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 language | English (US) |
|---|---|
| Pages (from-to) | 75-79 |
| Number of pages | 5 |
| Journal | Journal of Immunological Methods |
| Volume | 351 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Dec 31 2009 |
Fingerprint
Keywords
- Azide
- Epitope
- Islet autoantibody assay
- Protein tyrosine phosphatase
- Tween-20
- Type 1 diabetes
Cite this
Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility. / Williams, Alistair J.K.; Somerville, Michelle; Rokni, Saba; Bonifacio, Ezio; Yu, Liping; Eisenbarth, George; Akolkar, Beena; Steffes, Michael; Bingley, Polly J.
In: Journal of Immunological Methods, Vol. 351, No. 1-2, 31.12.2009, p. 75-79.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility
AU - Williams, Alistair J.K.
AU - Somerville, Michelle
AU - Rokni, Saba
AU - Bonifacio, Ezio
AU - Yu, Liping
AU - Eisenbarth, George
AU - Akolkar, Beena
AU - Steffes, Michael
AU - Bingley, Polly J.
PY - 2009/12/31
Y1 - 2009/12/31
N2 - 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.
AB - 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.
KW - Azide
KW - Epitope
KW - Islet autoantibody assay
KW - Protein tyrosine phosphatase
KW - Tween-20
KW - Type 1 diabetes
UR - http://www.scopus.com/inward/record.url?scp=70350616188&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70350616188&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2009.10.004
DO - 10.1016/j.jim.2009.10.004
M3 - Article
C2 - 19837075
AN - SCOPUS:70350616188
VL - 351
SP - 75
EP - 79
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -