用于 DASP 2010 的高通量 LIPS 胰岛自身抗体检测。

LIPS islet autoantibody assays in high-throughput format for DASP 2010.

机构信息

Pacific Northwest Diabetes Research Institute, Seattle, WA 98122, USA.

出版信息

Diabetes Metab Res Rev. 2011 Nov;27(8):891-4. doi: 10.1002/dmrr.1268.

DOI:10.1002/dmrr.1268

PMID:22069280

Abstract

BACKGROUND

For cost-effective population-based diabetes prediction and confirmation, islet autoantibody assays must be made more economical.

METHODS

We evaluated glutamic acid decarboxylase (GAD)-Ruc (renilla luciferase) and IA2ic (also known as ICA512ic)-Ruc (renilla luciferase) fusion protein constructs in high-throughput islet antibody assay formats.

RESULTS

Antigen production via transfection onto COS cells in 100 mm culture dishes yielded sufficient antigen to assay 375 and 535 serum samples for GAD and IA2ic per dish, respectively. Antigen was usably stable after -80 °C storage for 40-80 days after which luciferase activity decreased. The mean signal-to-noise ratios for luciferase-based immunoprecitation system (LIPS) GAD and LIPS IA2ic were 88±24 and 219±89, respectively, comparing favourably to radio-binding assays (RBA) in the same format. However, the coefficient of variation among triplicate wells was higher for IA2ic than for GAD in LIPS, similar to findings in RBA format. Correlation coefficients between autoantibody indices determined from the RBA and LIPS methods were only R2=0.79 and R2=0.75 for GAD and IA2ic, respectively, raising the possibility that different epitopes were favoured in the two different assay formats. Nevertheless, overall concordance for the two assay types was high, at 228/240=95.0% for GAD and 494/521=94.8% for IA2ic. Using optimal cutoffs, Diabetes Autoantibody Standardization Program (DASP) 2010 sensitivity/specificity was 80/99% for GAD RBA, 80/99% for GAD LIPS, 70/98% for IA2 RBA, and 72/99% for IA2 LIPS.

CONCLUSION

The LIPS assays for islet autoantibodies to GAD and IA2ic performed as well as RBA in DASP 2010. With further refinements in expression and storage, these assays may be more economical than current methods to measure islet autoantibodies in type 1 diabetes.

摘要

背景

为了在具有成本效益的基础上进行人群糖尿病预测和确诊，胰岛自身抗体检测必须更加经济实惠。

方法

我们评估了谷氨酸脱羧酶（GAD）-Ruc（海肾荧光素酶）和 IA2ic（也称为 ICA512ic）-Ruc（海肾荧光素酶）融合蛋白在高通量胰岛抗体检测中的构建。

结果

通过转染到 100mm 培养皿中的 COS 细胞产生足够的抗原，可分别对每皿 375 和 535 个血清样本进行 GAD 和 IA2ic 的检测。抗原在 -80°C 储存 40-80 天后仍可使用，之后荧光素酶活性降低。基于荧光素酶的免疫沉淀系统（LIPS）GAD 和 LIPS IA2ic 的平均信噪比分别为 88±24 和 219±89，与相同格式的放射结合测定（RBA）相比具有优势。然而，在 LIPS 中，IA2ic 的三复孔间变异系数高于 GAD，与 RBA 格式的发现相似。RBA 和 LIPS 方法确定的自身抗体指数之间的相关系数仅分别为 GAD 的 R2=0.79 和 IA2ic 的 R2=0.75，这表明在两种不同的检测方法中可能存在不同的表位。尽管如此，两种检测类型的总体一致性仍然很高，GAD 为 228/240=95.0%，IA2ic 为 494/521=94.8%。使用最佳截断值，糖尿病自身抗体标准化计划（DASP）2010 年的 GAD RBA 的灵敏度/特异性为 80/99%，GAD LIPS 为 80/99%，IA2 RBA 为 70/98%，IA2 LIPS 为 72/99%。