人血清白蛋白抗体的制备及多种免疫学方法的初步建立

发布时间：2018-01-19 20:55

本文关键词： 尿微量白蛋白单克隆抗体多克隆抗体荧光免疫检测酶联免疫检测　出处：《长春理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 尿微量白蛋白(MA)作为糖尿病、肾病、心血管疾病等慢性病的早期诊断指标,对其定量检测有广泛的临床意义。因此,建立特异、灵敏的检测尿微量白蛋白的方法具有重要意义。本研究利用人血清白蛋白(HSA)制备抗HSA的兔多抗和小鼠单抗,并初步建立了基于尿微量白蛋白的双抗体夹心免疫荧光及ELISA检测方法。采用HSA免疫大耳白兔制备免疫兔血清,并采用蛋白A亲和层析柱纯化,获得高纯度的兔多抗,效价测定达到1×10~6;同时用HSA免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经克隆和间接ELISA筛选,筛选到六株能分泌抗HSA单克隆抗体的杂交瘤细胞株,其中三株效价高的杂交瘤株3F4、6F2和8B5制备腹水,经过鉴定:三株单抗都识别同一抗原表位,且3F4的单抗亚类是IgG2b(k型);采用蛋白G亲和层析柱纯化腹水,测定效价均达到1×10~7。以兔多抗和鼠单抗为基础,初步建立了尿微量白蛋白的双抗体夹心免疫荧光检测方法和ELISA检测方法。免疫荧光与ELISA法的线性范围范围分别为:50～1000ng/ml、10～400ng/ml。经敏感性实验、特异性实验、重复性实验,结果表明,该两种方法具有特异性好、灵敏度高和重复性好的特点,为建立科学完善的尿微量白蛋白检测方法提供基础。
[Abstract]:As an early diagnostic index of diabetes, nephropathy, cardiovascular diseases, urinary microalbuminuria (MAA) has a wide range of clinical significance in quantitative detection. Therefore, the establishment of a specific. A sensitive method for the detection of urinary microalbumin is of great significance. In this study, rabbit polyclonal antibody and mouse monoclonal antibody against HSA were prepared by using human serum albumin (HSA), and a double antibody sandwich immunofluorescence and ELISA detection method based on urinary microalbumin was established. Rabbit serum was prepared by immunizing big ear white rabbits with HSA and purified by protein A affinity chromatography. The high purity rabbit polyclonal antibody was obtained and the titer was 1 脳 10 ~ (6). At the same time, BALB/c mice were immunized with HSA, and the spleen cells were fused with SP2/0 myeloma cells, and were screened by clone and indirect ELISA. Six hybridoma cell lines secreting monoclonal antibodies against HSA were screened, and three hybridoma strains with high titer 3F4, 6F2 and 8B5 were selected to produce ascites. All the three McAbs recognize the same epitope, and the McAb subclass of 3F4 is IgG2b(k type. Ascites were purified by affinity chromatography with protein G, and the titers reached 1 脳 10 ~ (7). It was based on rabbit polyclonal antibody and mouse monoclonal antibody. A double antibody sandwich immunofluorescence assay and a ELISA method for detection of urinary microalbumin were established. The linear ranges of immunofluorescence and ELISA were as follows:. 50ng / ml. The sensitivity, specificity and repeatability of the two methods were tested. The results showed that the two methods had good specificity, high sensitivity and good reproducibility. It provides a basis for the establishment of a scientific and perfect method for the detection of urinary microalbumin.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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