Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera
Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera
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DOI：
10.1515/cclm-2019-0087
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发表时间：
2019-11-01
影响因子：
6.8
通讯作者：
Andrade, Luis E. C.
中科院分区：
文献类型：
--
作者：
Dellavance, Alessandra;Baldo, Danielle C.;Andrade, Luis E. C.
工作背景：国际自身抗体标准，传统上基于从单个受试者的血浆置换获得的材料，代表个体免疫应答，可能不理解一般人群的异质性。抗DFS 70自身抗体在HEp-2细胞上的间接免疫荧光测定（HEp-2 IFA）上产生特征性的致密细斑点（DFS）核模式，并且与自身免疫性相矛盾。我们提出了一种新的策略来发展自身抗体参考标准，基于从数百个具有抗DFS 70抗体的个体中逐步汇集血清样品。方法：在2年的时间内，根据以下标准从常规HEp-2 IFA中选择血清样品：DFS HEp-2 IFA模式在滴度≥ 1：640;在三种分析物特异性测试（Western印迹[WB]、酶联免疫吸附测定[ELISA]和荧光免疫测定[CLIA]）中检测抗DFS 70反应性。将各个样品的等分试样合并为逐渐增大的样本池，并逐步验证中间样本池（如各个样品）。经验证的中间池合并成最终池用于冻干。结果：总共741个经验证的样品产生750 mL最终池，其被冻干成数千个200 μ L的等分试样。复溶等分试样在ELISA、CLIA和WB中产生预期的抗DFS 70反应性，以及高滴度DFS HEp-2 IFA模式。适当的抗DFS 70反应的冻干池被确认由7个国际专家中心，使用HEp-2 IFA，ELISA，WB和immunoprecipitation.Conclusions：这个概念验证研究提供了一个创新的和有效的策略，建立血清参考标准的自身抗体检测。抗-DFS 70标准品将整合自身抗体标准化委员会（ASC，www.autoab.org）的标准品组，有助于教育正确的测定验证和DFS模式和其他HEp-2 IFA模式的解释。
Background: International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies.Methods: Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer >= 1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization.Results: A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 mu L-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate antiDFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation.Conclusions: This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org ), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.