双抗体夹心ELISA测定人血浆可溶性血管内皮钙粘蛋白方法的建立及其应用研究

发布时间：2018-12-10 17:55

【摘要】：目的:建立测定人血浆可溶性血管内皮钙粘蛋白(sVE-cadherin)浓度的双抗体夹心酶联免疫吸附试验(ELISA)方法,并探讨其临床应用价值。方法:应用原核表达的人VE-cadherin重组蛋白作为抗原免疫BALB/c小鼠制备单克隆抗体,采用棋盘格滴定法筛选出最佳双抗体组合,其中检测抗体采用辣根过氧化物酶(HRP)标记,建立检测人血浆sVE-cadherin双抗体夹心ELISA方法,并进行方法学评价。同时采用该法检测28名健康体检者和60例肿瘤患者的血浆sVE-cadherin浓度。结果:经筛选共获得6株单克隆抗体,通过筛查配对,成功建立检测人血浆可溶性VE-cadherin的双抗体夹心ELISA方法。该方法的检测限为24.7 pg/ml,批内变异系数(CV)为4.1%-7.7%,批间CV为8.7%-10.8%,平均回收率为96.7%。用该法检测28名健康体检者血浆VE-cadherin浓度为262.1±11.75 pg/ml,而用该方法检测24例白血病患者血浆VE-cadherin浓度为173.9±17.98 pg/ml,显著低于正常人(P0.01),14例胃癌患者血浆VE-cadherin浓度为311.7±25.24 pg/ml(P0.05),11例肺癌患者血浆VEcadherin浓度为206.8±25.01 pg/ml,均低于正常人(P0.05),11例其他肿瘤(9例乳腺癌,1例胶质瘤,1例肝癌)患者血浆VE-cadherin浓度为310.7±11.82 pg/ml(P0.05)。结论:建立的双抗体夹心ELISA试剂盒有较好的灵敏度及特异性,可用于人血浆中可溶性VE-cadherin含量的检测,并可作为肿瘤诊断的一种新检测指标。
[Abstract]:Aim: to establish a double antibody sandwich enzyme linked immunosorbent assay (ELISA) method for the determination of soluble vascular endothelial cadherin (sVE-cadherin) in human plasma and to explore its clinical value. Methods: Monoclonal antibodies were prepared by immunizing BALB/c mice with prokaryotic expressed human VE-cadherin recombinant protein. The best combination of double antibodies was screened by chessboard titration. The detection antibody was labeled with horseradish peroxidase (HRP). A method for the detection of double antibody sandwich ELISA (ELISA) in human plasma sVE-cadherin was established and evaluated. Plasma sVE-cadherin levels were measured in 28 healthy persons and 60 tumor patients. Results: six monoclonal antibodies were obtained and a double antibody sandwich ELISA method was successfully established for the detection of soluble VE-cadherin in human plasma by screening and pairing. The detection limit of this method was 24.7 pg/ml, within-run variation coefficient (CV) of 4.1-7.7.The CV between batches was 8.7- 10.8.The average recovery was 96.7. The plasma VE-cadherin concentration was 262.1 卤11.75 pg/ml, in 28 healthy controls and 173.9 卤17.98 pg/ml, in 24 leukemia patients (P0.01). The plasma VE-cadherin concentration of 14 patients with gastric cancer was 311.7 卤25.24 pg/ml (P0.05), the plasma VEcadherin concentration of 11 patients with lung cancer was 206.8 卤25.01 pg/ml, lower than that of normal controls (P0.05), 11 patients with other tumors (9 patients with breast cancer), 11 patients with other tumors (9 patients with breast cancer, 9 patients with breast cancer), The plasma VE-cadherin concentration in 1 case of glioma and 1 case of liver cancer was 310.7 卤11.82 pg/ml (P0.05). Conclusion: the double antibody sandwich ELISA kit has good sensitivity and specificity. It can be used for the detection of soluble VE-cadherin in human plasma, and can be used as a new detection index for tumor diagnosis.
【作者单位】：苏州大学附属第一医院检验科;江苏省血液研究所;苏州大学唐仲英血液学研究中心;
【基金】：江苏高校优势学科建设工程资助项目(YX21100214) 苏州市第十五批科技发展项目(SYS201409)
【分类号】：R446.6

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