R&D Division, AroCell AB, Stockholm, Sweden.
Future Diagnostics, Wijchen, The Netherlands.
PLoS One. 2022 Oct 6;17(10):e0275444. doi: 10.1371/journal.pone.0275444. eCollection 2022.
Thymidine Kinase 1 (TK1) plays an important role in DNA precursor synthesis and serum TK1 activity has been used as a biomarker for prognosis and therapy monitoring of different malignancies. AroCell has developed a dual monoclonal antibody ELISA for determination of TK1 protein in clinical samples. The purpose of the study is to validate the ELISA analytically in relation to the gold standard, [3H]-deoxythymidine (dThd) phosphorylation assay for TK1 activity using sera from patients with different malignancies. The colorimetric TK 210 ELISA was validated analytically by assessment of precision, linearity, interfering substances, and stability. For the clinical validation, serum samples from patients with hematological malignancies (n = 100), breast cancer (n = 56), prostate cancer (n = 70) and blood donors (n = 159) were analyzed using TK 210 ELISA and TK1 activity by [3H]-deoxythymidine (dThd) phosphorylation assay. The sandwich TK 210 ELISA was highly specific for TK1 protein having a detection limit of 0.12 ng/mL, with a functional sensitivity of 0.25 ng/mL. Within-run CVs ranged from 5.5% to 10% and between-run CVs ranged from 5% to 15%. The ratio of observed to expected dilutional parallelism of 5 serum samples was in the range of 80-120%. Samples exhibited stability through four freeze/thaw cycles and 5 days at 4°C. Further, the ROC curve analysis showed that TK 210 ELISA and [3H]-dThd phosphorylation assay had similar sensitivity (62% vs 59%) in hematological malignancies. However, in the case of breast and prostate cancer sera, TK 210 ELISA had higher sensitivity (59% and 44%) compared to [3H]-dThd phosphorylation assay (47% and 25%) at a specificity of 98%. These data demonstrate that the dual monoclonal antibody based AroCell TK 210 ELISA is a robust, accurate and precise tool for measuring TK1 protein in different malignancies that can improve the clinical applications of TK1 as a biomarker in cancer management.
胸苷激酶 1（TK1）在 DNA 前体合成中发挥着重要作用，血清 TK1 活性已被用作不同恶性肿瘤的预后和治疗监测的生物标志物。AroCell 开发了一种用于测定临床样本中 TK1 蛋白的双单克隆抗体 ELISA。本研究的目的是使用来自不同恶性肿瘤患者的血清，相对于金标准 [3H]-胸苷（dThd）磷酸化测定法，对 ELISA 进行分析验证。比色 TK210 ELISA 通过评估精密度、线性、干扰物质和稳定性进行了分析验证。为了进行临床验证，使用 TK210 ELISA 和 [3H]-胸苷（dThd）磷酸化测定法分析了来自血液系统恶性肿瘤患者（n=100）、乳腺癌患者（n=56）、前列腺癌患者（n=70）和献血者（n=159）的血清样本。夹心 TK210 ELISA 对 TK1 蛋白具有高度特异性，检测限为 0.12ng/mL，功能灵敏度为 0.25ng/mL。批内 CV 范围为 5.5%至 10%，批间 CV 范围为 5%至 15%。5 份血清样本的观察稀释度与预期稀释度的比值在 80-120%范围内。样品经过四次冻融循环和 4°C 下 5 天的稳定性测试。此外，ROC 曲线分析表明，在血液系统恶性肿瘤中，TK210 ELISA 和 [3H]-dThd 磷酸化测定法的灵敏度相似（62%对 59%）。然而，在乳腺癌和前列腺癌血清的情况下，与 [3H]-dThd 磷酸化测定法（47%和 25%）相比，TK210 ELISA 的灵敏度更高（59%和 44%），特异性为 98%。这些数据表明，基于双单克隆抗体的 AroCell TK210 ELISA 是一种强大、准确和精确的工具，可用于测量不同恶性肿瘤中的 TK1 蛋白，从而提高 TK1 作为癌症管理中生物标志物的临床应用。
