中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白（neutrophil gelatinase-associated lipocalin）
發(fā)布時(shí)間：2019-03-04 19:30
【摘要】：中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白(neutrophil gelatinase-associated lipocalin, NGAL)是lipocalin超家族的成員,其分子量為25KD,在炎癥或慢性惡性腫瘤環(huán)境中,人體器官組織(如子宮、唾液腺、腎、乳腺、消化道上皮等)均可誘導(dǎo)產(chǎn)生NGAL,正常的腎臟、肺、胃、大腸等組織的NGAL表達(dá)水平較低,但當(dāng)這些上皮細(xì)胞受損時(shí),NGAL被誘導(dǎo)而大量表達(dá)。NGAL分子量小、不容易降解,在尿中比較容易檢測(cè)。近年來(lái)國(guó)外大量報(bào)道NGAL是AKI早期的一種敏感性及特異性較高的生物學(xué)指標(biāo),其評(píng)價(jià)腎臟急性受損的價(jià)值優(yōu)于肌酐、血尿素、尿酸和內(nèi)生肌酐清除率。但目前國(guó)內(nèi)用于實(shí)驗(yàn)的NGAL的單克隆抗體絕大多數(shù)為進(jìn)口抗體,其價(jià)格昂貴,而國(guó)內(nèi)生產(chǎn)的的多為多克隆抗體,其特異性較差,上述原因均制約了關(guān)于NGAL的研究。 目的： 采用雜交瘤單克隆抗體技術(shù)制備NGAL單克隆抗體,建立能夠穩(wěn)定分泌NGAL單克隆抗體的雜交瘤細(xì)胞株,從而大量制備NGAL單克隆抗體,初步建立抗NGAL雙抗體夾心ELSIA檢測(cè)方法。 方法： 用純化重組的NGAL蛋白免疫Balb/c小鼠,取免疫小鼠脾細(xì)胞與骨髓瘤細(xì)胞SP2/0進(jìn)行融合,經(jīng)過(guò)克隆化和篩選,建立穩(wěn)定分泌抗NGAL的雜交瘤細(xì)胞株,并通過(guò)動(dòng)物體內(nèi)誘生腹水大量制備單克隆抗體,利用ELISA、Western Blot等方法鑒定單克隆抗體的性質(zhì)。將抗體行硫酸銨沉淀及親和純化,SDS-PAGE染色鑒定純化后抗體的純度,同時(shí)對(duì)純化后抗體HRP標(biāo)記后行抗體配對(duì)實(shí)驗(yàn)。 結(jié)果： 1、采用雜交瘤技術(shù),篩選出4株穩(wěn)定分泌抗重組蛋白抗體的雜交瘤細(xì)胞株,分別命名為5B1、7B6、9A11、9F1。將這4株細(xì)胞注射于小鼠腹腔得到了富含NGAL單克隆抗體的小鼠腹水。 2、SDS-PAGE染色表明純化后的抗體純度高 3、抗體配對(duì)實(shí)驗(yàn)表明9A11和7B6以及5B1和7B6能配對(duì)成功。 結(jié)論： 1、利用重組的NGAL抗原成功制備了鼠抗人NGAL單克隆抗體。 2、抗NGAL雙抗體夾心ELSIA檢測(cè)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
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692
本文編號(hào)：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
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692
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