title: Sensitive non-isotopic assays for autoantibodies to IA-2 and to a combination of both IA-2 and GAD65.
abstract: BACKGROUND
  A sensitive ELISA for measurement of IA-2 autoantibodies has been developed and assessed. Also, a combination ELISA for detection of both GAD65 autoantibodies and IA-2 autoantibodies is described.
  
  
  METHODS
  The IA-2 autoantibody assay is based on the ability of IA-2 autoantibodies to form a bridge between IA-2 intracellular fragment coated onto ELISA plate wells and liquid-phase IA-2 labelled with biotin. The combination ELISA uses plates coated with both IA-2 and GAD65 and a mixture of IA-2-biotin and GAD65-biotin. Assay sensitivity was assessed using the WHO reference (NIBSC 97/550) for islet cell antibodies. IA-2 autoantibody measurements by ELISA were compared with measurements in immunoprecipitation assays (IPAs) based on 125I or 35S labelled IA-2. Combination ELISA results were compared with results obtained for individual autoantibodies.
  
  
  RESULTS
  As little as 15 units/mL of NIBSC 97/550 was detectable in the IA-2 autoantibody ELISA compared to 125 units/mL by 125I-IA-2 IPA. 110/216(51%) sera from patients with type 1 DM were positive in the IA-2 autoantibody ELISA while 97/216 (45%) and 91/216 (42%) were positive in the 125I-IA2 and 35S-IA-2 IPAs, respectively. The IA-2 autoantibody ELISA showed 100% specificity for type 1 DM. The combination ELISA was able to detect GAD65 and/or IA-2 autoantibodies in 183/216 (85%) diabetic sera and 183/216 (85%) were also found positive for autoantibodies to IA-2 and/or to GAD65 in the assays for individual antibodies. Autoantibody measurements in the individual autoantibody assays and in the combination ELISA showed good agreement by Pearson correlation (r=0.92, n=216, p<0.001) and by Bland and Altman analysis.
  
  
  CONCLUSIONS
  Sensitive and specific ELISAs for measurement of autoantibodies to IA-2 and to a combination of IA-2 and GAD65 have been developed. These assays are suitable for screening large numbers of samples in diabetes prediction and prevention trials.
authors.affiliation: FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK. firs@rsltd.eclipse.co.uk
authors.forename: Shu
authors.initials: S
authors.lastname: Chen
authors.forename: Jinny
authors.initials: J
authors.lastname: Willis
authors.forename: Clare
authors.initials: C
authors.lastname: Maclean
authors.forename: Rossitza
authors.initials: R
authors.lastname: Ananieva-Jordanova
authors.forename: Marie-Andrée
authors.initials: MA
authors.lastname: Amoroso
authors.forename: Helen
authors.initials: H
authors.lastname: Brooking
authors.forename: Michael
authors.initials: M
authors.lastname: Powell
authors.forename: Anne
authors.initials: A
authors.lastname: Collins
authors.forename: Stuart
authors.initials: S
authors.lastname: Bennett
authors.forename: Sarah
authors.initials: S
authors.lastname: Mitchell
authors.forename: Peter
authors.initials: P
authors.lastname: Burne
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Furmaniak
authors.forename: Bernard Rees
authors.initials: BR
authors.lastname: Smith
mesh_terms: D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D003920:Diabetes Mellitus / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005968:Glutamate Decarboxylase / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007527:Isoenzymes / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology* / Q000378:metabolism; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D008565:Membrane Proteins; D001710:Biotin; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2