کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2089301 | 1545786 | 2006 | 4 صفحه PDF | دانلود رایگان |

Insulin autoantibody (IAA) microassays are widely used for predicting type 1 diabetes. As levels of IAA are often low in type 1 diabetes, non-specific binding (NSB) needs to be minimised if assays are to achieve high analytical sensitivity. IAA microassays use protein A Sepharose (PAS) or protein G Sepharose (PGS) to isolate the antibody-bound label, but NSB by the gel can differ between commercially-produced batches. We investigated whether pre-incubation of gel with glycine or ethanolamine could overcome this problem. Batches of PAS/PGS shown to have high NSB (0.3–3.2%) were incubated with glycine or ethanolamine at various pHs between 8 and 10.6 for 2–18 h at 4 °C or room temperature. Treating PAS at pH 10.6 with 0.2 M glycine overnight at room temperature reduced NSB by > 84%, with minimal reduction in specific binding (< 5%). Treating PGS at pH 10.6 with 0.2 M ethanolamine overnight at 4 °C reduced background by > 95%, with minimal reduction in specific binding by most sera. Treatment at high pH was critical in reducing NSB to both PAS and PGS, with slight reduction at pH 8, but a major reduction at pH 10.6. Pre-treatment with glycine or ethanolamine allows “poor” batches of PAS or PGS to be used in sensitive IAA assays, improving both consistency and performance.
Journal: Journal of Immunological Methods - Volume 314, Issues 1–2, 31 July 2006, Pages 170–173