This work is supported by Guangdong Basic and Applied Basic Research Foundation (2020A1515110840), and Shenzhen Basic Research Fund (JCYJ20190807170801656)
凝血酶-抗凝血酶复合物(thrombin-antithrombin complex，TAT)是人体凝血和抗凝血平衡的产物，反映人体的凝血状态，其检测结果可用于血栓性疾病的辅助诊断。该研究建立了人血浆样本中 TAT 化学发光免疫分析检测方法，并对其性能进行评估。该研究以 TAT 为免疫原制备杂交瘤抗体，以双抗体夹心模式建立 TAT 化学发光免疫分析检测方法。经过反应优化，磁珠浓度和吖啶酯抗体浓度确定为 0.20 g/L 和 0.2 mg/L，样本量为 50 μL，在 37 ℃ 条件下，磁珠抗体和待测物孵育 5 min，再与吖啶酯标记抗体孵育 5 min。结果表明，该方法与含有凝血酶原(0.20 mg/mL)和抗凝血酶 Ⅲ(0.31 mg/mL)的样本没有交叉反应，且与日本希森美康的 TAT 试剂的检测结果相关性较高(r＞0.95)。此外，该方法的各项性能指标良好：空白限 LoB≤0.20 ng/mL，检出限 LoD≤0.40 ng/mL，在 0.40～120 ng/mL 范围内的线性相关系数为 0.998，准确度在 ±8% 的范围内，可满足临床上对血栓性疾病进行辅助诊断的需求。
Thrombin-antithrombin complex (TAT) is the product of coagulation and anticoagulation balance. Its detection reflects thrombin status and can be used as an auxiliary in the diagnosis of thrombotic diseases. A chemiluminescence immunoassay method was established to analyze TAT in human plasma samples, and its analytical performance was evaluated. The TAT was used as immunogen for monoclonal antibody preparation. TAT measurement method was established using double antibody sandwich format. After the optimization of reaction, the bulk reagent concentrations of magnetic microparticle coated with antibody and acridinium labeled antibody were 0.20 g/L and 0.2 mg/L, respectively, and the sample size was 50 μL. The reaction of magnetic particle coated antibody and sample were incubated for 5 min at 37 ℃, and 5 min for subsequent reacting with the antibody labeled with acridinium. There was no cross-reaction with samples containing 0.20 mg/mL prothrombin or 0.31 mg/mL antithrombin Ⅲ. Besides, there was a high correlation (r＞0.95) between this method and TAT test kit of Sysmex. LoB is less than or equal to 0.20 ng/mL. LoD is less than or equal to 0.40 ng/mL. The linear correlation coefficient is 0.998 in the detection interval of 0.40 to 120 ng/mL, and the accuracy is within the range of ±8%. A quantitative chemiluminescence immunoassay method for TAT measurement has been established, and the performance meets customer needs for clinical utility.
安 倩,余枝广,柴 凡,等.凝血酶-抗凝血酶复合物化学发光免疫分析检测方法的建立 [J].集成技术,2023,12(6):72-82
AN Qian, YU Zhiguang, CHAI Fan, et al. Establishment of Chemiluminescence Immunoassay Method for Determination of Thrombin-Antithrombin Complex[J]. Journal of Integration Technology,2023,12(6):72-82