DAB2IP检测方法的建立及在不同临床中医证型肺癌中的应用

发布时间：2018-03-31 17:07

本文选题：DAB2IP　切入点：荧光免疫层析　出处：《福建中医药大学》2014年硕士论文

【摘要】：目的：研发DAB2IP蛋白的荧光免疫层析检测试剂盒,探讨不同中医证型肺癌与DAB2IP蛋白表达量的相关性。方法：应用B细胞杂交瘤技术建立分泌抗人DAB2IP单克隆抗体的杂交瘤细胞株；采用鼠源性单克隆抗体亚型检测试剂盒鉴定单抗的亚类；体内诱生腹水法制备单抗；免疫亲和层析法纯化抗体；Western blot分析抗体特异性；荧光免疫层析法研制DAB2IP荧光免疫试剂盒；在此基础上,应用DAB2IP荧光免疫试剂盒检测不同临床中医证型肺癌中DAB2IP蛋白的表达。结果：获取两株稳定分泌抗人DAB2IP单抗的杂交瘤(2A4、2G8)；鼠源性单克隆抗体亚型检测试剂盒鉴定2A4、2G8细胞株均属小鼠IgGl亚类；经体内诱生腹水法制备单抗,小鼠腹水形成的阳性率为95%以上,腹水的收获量平均为5.Oml/只小鼠；经免疫亲和层析法纯化后的抗体跑胶结果显示其分子量约为150kD; Western blot显示2A4单抗能与蛋白发生特异性反应,特异性良好；成功研制DAB2IP分子的荧光免疫层析检测试剂盒,灵敏度为ing/m1,精密度为6.08%,线性范围在1.00-500.00ng/mL,提示该检测系统具有良好的灵敏度、精密性和准确性。应用该试剂盒检测不同临床中医证型肺癌中的DAB21P分子的表达量,显示气阴两虚组与其它组间差别有显著性(P0.05),具有明显统计学意义。结论：本研究成功制备了具有自主知识产权的两株抗人DAB2IP单抗和DAB2IP荧光免疫层析检测试剂盒。该试剂盒通过对血清中DAB2IP含量进行半定量分析,为其在肿瘤基础研究和临床中的应用提供了实验依据,为肺癌的靶向治疗提供了新的思路和途径。由于肺癌气阴两虚组与其它组在DAB2IP分子表达上存在显著差异(P0.05),提示,采用望闻问切与现代免疫学方法相结合,可能使中医分型更加客观、更加深入,该研究对于中医证型现代化研究具有一定参考价值。
[Abstract]:Objective : To develop a fluorescent immunochromatography assay kit for the research and research on the correlation between different types of traditional Chinese medicine syndrome type lung cancer ( NSCLC ) and the expression level of DAB 2IP protein .

Methods : A hybridoma cell line secreting anti - human DAB 2IP monoclonal antibody was established by B - cell hybridoma technique .
a murine monoclonal antibody subtype detection kit is adopted to identify the subclass of the monoclonal antibody ;
preparing monoclonal antibody by in vivo induced ascites method ;
purifying the antibody by immunoaffinity chromatography ;
The specificity of antibody was analyzed by Western blot .
Development of fluorescence immunoassay kit by fluorescence immunochromatography
On the basis of this , the expression of DAB 2IP protein in lung cancer of different clinical syndrome types was detected by the fluorescence immunoassay kit .

Results : Two hybridoma clones ( 2A4 , 2G8 ) were obtained .
The mouse - derived monoclonal antibody subtype detection kit was used to identify the 2A4 and 2G8 cell strains , which belong to the mouse IgG1 subclass ;
The positive rate of ascites formation in mice was more than 95 % , and the average recovery of ascites was 5.Oml / mouse ;
The results showed that the molecular weight was about 150 kD after purified by immunoaffinity chromatography . Western blot showed that the monoclonal antibody of 2A4 could specifically react with the protein , and its specificity was good ;
The sensitivity is ing / m1 , the precision is 6.08 % and the linear range is 1.00 - 500.00ng / mL . It is suggested that the detection system has good sensitivity , precision and accuracy .

Conclusion : This study has successfully prepared two kinds of anti - human DAB 2IP monoclonal antibodies with independent intellectual property . The kit has provided an experimental basis for its application in tumor basic research and clinical application by semi - quantitative analysis of the content in serum . It suggests that the combination of the two virtual groups of lung cancer and modern immunology may make the classification of traditional Chinese medicine more objective and deeper , and the research has certain reference value for the modern research of TCM syndrome type .

【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R734.2

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