Department of Hepatitis C and Drug-Induced Liver Disease, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Center of Minimally Invasive Interventional Therapy, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Braz J Med Biol Res. 2019 Sep 30;52(10):e8845. doi: 10.1590/1414-431X20198845. eCollection 2019.
Regucalcin is a soluble protein that is principally expressed in hepatocytes. Studies of regucalcin have mainly been conducted in animals due to a lack of commercially available kits. We aimed to develop an enzyme-linked immunosorbent assay (ELISA) to quantify serum regucalcin in patients with hepatitis B virus (HBV)-related disease. High-titer monoclonal antibodies and a polyclonal antibody to regucalcin were produced, a double-antibody sandwich ELISA method was established, and serum regucalcin was determined in 47 chronic hepatitis B (CHB) patients, 91 HBV-related acute-on-chronic liver failure (HBV-ACLF) patients, and 33 healthy controls. The ELISA demonstrated an appropriate linear range, and high levels of reproducibility, sensitivity, specificity, accuracy, and stability. The median serum regucalcin concentrations in HBV-ACLF and CHB patients were 5.46 and 3.76 ng/mL, respectively (P<0.01), which were much higher than in healthy controls (1.72 ng/mL, both P<0.01). For the differentiation of CHB patients and healthy controls, the area under curve (AUC) was 0.86 with a cut-off of 2.42 ng/mL, 85.7% sensitivity, and 78.8% specificity. In contrast, the AUC of alanine aminotransferase (ALT) was lower (AUC=0.80, P=0.01). To differentiate ACLF from CHB, the AUC was 0.72 with a cut-off of 4.26 ng/mL, 77.0% sensitivity, and 61.2% specificity while the AUC of ALT was 0.41 (P=0.07). Thus, we have developed an ELISA that is suitable for measuring serum regucalcin and have shown that serum regucalcin increased with the severity of liver injury due to HBV-related diseases, such that it appears to be more useful than ALT as a marker of liver injury.
钙调节蛋白是一种主要在肝细胞中表达的可溶性蛋白。由于缺乏商业上可获得的试剂盒，钙调节蛋白的研究主要在动物中进行。我们旨在开发一种酶联免疫吸附测定（ELISA）来定量乙型肝炎病毒（HBV）相关疾病患者的血清钙调节蛋白。制备了高滴度的单克隆抗体和多克隆抗体，建立了双抗体夹心 ELISA 方法，并在 47 例慢性乙型肝炎（CHB）患者、91 例 HBV 相关慢加急性肝衰竭（HBV-ACLF）患者和 33 名健康对照者中测定了血清钙调节蛋白。该 ELISA 显示出适当的线性范围和较高的重现性、灵敏度、特异性、准确性和稳定性。HBV-ACLF 和 CHB 患者的血清钙调节蛋白中位数浓度分别为 5.46 和 3.76ng/ml（均 P<0.01），明显高于健康对照组（1.72ng/ml，均 P<0.01）。对于 CHB 患者和健康对照者的鉴别，曲线下面积（AUC）为 0.86，截断值为 2.42ng/ml，灵敏度为 85.7%，特异性为 78.8%。相比之下，丙氨酸氨基转移酶（ALT）的 AUC 较低（AUC=0.80，P=0.01）。对于 ACLF 与 CHB 的鉴别，AUC 为 0.72，截断值为 4.26ng/ml，灵敏度为 77.0%，特异性为 61.2%，而 ALT 的 AUC 为 0.41（P=0.07）。因此，我们开发了一种适合于测量血清钙调节蛋白的 ELISA，并表明血清钙调节蛋白随着乙型肝炎相关疾病导致的肝损伤严重程度而增加，因此其作为肝损伤标志物似乎比 ALT 更有用。
