ELISA 中抗 ADAMTS-13 自身抗体滴度的测定：ADAMTS-13 呈现和自身抗体检测的影响。
Determination of anti-ADAMTS-13 autoantibody titers in ELISA: Influence of ADAMTS-13 presentation and autoantibody detection.
机构信息
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.
Icosagen Cell Factory OÜ, Kambja vald, Tartumaa, Estonia.
出版信息
J Thromb Haemost. 2021 Sep;19(9):2248-2255. doi: 10.1111/jth.15297. Epub 2021 Jul 14.
BACKGROUND
Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by inhibitory and/or clearing anti-ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) autoantibodies. To determine the presence and total level of anti-ADAMTS-13 autoantibodies, commercial and in-house developed ELISAs are performed. However, different ELISA methods vary in relation to the presentation of recombinant (r)ADAMTS-13 and the detection method of the anti-ADAMTS-13 autoantibodies. Currently, the influence of those different approaches on anti-ADAMTS-13 autoantibody titers is not known.
OBJECTIVES
To assess the influence of different ADAMTS-13 presentation- and autoantibody detection methods on anti-ADAMTS-13 autoantibody titers in ELISA.
MATERIALS/METHODS: Anti-ADAMTS-13 autoantibody titers from 18 iTTP patients were determined using four different set-ups of anti-ADAMTS-13 autoantibody ELISAs. The ELISAs varied in the used presentation of rADAMTS-13 (directly coated full-length rADAMTS-13, directly coated rMDTCS and rT2C2, or antibody-captured full-length rADAMTS-13) and the detection antibodies (polyclonal anti-human IgG or monoclonal anti-human IgG antibodies).
RESULTS
Strong correlations between the different anti-ADAMTS-13 autoantibody ELISA approaches were observed, when using polyclonal anti-human IgG detection antibodies recognizing all IgG subclasses similarly, independent of the method of rADAMTS-13 presentation. Anti-ADAMTS-13 autoantibody titers correlated less when using a mixture of monoclonal anti-human IgG , because not all IgG subclasses were recognized with similar affinities.
CONCLUSION
Anti-ADAMTS-13 autoantibody levels using different methods of rADAMTS-13 presentation strongly correlate. However, the levels of anti-ADAMTS-13 autoantibodies are highly dependent on the detection antibody used, which should detect all IgG subclasses (IgG ) equally well.
背景
免疫介导的血栓性血小板减少性紫癜（iTTP）是由抑制性和/或清除抗 ADAMTS-13（一种具有血小板反应素 1 型重复的解整合素和金属蛋白酶，成员 13）自身抗体引起的。为了确定抗 ADAMTS-13 自身抗体的存在和总水平，需要进行商业和内部开发的 ELISA。然而，不同的 ELISA 方法在重组（r）ADAMTS-13 的呈现方式和抗 ADAMTS-13 自身抗体的检测方法上存在差异。目前，这些不同方法对抗 ADAMTS-13 自身抗体滴度的影响尚不清楚。
目的
评估不同 ADAMTS-13 呈现方式和自身抗体检测方法在 ELISA 中对抗 ADAMTS-13 自身抗体滴度的影响。
材料/方法：使用四种不同的抗 ADAMTS-13 自身抗体 ELISA 方案，测定 18 例 iTTP 患者的抗 ADAMTS-13 自身抗体滴度。ELISA 方案在 rADAMTS-13 的使用方式（直接包被全长 rADAMTS-13、直接包被 rMDTCS 和 rT2C2、或抗体捕获全长 rADAMTS-13）和检测抗体（多克隆抗人 IgG 或单克隆抗人 IgG 抗体）上有所不同。
结果
当使用多克隆抗人 IgG 检测抗体识别所有 IgG 亚类时，观察到不同的抗 ADAMTS-13 自身抗体 ELISA 方法之间具有很强的相关性，而与 rADAMTS-13 呈现方法无关。当使用混合的单克隆抗人 IgG 时，抗 ADAMTS-13 自身抗体滴度相关性较差，因为并非所有 IgG 亚类都以相似的亲和力被识别。
结论
使用不同的 rADAMTS-13 呈现方法的抗 ADAMTS-13 自身抗体水平具有很强的相关性。然而，抗 ADAMTS-13 自身抗体的水平高度依赖于所使用的检测抗体，该抗体应能同等良好地识别所有 IgG 亚类（IgG）。
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