Bazzini Cecilia, Begré Nadja, Favre Bertand, Hashimoto Takashi, Hertl Michael, Schlapbach Christoph, Borradori Luca
Department of Dermatology, Inselspital, Hospital and University of Bern, Bern, Switzerland.
Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan.
J Dermatol Sci. 2020 Jun;98(3):173-178. doi: 10.1016/j.jdermsci.2020.04.005. Epub 2020 May 7.
Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies.
To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera.
We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates.
A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values.
Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.
副肿瘤性天疱疮（PNP）是一种严重的自身免疫性多器官综合征，与针对多种自身抗原的自身抗体有关，包括α-2-巨球蛋白样-1（A2ML1）。高达70%的PNP血清可识别A2ML1。目前推荐的PNP血清学诊断技术不足以检测抗A2ML1抗体。
开发新的检测方法，以便轻松、可靠地检测PNP血清中的抗A2ML1自身抗体。
我们在转染的人胚肾293T细胞中产生了与增强型绿色荧光蛋白融合的全长A2ML1（EGFP-A2ML1）。该重组蛋白用作免疫沉淀研究的荧光配体。我们通过将EGFP-A2ML1固定在96孔板上进一步开发了一种酶联免疫吸附测定（ELISA）。
A2ML1阳性的PNP血清能够免疫沉淀EGFP-A2ML1。免疫沉淀物中荧光的直接测量与PNP血清中抗A2ML1抗体的相对水平相关。通过基于确定的最佳临界值的新型ELISA，在检测的36份PNP血清中，61%为A2ML1阳性，特异性为88.9%，敏感性为95%。20份检测的正常血清（NHS）为阴性，而20份寻常型天疱疮血清中的2份（10%）和20份大疱性类天疱疮血清中的3份（15%）显示临界值。
我们的新型免疫测定法能够对PNP患者进行快速分层。利用活性真核A2ML1的新型基于绿色荧光蛋白的ELISA高度敏感且可靠，因此有助于更好地理解PNP的免疫背景。这种方法可轻松应用于快速检测针对各种其他抗原的抗体。
