Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility

Alistair J.K. Williams, Michelle Somerville, Saba Rokni, Ezio Bonifacio, Liping Yu, George Eisenbarth, Beena Akolkar, Michael Steffes, Polly J. Bingley

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1% azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41% (range, 78 to - 33%, p < 0.001). The effect on binding was epitope specific; median IA-2A binding by 14 sera with antibodies to the protein tyrosine phosphatase region of IA-2 was reduced by 48% (range, 11 to 78%, p < 0.001), while binding by 4 sera with antibodies specific to only the juxtamembrane region of IA-2 showed no change (median increase 16% (range 6 to 33%, p = 0.125). When the Tween-20 concentration was reduced from 1% to 0.15% the median reduction in IA-2A binding with azide by the 18 sera was only 10% (range, - 12 to 41%, p < 0.001). Tween-20 also exerted an independent effect, since median IA-2A binding increased by 23% (range 3% to 86%, p < 0.001) when Tween-20 concentration was reduced from 1% to 0.15% in the absence of azide. We conclude that common assay reagents such as azide and Tween-20 can strongly influence IA-2A binding in an epitope-related manner, and their use may explain some of the differences between laboratories in IA-2A measurement.

Original languageEnglish (US)
Pages (from-to)75-79
Number of pages5
JournalJournal of Immunological Methods
Volume351
Issue number1-2
DOIs
StatePublished - Dec 31 2009

Bibliographical note

Funding Information:
This study was supported by funding from the National Institute of Diabetes and Digestive and Kidney Diseases . The Munich family study is supported by grants from the European Union ( EP7-HEALTH-2007 , DIAPREPP N202013 ), the Federal Ministry of Education and Research of Germany (Kompetenznetz Diabetes mellitus, FKZ 01GI0805-07 ), and the DFG-Center for Regenerative Therapies Dresden, Cluster of Excellence ( FZ 111 ). The BOX family study was funded by Diabetes UK .

Keywords

  • Azide
  • Epitope
  • Islet autoantibody assay
  • Protein tyrosine phosphatase
  • Tween-20
  • Type 1 diabetes

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