晚期氧化蛋白产物单克隆抗体的制备和初步应用

发布时间：2018-05-19 19:50

本文选题：AOPP + 单克隆抗体　；参考：《南方医科大学》2011年硕士论文

【摘要】：晚期氧化蛋白产物(Advanced oxidation protein products, AOPP)是体内氧化应激过程中生成的一类含双酪氨酸的蛋白质交联物,血浆AOPP以次氯酸(HOCl)氧化修饰的白蛋白为主。循环AOPP水平增高最先在慢性肾功能衰竭、腹膜透析的患者中被发现,随后在糖尿病、肥胖或代谢综合征、动脉粥样硬化等多种常见疾病中被证实。AOPP增高还见于类风湿性关节炎、溃疡性结肠炎等免疫炎症性疾病和恶性肿瘤(结肠、直肠癌、乳腺癌等),因此AOPP被认为是体内氧化应激的敏感生物学标志。近年不断增加的研究证据表明,血浆AOPP水平增高促使该分子在肾脏和血管等组织中沉积并与慢性肾脏病(chronic kidney disease, CKD)及动脉粥样硬化病变的发生发展密切相关。慢性AOPP在体内堆积促进糖尿病动物模型的肾脏的炎症反应和肾组织损伤,促使CKD动物模型的肾脏炎症和纤维化,并促使高脂血症动物发生动脉粥样硬化性病变。这些研究结果均表明,AOPP潴留不仅是反映体内氧化应激的指标,其本身可能是一类促进组织炎症和纤维化的生物致病分子。深入探讨AOPP在组织中的分布及其与病变的关系对于阐明AOPP致病的分子基础至关重要,并有可能为上述疾病的发生发展提供新的生物学标志或干预靶标。然而,目前检测组织中AOPP水平尚缺乏理想方法。检查外周血AOPP则通常采用比色分析法以340nm处吸光度值以反映标本中AOPP水平,这是基于AOPP结构含双酪氨酸,在酸性条件下于340nm处有一特异吸收峰。此法虽然简便,但易受血脂、纤维蛋白的干扰而影响其准确性；且不能用于对病变组织或细胞中的AOPP定位。我们拟建立免疫学检查方法克服上述缺点。采用体外次氯酸修饰的白蛋白作为抗原,成功制备了特异性识别AOPP的单克隆抗体,通过Western Blot、免疫组织化学染色首次证实单抗识别血清和沉积于肾脏中的AOPP,初步建立双单抗、单多抗及多单抗夹心ELISA法鉴定人工制备的AOPP,为深入研究AOPP这种内源性致病分子提供了必要工具。此外,我们利用噬菌体肽库筛选技术对AOPP模拟表位进行了初步探讨。第一章特异性识别天然AOPP单克隆抗体制备和鉴定以次氯酸(HOCl)氧化不同种属血白蛋白(包括人血清白蛋白HSA、小鼠血清白蛋白MSA、牛血清白蛋白BSA、兔血清白蛋白RSA)和人纤维蛋白原、人IgG、人低密度脂蛋白(LDL),按蛋白：HOCl摩尔比为1：140等体积混合,室温放置30分钟。制备的AOPP在PBS中透析24小时,除去游离的HOCl。AOPP含量通过测定酸性条件下340nm的吸光度,以氯胺T为标准测得。用AOPP-MSA为免疫原加佐剂免疫BALB/c小鼠,取小鼠脾细胞与骨髓瘤细胞(NS-1)融合,按常规方法制备杂交瘤。以AOPP-HSA为抗原包被酶标板,并以未氧化正常HAS做平行筛选以排除与正常蛋白的交叉反应。间接ELISA鉴定筛选特异性识别AOPP-HSA的杂交瘤克隆,所获两株命名为3F2和4C5；扩大培养后制备腹水,用辛酸硫酸铵沉淀法、HItrap Protein G亲和层析柱从腹水中纯化抗体。抗体类别鉴定显示3F2为IgG1,4C5为IgG2a。间接ELISA结果显示2株单抗均能与不同种属来源的氧化型白蛋白(AOPP-HSA、AOPP-BSA、AOPP-MSA、AOPP-RSA)、HOCl-人纤维蛋白原、HOCl-人IgG、HOCl-LDL特异性结合,而与未氧化蛋白及Cu2+氧化的LDL、糖基化蛋白均无交叉反应。竞争ELISA鉴定表明这两株单抗分别识别AOPP不同位点。Western Blot结果显示,在变性与非变性条件下,3F2可特异性结合人工制备的AOPP-HSA、HOCl-IgG、HOCl-人纤维蛋白原。单抗3F2亦可识别天然血浆与组织中的AOPP。能在一定程度上区分血液透析患者和正常人血浆中AOPP含量的差别以及慢性肾衰患者透析前后血浆AOPP含量的不同。免疫组织化学法检查显示3F2可特异性结合沉积于大鼠模型及各种慢性肾脏病患者肾组织中的AOPP,这种结合可被外源AOPP阻断。此外,3F2可阻断AOPP诱导小鼠单核细胞株RAW264.7细胞胞内氧化反应簇(ROS)生成。第二章建立双抗体夹心ELISA检测血浆AOPP的尝试以HItrap Protein G柱纯化抗体3F2和4C5,纯度达95%以上并按照操作流程标记生物素。分别以双单抗、多单抗夹心、单多抗夹心ELISA方法检测人工制备的AOPP并进行了体系的优化。在抗体包被浓度为1μg/ml,封闭液为0.25%酪蛋白,洗液与抗体稀释液均为0.5%PBST,检测抗体浓度为0.5μg/ml时即可良好检测标准品。之后,我们又试用了竞争ELISA的方法,以人工制备的AOPP-HSA 1.25μg/ml包被酶标板,倍比稀释AOPP-HSA (0-4μg/ml)、慢性肾衰患者、健康成人血清与0.5μg/ml Bio-3F2等体积混匀后加入板孔中,最后加入HRP-亲和素。标准曲线的拟合度良好,而且各种抗体组合的双夹心ELISA均可特应性地检出人工体外制备的AOPP,且敏感度可达ng/ml水平但是肾衰患者与正常人的测定值之间没有统计学意义。因此又尝试用敏感性更强的时间分辨荧光检测法检测患者血清AOPP,按照普通夹心ELISA的条件摸索,即以4C51μg/ml包被酶标板,酪蛋白封闭后,加入不同浓度的标准品AOPP-HSA,反应30min后加入Bio-3F2 0.5μg/ml,30min后加入铕标记的亲和素,15min后加入增强液,37℃反应5min后多功能酶标仪测值。所建立的标准曲线已经符合临床检测的需要,但是在血清样本的检测上面,还是存在一定的问题,最佳的包被与检测条件还要改变。第三章AOPP表位模拟序列的筛选鉴于已确定的自然和人工制备的AOPP均为在体内外次氯酸氧化的白蛋白、脂蛋白或其他蛋白,即已形成非天然抗原表位。本章工作的目的旨在探索体内是否有自然存在的类似于AOPP表位的序列,如果存在,将会对机体产生何种影响。我们以亲和层析纯化的抗AOPP多克隆抗体为靶标,对噬菌体随机十二肽库进行了三轮筛选后,鉴定出18个阳性克隆,均可与多抗产生较强的结合,且不为吸板克隆。挑选6个阳性克隆经DNA测序得到三条序列：GWLEENLFDHTR、SSALNDMLRDQR、DSLQDMLADEWQ。上述序列中具有近似的LXDMLXD (X为任意氨基酸)核心序列,提示这一核心序列有可能是模拟AOPP共同表位的优势序列。其次又以抗AOPP单克隆抗体3F2为靶标,筛选噬菌体十二肽库,经三轮筛选、ELISA鉴定获得18个与3F2特异结合的噬菌体克隆,但这些阳性克隆与抗AOPP多抗和4C5单抗无交叉反应。DNA测序得到SSYSQDAATLRL、LNTPYRNQLAYP、LPYFDSYDSALP AHPMPMTQLFTS四条序列中未发现保守序列。最后,在以单抗4C5为靶分子筛选噬菌体十二肽库,经ELISA鉴定得到的12个克隆与4C5特异结合,其中一个克隆(No.2)与多抗及单抗3F2较强结合。DNA测序得到的三种序列,LSPLPHPLTRTV、QNLPEWLALHLD、RASPDLLEHQLM,但未发现保守序列。采用NCBI数据库(http://www.ncbi.nlm.nih.gov/blast)的Blastp程序在蛋白质水平上对筛选的所有序列分别进行同源性搜索,其结果显示这些序列存在于某些真菌属、藻类的蛋白分子上,而并不存在于正常的人类与哺乳动物蛋白分子中。即模拟AOPP抗原表位的序列并不存在于已知的正常人与哺乳动物蛋白分子中,其意义在于避免了与AOPP表位相似而导致的后续事件,维持了机体内环境的稳定；也由此支持AOPP并非人体与动物的正常结构。
[Abstract]:The advanced oxidation protein product (Advanced oxidation protein products, AOPP) is a class of protein crosslinked with dityrosine produced in the process of oxidative stress in the body, and the plasma AOPP is dominated by hypochloric acid (HOCl) oxidized albumin in plasma. The increased level of circulating AOPP is the first in patients with chronic renal failure and peritoneal dialysis, which is found in patients with peritoneal dialysis. In many common diseases, such as diabetes, obesity or metabolic syndrome, atherosclerosis, and other common diseases, the increase of.AOPP is also found in rheumatoid arthritis, ulcerative colitis and other immune inflammatory diseases and malignant tumors (colon, rectal cancer, breast cancer, etc.), so AOPP is recognized as a sensitive biological marker of oxidative stress in the body.
Increasing research evidence in recent years suggests that the increase in plasma AOPP levels causes the molecules to be deposited in the kidneys and blood vessels and is closely related to the development of chronic kidney disease (chronic kidney disease, CKD) and atherosclerotic lesions. Chronic AOPP is accumulated in the kidney of the diabetic animal model by accumulation of chronic AOPP in the body. Renal tissue injury leads to renal inflammation and fibrosis in the CKD animal model and causes atherosclerotic lesions in hyperlipidemia animals. These results show that AOPP retention is not only an indicator of oxidative stress in the body, but also may be a kind of biological pathogeny to promote tissue inflammation and fibrosis. The deep study of AOPP The distribution in the tissue and its relationship with the lesions are essential to elucidate the molecular basis of AOPP's pathogenicity, and may provide new biological markers or intervention targets for the development of the above diseases.
However, there is no ideal method for detecting the level of AOPP in the tissue at present. The detection of AOPP in peripheral blood is usually used to reflect the AOPP level at 340nm by colorimetric analysis. This is based on the AOPP structure containing double tyrosine and a specific absorption peak at 340nm under the acidic condition. This method is simple but easy to be affected by blood lipids and fibrin Interference can affect its accuracy, and can not be used to locate AOPP in pathological tissues or cells.
We intend to establish an immunological method to overcome these shortcomings. The monoclonal antibodies specific to AOPP are successfully prepared by using the albumin modified with hypochloric acid as antigen. Through the Western Blot, the immuno histochemical staining for the first time for the identification of the serum and the AOPP in the kidney of the kidney is first confirmed by immunohistochemical staining. The identification of artificial AOPP by the sandwich ELISA method of monoclonal antibody has provided a necessary tool for the in-depth study of AOPP endogenous pathogenic molecules. In addition, we used phage peptide library screening technique to investigate the AOPP mimic epitopes. Chapter 1 specifically identified the preparation and identification of the natural AOPP monoclonal antibody.
Hypochloric acid (HOCl) was used to oxidize different species of blood albumin (including human serum albumin HSA, mouse serum albumin MSA, bovine serum albumin BSA, rabbit serum albumin RSA) and Human Fibrinogen, human IgG, human low density lipoprotein (LDL), mixed in volume of protein: HOCl mole ratio of 1:140, at room temperature for 30 minutes. The prepared AOPP was permeable in PBS. After 24 hours, the free HOCl.AOPP content was removed by the determination of the absorbance of 340nm under acid condition. The standard of chloramine T was measured with the standard of chloramine T. AOPP-MSA was used as immunogen to immunization BALB/c mice, and the spleen cells of mice were fused with myeloma cells (NS-1), and the hybridoma was prepared by the conventional method. HAS was used in parallel screening to eliminate cross reaction with normal proteins. Indirect ELISA identification and screening of hybridoma clones specifically identified AOPP-HSA, two named 3F2 and 4C5; expanded culture to prepare ascites, ammonium sulfate precipitation, HItrap Protein G affinity chromatography column to purify antibodies from ascites. Antibody category identification showed 3F2 IgG1,4C5 is IgG2a.
The results of indirect ELISA showed that 2 mAbs could be combined with AOPP-HSA, AOPP-BSA, AOPP-MSA, AOPP-RSA, HOCl- Human Fibrinogen, HOCl- human IgG, HOCl-LDL specificity, but no cross reaction with the oxidized protein and Cu2+ oxidation LDL, and glycosylated proteins. Competitive ELISA identification showed that these two monoclonal antibodies were respectively The identification of.Western Blot at different sites of AOPP showed that under denaturing and non denaturing conditions, 3F2 could be specifically combined with artificial AOPP-HSA, HOCl-IgG, and HOCl- Human Fibrinogen.
The monoclonal antibody 3F2 can also identify the difference in the content of AOPP in the plasma and the normal human plasma in a certain degree between the natural plasma and the tissue AOPP., and the difference in the plasma AOPP content before and after the dialysis in the patients with chronic renal failure. The immuno histochemical examination shows that 3F2 can be specifically deposited in the rat model and various chronic renal diseases. AOPP in the renal tissue of the patient can be blocked by exogenous AOPP. In addition, 3F2 can block the formation of the intracellular oxidation reaction cluster (ROS) in the RAW264.7 cells of the mononuclear cell line of mice induced by AOPP.
The second chapter is to establish a double antibody sandwich ELISA for the detection of plasma AOPP.
The purified antibody 3F2 and 4C5 were purified with HItrap Protein G column. The purity of the antibody was over 95% and the biotin was marked in accordance with the operation process. The artificial AOPP was detected by double monoclonal antibody, multi monoclonal antibody sandwich and single multi antibody sandwich ELISA. The antibody package was concentrated in 1 u g/ml, the sealing solution was 0.25% casein, and the lotion and antibody diluent were all For 0.5%PBST, the standard samples were detected when the antibody concentration was 0.5 u g/ml. After that, we tried to try the competitive ELISA method. The artificial AOPP-HSA 1.25 mu g/ml package was mixed with the enzyme standard plate, the double dilution AOPP-HSA (0-4 u g/ml), the chronic renal failure patients, the healthy adult blood clear and 0.5 u g/ml Bio-3F2. After adding HRP- avidin, the standard curve is well fitted, and the double sandwich ELISA of various antibody combinations can detect the AOPP prepared in vitro, and the sensitivity can reach ng/ml level, but there is no statistical significance between the renal failure patients and the normal people. Test the patient serum AOPP, in accordance with the condition of ordinary sandwich ELISA, that is to pack the enzyme label with 4C51 mu g/ml, after the casein is closed, adding different concentration of standard AOPP-HSA, 30min after 30min adding Bio-3F2 0.5 u g/ml, 30min after adding europium avidin, 15min after adding the enhancement solution, and the reaction 5min after 37 degrees. The standard curve set up is in line with the needs of clinical testing, but there are still some problems in the detection of serum samples, and the best package is changed with the testing conditions.
Third chapter selection of AOPP epitope simulation sequence
Given that both naturally and artificially prepared AOPP are albumin, lipoprotein, or other proteins that are oxidized by hypochloric acid in vivo and in vivo, that is, the epitopes of non natural antigens have been formed. The purpose of this chapter is to explore whether there is a natural existence of a sequence similar to the AOPP epitope in the body, and what effect it will have on the body if it exists.
The anti AOPP polyclonal antibody purified by affinity chromatography was used as a target. After three rounds of screening of the phage random twelve peptide library, 18 positive clones were identified, which could produce a strong combination with polyclonal and not to be cloned. 6 positive clones were selected and three sequences were sequenced by DNA sequencing: GWLEENLFDHTR, SSALNDMLRDQR, DSLQDMLADEWQ. The above sequence has an approximate core sequence of LXDMLXD (X as any amino acid), suggesting that this core sequence may be the dominant sequence to simulate the common epitopes of AOPP. Secondly, the phage twelve peptide library is screened with anti AOPP monoclonal antibody 3F2 as the target, and 18 specific phage clones which are specifically combined with 3F2 have been obtained by three rounds of screening and ELISA identification. These positive clones were sequenced with anti AOPP polyclonal and 4C5 McAb free reaction.DNA sequencing, and no conservative sequences were found in the four sequences of SSYSQDAATLRL, LNTPYRNQLAYP, LPYFDSYDSALP AHPMPMTQLFTS. Finally, the phage twelve peptide library was screened with mAb 4C5 as the target molecule, and 12 clones identified by ELISA were specifically combined with 4C5, one of which was cloned. No.2) three sequences, LSPLPHPLTRTV, QNLPEWLALHLD, RASPDLLEHQLM, were obtained with strong combination of mAb and MAb 3F2, but no conservative sequence was found. The Blastp program using the NCBI database (http://www.ncbi.nlm.nih.gov/blast) was homologous to the selected sequences at the protein level. The results showed that these sequences were homologous to the selected sequences at the protein level. The sequence exists on some fungi, the protein molecules of algae, and not in normal human and mammalian protein molecules. That is, the sequence of analog AOPP epitopes does not exist in known normal and mammalian protein molecules, the significance of which is to avoid subsequent events similar to the AOPP epitopes and maintain the machine. The stability of the body environment also supports that AOPP is not the normal structure of human beings and animals.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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