Research and Development Division, Fleury Medicine and Health Laboratory, São Paulo, Brazil.
Department of Oral Biology, University of Florida, Gainesville, FL, USA.
Clin Chem Lab Med. 2019 Oct 25;57(11):1754-1763. doi: 10.1515/cclm-2019-0087.
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 μL-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 anti-DFS70 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.
国际自身抗体标准传统上基于从单个人体血浆中提取的材料，代表个体免疫反应，可能无法涵盖人群的异质性。抗 DFS70 抗体在间接免疫荧光法（HEp-2 IFA）上产生特征性的密集细点状（DFS）核模式，提示自身免疫。我们提出了一种新的自身抗体参考标准开发策略，基于对数百名具有抗 DFS70 抗体的个体血清样本进行逐步汇集。
在两年的时间内，根据以下标准从常规 HEp-2 IFA 中选择血清样本：DFS HEp-2 IFA 滴度≥1:640；在三种分析物特异性检测（免疫印迹 [WB]、酶联免疫吸附试验 [ELISA]和化学发光免疫分析 [CLIA]）中具有抗 DFS70 反应性。将个体样本的等分试样合并到较大的池，逐步验证中间池与个体样本一样。验证后的中间池合并到最终用于冻干的池。
总共 741 个验证样本产生了 750 毫升的最终池，该池冻干成数千个 200 μL 等分试样。重新配制的等分试样在 ELISA、CLIA 和 WB 中产生了预期的抗 DFS70 反应性，以及高滴度 DFS HEp-2 IFA 模式。通过七个国际专家中心使用 HEp-2 IFA、ELISA、WB 和免疫沉淀来确认冻干池的适当抗 DFS70 反应性。
这项概念验证研究提供了一种创新且高效的策略，用于构建自身抗体检测的血清参考标准。抗 DFS70 标准将整合自身抗体标准化委员会（ASC，www.autoab.org）的标准面板，有助于正确验证检测和解释 DFS 模式和其他 HEp-2 IFA 模式。
