Central Sydney Immunology Laboratory, Royal Prince Alfred Hospital, NSW Health Pathology, Sydney, NSW, Australia.
Sutherland Centre of Immunology, NSW Health Pathology, Sydney, NSW, Australia.
Pathology. 2023 Oct;55(6):843-849. doi: 10.1016/j.pathol.2023.04.002. Epub 2023 May 29.
The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.
间接免疫荧光法（IIFA）在 Hep-2 细胞上的应用已广泛用于筛查与系统性自身免疫性风湿病（SARD）相关的抗核抗体（ANA）。含有 ANA 的血清在 Hep-2 细胞上显示出多种不同的荧光模式。其中，由抗-DFS70 抗体引起的密集细点状（DFS）模式在健康个体中的患病率高于 SARD 患者。这种 DFS 模式通常难以与其他与 SARD 相关的 ANA 模式区分，特别是混合均匀和点状模式。此外，强烈的 DFS 模式可以掩盖其他与 SARD 相关的模式。因此，我们开发了一种新的免疫沉淀方法，使用磁珠在进行 IIFA 之前去除血清中的抗-DFS70 抗体。我们还旨在确认抗-DFS70 的存在，并发现任何被强烈 DFS 模式掩盖的与 SARD 相关的 ANA 模式。我们研究中使用的血清来自 70 名接受常规 ANA 筛查的个体，其中 35 份血清具有与单特异性抗-DFS70 抗体一致的孤立 DFS 模式，这些抗体已通过互补检测得到证实，35 份为无 DFS 模式的对照血清。开发了一种使用重组人全长 DFS70 蛋白包被的磁珠免疫沉淀方法。与使用相同 DFS70 蛋白的免疫吸附方法相比，评估了这种新方法的性能。我们新开发的免疫沉淀方法在确认与抗-DFS70 相关的 DFS 模式方面表现出优异的灵敏度（91.4%）和特异性（100%）。此外，该方法能够去除抗-DFS70 并揭示被强烈 DFS 模式掩盖的与 SARD 相关的 ANA 模式。与免疫吸附方法相比，它在 IIFA 上显示出更清晰的背景。新型基于磁珠的免疫沉淀方法在去除抗-DFS70 而不干扰其他抗体检测方面表现出令人满意的性能。它可以很容易地与 IIFA 集成，以确认与抗-DFS70 相关的 DFS 模式。此外，它可以同时揭示其他 ANA 模式，而常规方案需要进行互补的抗-DFS70 特异性检测才能实现这一点。因此，该新方法为 ANA 筛查提供了更有效和准确的解决方案。
