抗exendin-4单克隆抗体的制备及其ELISA检测方法的建立
发布时间：2018-11-26 14:18
【摘要】：Exendin-4是由39个氨基酸组成的多肽,与胰高血糖素样肽-1(GLP-1)有53%的同源性,是一种有效的GLP-1受体激动剂,已作为II型糖尿病治疗药物在临床应用。目前其检测方法主要包括高效液相色谱法,液相-质谱法和免疫分析法。色谱法无法同时检测大批量样品,而且需要专业仪器设备;虽然免疫分析法已有商品化的检测试剂盒,但其价格昂贵,检测准确度欠佳。因此本文设计利用抗exendin-4的单克隆抗体构建一种夹心的酶联免疫吸附法(ELISA)用于检测exendin-4。以exendin-4免疫雌性的BALB/c小鼠,经过3次免疫检测血清效价后,取脾细胞与SP2/0骨髓瘤细胞融合。用有限稀释法将阳性细胞单克隆化筛选,经过4轮筛选共得到15株单克隆杂交瘤细胞。收集其中1株细胞培养基上清,经纯化得到单克隆抗体2-B7。将单抗2-B7与生物素标记的多抗联合构建了夹心的ELISA检测方法,并对其进行了方法学考察。该方法的可检测最低浓度为0.25ng/ml,特异性良好,批内变异系数和批间变异系数均小于5%,具有很好的重复性,可以用于本实验室相关研究样品的检测。在上述研究基础上,提出进一步优化ELISA检测方法的设想,并完成了一部分工作,包括半抗原偶联载体的免疫和特异性识别exendin-4 C末端和N末端的阳性细胞筛选。
[Abstract]:Exendin-4 is a 39 amino acid peptide with 53% homology with glucagon like peptide-1 (GLP-1). It is an effective GLP-1 receptor agonist and has been used as a therapeutic drug for type II diabetes. At present, the main detection methods include high performance liquid chromatography, liquid-mass spectrometry and immunoassay. Chromatography can not detect large quantities of samples at the same time, and requires professional instruments and equipment. Although commercial detection kits have been developed for immunoassay, their prices are expensive and the detection accuracy is poor. So we designed a sandwich enzyme linked immunosorbent assay (ELISA) using monoclonal antibody against exendin-4 to detect exendin-4.. Female BALB/c mice were immunized with exendin-4. After three times of immunoassay, spleen cells were fused with SP2/0 myeloma cells. A total of 15 monoclonal hybridoma cells were obtained after four rounds of screening by monoclonal screening with limited dilution method. One cell culture medium supernatant was collected and purified to obtain monoclonal antibody 2-B 7. A sandwich ELISA detection method was constructed by combining monoclonal antibody 2-B7 with biotinylated polyclonal antibody, and its methodology was investigated. The lowest detectable concentration of this method is 0.25 ng / ml, the specificity is good, the coefficient of variation within and between batches is less than 5, and the method has good repeatability. It can be used for the detection of related samples in our laboratory. Based on the above studies, the idea of further optimization of ELISA detection method is proposed, and part of the work is completed, including the immunological screening of hapten coupling vectors and the specific identification of exendin-4 C-terminal and N-terminal positive cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.11
本文编号：2358795
[Abstract]:Exendin-4 is a 39 amino acid peptide with 53% homology with glucagon like peptide-1 (GLP-1). It is an effective GLP-1 receptor agonist and has been used as a therapeutic drug for type II diabetes. At present, the main detection methods include high performance liquid chromatography, liquid-mass spectrometry and immunoassay. Chromatography can not detect large quantities of samples at the same time, and requires professional instruments and equipment. Although commercial detection kits have been developed for immunoassay, their prices are expensive and the detection accuracy is poor. So we designed a sandwich enzyme linked immunosorbent assay (ELISA) using monoclonal antibody against exendin-4 to detect exendin-4.. Female BALB/c mice were immunized with exendin-4. After three times of immunoassay, spleen cells were fused with SP2/0 myeloma cells. A total of 15 monoclonal hybridoma cells were obtained after four rounds of screening by monoclonal screening with limited dilution method. One cell culture medium supernatant was collected and purified to obtain monoclonal antibody 2-B 7. A sandwich ELISA detection method was constructed by combining monoclonal antibody 2-B7 with biotinylated polyclonal antibody, and its methodology was investigated. The lowest detectable concentration of this method is 0.25 ng / ml, the specificity is good, the coefficient of variation within and between batches is less than 5, and the method has good repeatability. It can be used for the detection of related samples in our laboratory. Based on the above studies, the idea of further optimization of ELISA detection method is proposed, and part of the work is completed, including the immunological screening of hapten coupling vectors and the specific identification of exendin-4 C-terminal and N-terminal positive cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.11
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