title: Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility.
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.
authors.affiliation: Clinical Science at North Bristol, Southmead Hospital, Bristol, UK. A.J.K.Williams@bristol.ac.uk
authors.forename: Alistair J K
authors.initials: AJ
authors.lastname: Williams
authors.forename: Michelle
authors.initials: M
authors.lastname: Somerville
authors.forename: Saba
authors.initials: S
authors.lastname: Rokni
authors.forename: Ezio
authors.initials: E
authors.lastname: Bonifacio
authors.forename: Liping
authors.initials: L
authors.lastname: Yu
authors.forename: George
authors.initials: G
authors.lastname: Eisenbarth
authors.forename: Beena
authors.initials: B
authors.lastname: Akolkar
authors.forename: Michael
authors.initials: M
authors.lastname: Steffes
authors.forename: Polly J
authors.initials: PJ
authors.lastname: Bingley
mesh_terms: D001323:Autoantibodies / Q000097:blood; D001386:Azides; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D011136:Polysorbates; D011485:Protein Binding; D011869:Radioligand Assay; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000276:immunology* / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D001323:Autoantibodies; D001386:Azides; D000939:Epitopes; D011136:Polysorbates; C516862:PTPRN protein, human; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8