Institute of Technical Chemistry, Leibniz University Hannover, Callinstrasse 5, 30167 Hannover, Germany.
Analyst. 2021 Sep 7;146(17):5369-5379. doi: 10.1039/d1an01001h. Epub 2021 Aug 2.
Kidney is the most frequently transplanted among all solid organs worldwide. Kidney transplant recipients (KTRs) undergo regular follow-up examinations for the early detection of acute rejections. The gold standard for proving a T-cell mediated rejection (TCMR) is a biopsy of the renal graft often occurring as indication biopsy, in parallel to an increased serum creatinine that may indicate deterioration of renal transplant function. The goal of the current work was to establish a lateral flow assay (LFA) for diagnosing acute TCMR to avoid harmful, invasive biopsies. Soluble interleukin-2 (IL-2) receptor (sIl-2R) is a potential biomarker representing the α-subunit of the IL-2 receptor produced by activated T-cells, e.g., after allogen contact. To explore the diagnostic potential of sIL-2R as a biomarker for TCMR and borderline TCMR, plasma and urine samples were collected from three independent KTR cohorts with various distinct histopathological diagnostic findings according to BANFF (containing 112 rsp. 71 rsp. 61 KTRs). Samples were analyzed by a Luminex-based multiplex technique and cut off-ranges were determined. An LFA was established with two specific sIL-2R-antibodies immobilized on a nitrocellulose membrane. A significant association between TCMR, borderline TCMR and sIL-2R in plasma and between TCMR and sIL-2R in urine of KTRs was confirmed using the Mann-Whitney U test. The LFA was tested with sIL-2R-spiked buffer samples establishing a detection limit of 25 pM. The performance of the new LFA was confirmed by analyzing urine samples of the 2 and 3 patient cohort with 35 KTRs with biopsy proven TCMRs, 3 KTRs diagnosed with borderline TCMR, 1 mixed AMR/TCMR rsp. AMR/borderline TCMR and 13 control patients with a rejection-free kidney graft proven by protocol biopsies. The new point-of-care assay showed a specificity of 84.6% and sensitivity of 87.5%, and a superior estimated glomerular filtration rate (eGFR) at the time point of biopsy (specificity 30.8%, sensitivity 85%).
肾脏是全球所有实体器官中最常进行移植的器官。肾脏移植受者（KTR）需要定期进行随访检查，以早期发现急性排斥反应。证明 T 细胞介导的排斥反应（TCMR）的金标准是对肾移植物进行活检，通常作为指示性活检，同时血清肌酐升高可能表明肾移植功能恶化。目前的工作旨在建立一种用于诊断急性 TCMR 的侧向流动测定法（LFA），以避免进行有害的、有创性的活检。可溶性白细胞介素-2（IL-2）受体（sIl-2R）是一种潜在的生物标志物，代表活化 T 细胞产生的 IL-2 受体的α亚单位，例如在同种异体接触后。为了探索 sIL-2R 作为 TCMR 和边界 TCMR 生物标志物的诊断潜力，根据 BANFF（包含 112 例、71 例和 61 例 KTR），从三个独立的 KTR 队列中采集了具有不同明显组织病理学诊断发现的血浆和尿液样本。使用基于 Luminex 的多重技术对样本进行分析，并确定了截止范围。使用固定在硝酸纤维素膜上的两种特定 sIL-2R 抗体建立了 LFA。使用 Mann-Whitney U 检验证实了 KTR 血浆中的 TCMR、边界 TCMR 和 sIL-2R 之间以及 KTR 尿液中的 TCMR 和 sIL-2R 之间存在显著相关性。使用 sIL-2R 加标缓冲样品对 LFA 进行了测试，确定了 25 pM 的检测限。通过分析由 2 名和 3 名患者队列的尿液样本，对新的 LFA 进行了验证，这 35 名 KTR 中有活检证实的 TCMR、3 名诊断为边界 TCMR、1 名混合 AMR/TCMR 或 AMR/边界 TCMR 以及 13 名接受方案活检证实排斥反应阴性的对照患者。新的即时检测测定法显示出 84.6%的特异性和 87.5%的敏感性，并且在活检时的估计肾小球滤过率（eGFR）更高（特异性为 30.8%，敏感性为 85%）。
