBA) for ZnT8A; 2) to identify the individual ZnT8-R,-W,-QA reactivity and 3) to validate the triple mix ZnT8A RBA in children with newly diagnosed T1D.
  
  
  METHODS
  Serum samples were obtained from 2664 (56% males, n=1436) patients in the Swedish nationwide Better Diabetes Diagnosis (BDD) study representing patients with T1D (97%, n=2582), T2D (1.7%, n=46), MODY (1.0%, n=28) and secondary diabetes (0.3%, n=8). cDNA coding for the C-terminal end of each variant was prepared by site-directed mutagenesis and subcloned into a high efficiency in vitro transcription translation vector. The ZnT8 variants were labeled with 35S-methionine and used in a standard RBA separating free from autoantibody-bound autoantigen with Protein A-Sepharose.
  
  
  RESULTS
  ZnT8-TripleA was detected in 1678 (65%) patients with T1D, 4 (9%) T2D, 3 (11%) MODY and in none (0%) of the patients with secondary diabetes. Among the T1D patients ZnT8-RA was detected in 1351 (52%) patients, ZnT8-WA in 1209 (47%) and ZnT8-QA in 790 (31%) demonstrating that 1661 (64%) had one or several ZnT8A. The ZnT8-TripleA assay showed a false positive rate of 1.9% (n=49). Only 1.2% (n=32) of the T1D patients were false negative for ZnT8-TripleA compared to 0/46 (0%) of the T2D patients. The precision (intra assay CV) and reproducibility (inter assay CV) of the ZnT8-TripleA assay did not differ from the RBA of the individual ZnT8 variants.
  
  
  CONCLUSIONS
  We conclude that the ZnT8-TripleA assay had low false positive and false negative rates. The ZnT8-TripleA assay would therefore be highly suitable not only to analyze patient with newly diagnosed diabetes but also for screening the general population since this assay demonstrated high sensitivity and very high specificity.
authors.affiliation: Department of Clinical Sciences, Lund University, Malmö, Sweden. Fariba.vaziri_sani@med.lu.se
authors.forename: Fariba
authors.initials: F
authors.lastname: Vaziri-Sani
authors.forename: Ahmed J
authors.initials: AJ
authors.lastname: Delli
authors.forename: Helena
authors.initials: H
authors.lastname: Elding-Larsson
authors.forename: Bengt
authors.initials: B
authors.lastname: Lindblad
authors.forename: Annelie
authors.initials: A
authors.lastname: Carlsson
authors.forename: Gun
authors.initials: G
authors.lastname: Forsander
authors.forename: Sten A
authors.initials: SA
authors.lastname: Ivarsson
authors.forename: Johnny
authors.initials: J
authors.lastname: Ludvigsson
authors.forename: Claude
authors.initials: C
authors.lastname: Marcus
authors.forename: Åke
authors.initials: Å
authors.lastname: Lernmark
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000235:genetics; D001483:Base Sequence; D016022:Case-Control Studies; D027682:Cation Transport Proteins / Q000235:genetics* / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003920:Diabetes Mellitus / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology*; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D003924:Diabetes Mellitus, Type 2 / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007223:Infant; D007515:Islets of Langerhans / Q000276:immunology; D008297:Male; D008875:Middle Aged; D011863:Radioimmunoassay / Q000379:methods* / Q000706:statistics & numerical data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D055815:Young Adult; D000075703:Zinc Transporter 8
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; C492522:SLC30A8 protein, human; D000075703:Zinc Transporter 8; C024211:islet cell antibody