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.
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: C Wasserfall; E Montgomery; L Yu; A Michels; R Gianani; A Pugliese; C Nierras; J S Kaddis; D A Schatz; E Bonifacio; M A Atkinson Journal: Clin Exp Immunol Date: 2016-05-04 Impact factor: 4.330
Authors: Felipe de Jesus Cortez; David Gebhart; Peter V Robinson; David Seftel; Narges Pourmandi; Jordan Owyoung; Carolyn R Bertozzi; Darrell M Wilson; David M Maahs; Bruce A Buckingham; John R Mills; Matthew M Roforth; Sean J Pittock; Andrew McKeon; Kara Page; Wendy A Wolf; Srinath Sanda; Cate Speake; Carla J Greenbaum; Cheng-Ting Tsai Journal: PLoS One Date: 2020-11-13 Impact factor: 3.240