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A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes.

Vaziri Sani, Fariba LU ; Delli, Ahmed LU ; Larsson, Helena LU ; Lindblad, Bengt LU ; Carlsson, Annelie LU ; Forsander, Gun; Ivarsson, Sten LU ; Ludvigsson, Johnny; Marcus, Claude and Lernmark, Åke LU (2011) In Journal of Immunological Methods 371. p.25-37
Abstract
BACKGROUND AND AIMS: 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%,... (More)
BACKGROUND AND AIMS: 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. CONCLUSION: 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. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Journal of Immunological Methods
volume
371
pages
25 - 37
publisher
Elsevier
external identifiers
  • WOS:000294518100004
  • PMID:21708156
  • Scopus:79960956458
ISSN
1872-7905
DOI
10.1016/j.jim.2011.06.011
language
English
LU publication?
yes
id
799b045a-6437-4320-9344-3b011caa565b (old id 2007687)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/21708156?dopt=Abstract
date added to LUP
2011-07-06 11:35:21
date last changed
2016-11-20 04:23:44
@misc{799b045a-6437-4320-9344-3b011caa565b,
  abstract     = {BACKGROUND AND AIMS: 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. CONCLUSION: 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.},
  author       = {Vaziri Sani, Fariba and Delli, Ahmed and Larsson, Helena and Lindblad, Bengt and Carlsson, Annelie and Forsander, Gun and Ivarsson, Sten and Ludvigsson, Johnny and Marcus, Claude and Lernmark, Åke},
  issn         = {1872-7905},
  language     = {eng},
  pages        = {25--37},
  publisher    = {ARRAY(0xc33d7d8)},
  series       = {Journal of Immunological Methods},
  title        = {A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes.},
  url          = {http://dx.doi.org/10.1016/j.jim.2011.06.011},
  volume       = {371},
  year         = {2011},
}