小鼠高特异性/敏感性竞争性铕胰岛素自身抗体检测法
Murine high specificity/sensitivity competitive europium insulin autoantibody assay.
作者信息
Babaya Naru, Liu Edwin, Miao Dongmei, Li Marcella, Yu Liping, Eisenbarth George S
机构信息
Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center , Aurora, Colorado 80045-6511, USA.
出版信息
Diabetes Technol Ther. 2009 Apr;11(4):227-33. doi: 10.1089/dia.2008.0072.
BACKGROUND
Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing (125)I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays.
METHODS
To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay).
RESULTS
The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected.
CONCLUSIONS
Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format.
背景
大多数针对人类和动物模型的胰岛素自身抗体检测采用放射性检测方法，利用（125）I -胰岛素，但尽管是放射性检测方法，国际研讨会已证明各实验室之间在标准化方面存在困难。因此，需要更简单的不使用放射性的检测方法，同时保持放射性检测的高特异性和高灵敏度。
方法
为建立一种更简便的用于检测非肥胖糖尿病（NOD）小鼠胰岛素自身抗体的酶联免疫吸附测定（ELISA）方法，我们采用了ELISA形式，与未标记胰岛素竞争，结合铕 -抗生物素蛋白以及时间分辨荧光检测（竞争性铕胰岛素自身抗体检测）。
结果
在分析盲法研讨会血清时，将胰岛素自身抗体的竞争性铕检测应用于NOD小鼠血清时，与我们的标准胰岛素自身抗体放射性检测相比，具有高灵敏度和高特异性（灵敏度92%，特异性100%），而标准放射性检测的灵敏度为72%，特异性为100%。值得注意的是，尽管该检测对鼠胰岛素自身抗体具有极高的灵敏度，并且以人胰岛素作为靶自身抗原，但未检测到含有高水平胰岛素自身抗体的人血清。
结论
我们的结果清楚地表明，可以以类似ELISA的形式检测低水平的胰岛素自身抗体。将基于铕的ELISA与液相自身抗原竞争相结合，可应用于多种自身抗原，以在ELISA形式中实现高特异性和高灵敏度。
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