中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin）
发布时间：2019-03-04 19:30
【摘要】：中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)是lipocalin超家族的成员,其分子量为25KD,在炎症或慢性恶性肿瘤环境中,人体器官组织(如子宫、唾液腺、肾、乳腺、消化道上皮等)均可诱导产生NGAL,正常的肾脏、肺、胃、大肠等组织的NGAL表达水平较低,但当这些上皮细胞受损时,NGAL被诱导而大量表达。NGAL分子量小、不容易降解,在尿中比较容易检测。近年来国外大量报道NGAL是AKI早期的一种敏感性及特异性较高的生物学指标,其评价肾脏急性受损的价值优于肌酐、血尿素、尿酸和内生肌酐清除率。但目前国内用于实验的NGAL的单克隆抗体绝大多数为进口抗体,其价格昂贵,而国内生产的的多为多克隆抗体,其特异性较差,上述原因均制约了关于NGAL的研究。 目的： 采用杂交瘤单克隆抗体技术制备NGAL单克隆抗体,建立能够稳定分泌NGAL单克隆抗体的杂交瘤细胞株,从而大量制备NGAL单克隆抗体,初步建立抗NGAL双抗体夹心ELSIA检测方法。 方法： 用纯化重组的NGAL蛋白免疫Balb/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行融合,经过克隆化和筛选,建立稳定分泌抗NGAL的杂交瘤细胞株,并通过动物体内诱生腹水大量制备单克隆抗体,利用ELISA、Western Blot等方法鉴定单克隆抗体的性质。将抗体行硫酸铵沉淀及亲和纯化,SDS-PAGE染色鉴定纯化后抗体的纯度,同时对纯化后抗体HRP标记后行抗体配对实验。 结果： 1、采用杂交瘤技术,筛选出4株稳定分泌抗重组蛋白抗体的杂交瘤细胞株,分别命名为5B1、7B6、9A11、9F1。将这4株细胞注射于小鼠腹腔得到了富含NGAL单克隆抗体的小鼠腹水。 2、SDS-PAGE染色表明纯化后的抗体纯度高 3、抗体配对实验表明9A11和7B6以及5B1和7B6能配对成功。 结论： 1、利用重组的NGAL抗原成功制备了鼠抗人NGAL单克隆抗体。 2、抗NGAL双抗体夹心ELSIA检测方法初步建立
[Abstract]:Neutrophil gelatinase-associated lipid delivery protein (neutrophil gelatinase-associated lipocalin, NGAL) is a member of the lipocalin superfamily with a molecular weight of 25 KD. In inflammatory or chronic malignant tumor environments, human organs and tissues (such as uterus, salivary gland, kidney, mammary gland, etc.) The expression level of NGAL in normal kidney, lung, stomach, large intestine and other tissues was lower than that in normal kidney, lung, stomach and large intestine, but when these epithelial cells were damaged, NGAL was induced and expressed in a large amount. The molecular weight of NGAL, was small, and it was not easy to degrade. It is easier to detect in urine. In recent years, a large number of reports abroad reported that NGAL is a sensitive and specific biological index in the early stage of AKI, and its value is superior to creatinine, blood urea, uric acid and endogenous creatinine clearance rate in the evaluation of acute renal injury. But at present, most of the monoclonal antibodies used in the experiment of NGAL in our country are imported antibodies, which are expensive, but the polyclonal antibodies produced in our country are mostly polyclonal antibodies, whose specificity is poor. All these reasons restrict the research on NGAL. Objective: to prepare NGAL monoclonal antibody by hybridoma monoclonal antibody technique, and to establish hybridoma cell line which can stably secrete NGAL monoclonal antibody, so as to prepare NGAL monoclonal antibody in large quantities. A sandwich ELSIA assay for the detection of anti-NGAL double antibody was established. Methods: Balb/c mice were immunized with purified NGAL protein. Spleen cells of immunized mice were fused with myeloma cell line SP2/0. After cloning and screening, a hybridoma cell line stably secreting anti-NGAL was established. A large number of monoclonal antibodies were prepared by inducing ascites in animals. The properties of monoclonal antibodies were identified by ELISA,Western Blot and other methods. The purified antibody was purified by ammonium sulfate precipitation and affinity purification. The purity of the purified antibody was identified by SDS-PAGE staining. The purified antibody was labeled with HRP and then paired with the antibody. Results: 1. Four hybridoma cell lines stably secreting anti-recombinant protein antibodies were screened by hybridoma technique, which were named 5B1, 7B6,9A11,9F1. respectively. The ascites containing monoclonal antibody against NGAL was obtained by injecting these four cells into the abdominal cavity of mice. 2. The purity of purified antibody was higher than 3 by SDS-page. The results of antibody matching test showed that 9A11 and 7B6, 5B1 and 7B6 were able to pair successfully. Conclusion: 1. Mouse anti-human NGAL monoclonal antibody was successfully prepared by using recombinant NGAL antigen. 2. Establishment of sandwich ELSIA assay for detection of anti-NGAL double antibody
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
本文编号：2434563
[Abstract]:Neutrophil gelatinase-associated lipid delivery protein (neutrophil gelatinase-associated lipocalin, NGAL) is a member of the lipocalin superfamily with a molecular weight of 25 KD. In inflammatory or chronic malignant tumor environments, human organs and tissues (such as uterus, salivary gland, kidney, mammary gland, etc.) The expression level of NGAL in normal kidney, lung, stomach, large intestine and other tissues was lower than that in normal kidney, lung, stomach and large intestine, but when these epithelial cells were damaged, NGAL was induced and expressed in a large amount. The molecular weight of NGAL, was small, and it was not easy to degrade. It is easier to detect in urine. In recent years, a large number of reports abroad reported that NGAL is a sensitive and specific biological index in the early stage of AKI, and its value is superior to creatinine, blood urea, uric acid and endogenous creatinine clearance rate in the evaluation of acute renal injury. But at present, most of the monoclonal antibodies used in the experiment of NGAL in our country are imported antibodies, which are expensive, but the polyclonal antibodies produced in our country are mostly polyclonal antibodies, whose specificity is poor. All these reasons restrict the research on NGAL. Objective: to prepare NGAL monoclonal antibody by hybridoma monoclonal antibody technique, and to establish hybridoma cell line which can stably secrete NGAL monoclonal antibody, so as to prepare NGAL monoclonal antibody in large quantities. A sandwich ELSIA assay for the detection of anti-NGAL double antibody was established. Methods: Balb/c mice were immunized with purified NGAL protein. Spleen cells of immunized mice were fused with myeloma cell line SP2/0. After cloning and screening, a hybridoma cell line stably secreting anti-NGAL was established. A large number of monoclonal antibodies were prepared by inducing ascites in animals. The properties of monoclonal antibodies were identified by ELISA,Western Blot and other methods. The purified antibody was purified by ammonium sulfate precipitation and affinity purification. The purity of the purified antibody was identified by SDS-PAGE staining. The purified antibody was labeled with HRP and then paired with the antibody. Results: 1. Four hybridoma cell lines stably secreting anti-recombinant protein antibodies were screened by hybridoma technique, which were named 5B1, 7B6,9A11,9F1. respectively. The ascites containing monoclonal antibody against NGAL was obtained by injecting these four cells into the abdominal cavity of mice. 2. The purity of purified antibody was higher than 3 by SDS-page. The results of antibody matching test showed that 9A11 and 7B6, 5B1 and 7B6 were able to pair successfully. Conclusion: 1. Mouse anti-human NGAL monoclonal antibody was successfully prepared by using recombinant NGAL antigen. 2. Establishment of sandwich ELSIA assay for detection of anti-NGAL double antibody
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
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相关期刊论文 前3条
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