| 谢姣贵 王毅超 郭欣 宋朝君 赵宁 陈勇.抗Dysbindin-1多肽双抗夹心ELISA的建立及在肝癌中的应用[J].,2016,16(11):2022-2026 |
| 抗Dysbindin-1多肽双抗夹心ELISA的建立及在肝癌中的应用 |
| Establishment and Application DAS-ELISA of Antibody against Dysbindin-1 Polypeptides in Hepatocellular Carcinoma |
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| DOI： |
| 中文关键词: Dysbindin-1 单克隆抗体 DAS-ELISA 肝癌 |
| 英文关键词: Dysbindin-1 Monoclonal Antibodies ELISA Hepatocellular carcinoma |
| 基金项目:国家重点基础研究面上项目（81070363,81372607） |
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| 中文摘要: |
| 目的：制备抗人Dysbindin-1 特异性单克隆抗体，建立DAS-ELISA 检测体系，并初步应用于肝癌血清的检测。方法：采用合
成免疫原免疫BALB/c 小鼠，通过杂交瘤技术制备抗Dysbindin-1 单克隆抗体，用HRP 标记单克隆抗体，ELISA 和SDS-PAGE 电
泳法检测抗体的亚类、滴度；采用DAS-ELISA技术制备Dysbindin-1 检测试剂盒，检测正常、肝硬化及肝癌患者各30 例血清并比
较其差异。结果：细胞融合后获得了4 株稳定产生抗Dysbindin-1 单克隆抗体的杂交瘤细胞株(1C6A11、1E8H3、2D1C11、5B6D4)，
抗体亚类分别为IgG2b，IgG2b，IgG1和IgG2a，杂交瘤细胞诱生的腹水抗体效价达106 以上，通过抗体配对实验筛选确定2D1C11
作为包被抗体，1E8H3 作为酶标抗体。该ELISA 方法线性范围为62.5-1000 ng/mL，检测限为62.5 ng/mL；应用该方法检测显示正
常组与肝硬化组及肝癌组间差异显著（P<0.001），Dysbindin-1 诊断肝癌的敏感性和特异性分别为90 %和93.3 %。结论：Dysbindin-
1 可以成为新的候选的肝癌血清标志物，抗Dysbindin-1 多肽双抗夹心ELISA体系可以初步应用于肝癌的早期诊断。 |
| 英文摘要: |
| Objective:To prepare the monoclonal antibody against Dysbindin-1 and establish an ELISA kit of Dysbindin-1 for detection
of hepatocellular carcinoma (HCC).Methods:Hybridoma lines were obtained by fusing myeloma and spleen cells fromBALB /c
mice immunized with synthetic antigen Dysbindin-1-KLH. The mAbs from mice injected with hybridoma lines were purified and conjugated
to HRP. The subtype and titer of monoclonal antibodies were determined by ELISA and SDS-PAGE. Based on epitope of Dysbindin-
1 and Dysbindin-1 mAbs HRP-conjugation, the sandwich ELISA kit for Dysbindin-1 was developed, and used to detect the serum
of 30 healthy people, 30 patients with cirrhosis and 30 HCC patients.Results:Four clones derived hybridoma (1C6A11, 1E8H3, 2D1C11
and 5B6D4) were anti-Dysbindin-1 producers with higher antibody secretion. Each of them were producing antibodies from IgG2b,
IgG2b, IgG1 and IgG2a subclass, with titer of 106. According to our antibody pairing experiment result, 2D1C11 as Coating antibody,
1E8H3 as Detection antibody, hybridoma induced the ascites of antibody titer was more than 106. The linear range of the method was
62.5-1000 ng/mL with the detection limit of 62.5 ng/mL Sera. There was statistically significant difference in values of serum Dysbindin-
1 between healthy subjects and the patients with cirrhosis and HCC patients（P<0.001）. The sensitivity and specificity of peptide
in the diagnosis of HCC were 90%and 93.3 %, respectively.Conclusion:Dysbindin-1 may be a new candidate serumbiomarker for the
diagnosis of liver cancer, and anti- Dysbindin-1 polypeptide DAS-ELISA systemcan be used for early diagnosis of this disease. |
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