学校编码 :10384 分类号 密级 学号 : X2012192008 UDC 硕士学位论文 右旋糖酐定量检测试剂盒的研制及其初步 应用 Preparation and Preliminary application of Dextran Quantitative Detection Kit 王生育 指导教师 : 徐方成副教授 专业名称 : 化学工程 刘海波高级工程师 论文提交日期 : 2016 年 05 月 论文答辩时间 : 2016 年 06 月答辩委员会主席 : 评阅人 : 2016 年 06 月
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摘要 摘要 右旋糖酐 (Dextran), 又名葡聚糖, 由 Leuconostoc mesenteroides 等微生物 产生的酶聚合葡萄糖而成或葡萄糖通过化学聚合而成 右旋糖酐广泛应用于医 药 食品 化工等领域 在医学领域的应用主要包括 : 作为血浆体积扩张剂, 作为抗凝剂, 以及作为渗透压调节剂用于相关的疾病治疗 一般机体组织细胞并不能直接利用右旋糖酐, 主要从尿液排出, 右旋糖酐使用过量会引起急性肾衰竭 充血性心衰竭 肺水肿及血小板减少等毒副作用 因此, 监测血液 尿液的右旋糖酐含量是指导右旋糖酐用量的重要指标, 同时右旋糖酐也是诊断血液或尿道微生物感染的依据, 另外右旋糖酐的定量监测也是食品工业质控的一个重要指标与技术 本论文研究利用抗右旋糖酐单克隆抗体建立简便 快速的右旋糖酐竞争 ELISA 定量检测试剂盒 首先, 利用右旋糖酐与 BSA 交联物作为免疫原, 免疫 4~6 周龄雌性 BALB/c 小鼠, 通过细胞融合技术筛选获得 3 株 ( 命名为 D9,D24,D27) 稳定分泌抗右旋糖酐单克隆抗体杂交瘤细胞株 腹腔注射 D24 杂交瘤细胞制备腹水, 经 rprotein A 亲和柱纯化得到了纯度较高的抗体 ( 抗体纯度约为 95 %, 浓度约为 2 mg/ml); 间接 ELISA 结果表明抗体效价达到 2.5 10-5, 表明抗体具有较高的特异性和亲和力 其次, 利用抗右旋糖酐单克隆抗体建立右旋糖酐竞争 ELISA 定量检测方 法, 确定检测体系优化条件 :(1) 抗原包被浓度为 1.25 µg/ml;( 2) 包被时间 为 4 过夜 ;(3) 封闭液为 2 % BSA;( 4) 右旋糖酐单克隆抗体稀释度为 1: 8000;( 5) 竞争反应时间为 37,1 h;( 6) 底物作用时间为 10 min 该优化检测体系对 dextran 2000 标准品在浓度 19.53 ng/ml~1250 ng/ml 范围内线性关系良好, 相关系数 R 2 =0.9964; 体系最低检测限为 9.6 ng/ml; 标准品回收率在 90.7 %~115.6 % 之间 ; 批内 批间变异系数均小于 15 %, 表明该检测方法具有良好的灵敏度和较高的准确性 最后, 利用建立的定量检测方法测定临床样品, 结果表明,45 份人血清和尿液样品中,29 份未检出右旋糖酐 (<9.6 ng/ml); 有 16 份可检出右旋糖酐
摘要 ( 范围 :10.0±1.2 ng/ml~22.2±2.8 ng/ml), 其中有 2 份尿液样品出现较高浓度右旋糖酐, 比一般尿液右旋糖酐检出均值 (14.3 ng/ml) 高 5 倍以上, 提示这两份样品可能受到微生物的感染, 与临床诊断尿路感染相符 竞争 ELISA 测定的结果与双抗体夹心 ELISA 法测定的结果相似, 进一步证实竞争 ELISA 在临床上测定右旋糖酐含量的可行性 关键词 : 右旋糖酐 ; 单克隆抗体 ; 试剂盒 ; 应用 II
Abstract Abstract Dextran is a kind of polymer of macromolecule glucose, and is produced by glucose polymerizing with enzymes generated by microorganisms such as Leuconostoc mesenteroides or by chemical polymerization. Dextran is widely used in medicine, food, chemical industry, etc. The applications of dextran in medical fields mainly include: 1, as the plasma volume expander. 2, as the anticoagulant. 3, as the osmotic pressure regulating agent for the treatment of relevant diseases. Generally, dextran can not be used directly by the tissues and cells of the body, and it is excreted from urine. However, overdose of dextran may cause acute renal failure, congestive heart failure, pulmonary edema and thrombocytopenia side effects. Thus, monitoring of blood, urine dextran content is an important indicator of the amount of guidance, but also whether the diagnostic yield of the dextran microbial infection diagnosis is based on a quantitative monitoring of dextran, and food industry is an important indicator of quality control and technology. In this study, the preliminary aim is to establish a simple and rapid competitive ELISA kit for quantitative detection of dextran by anti-dextran monoclonal antibody. First of all, using dextran crosslinked with BSA as immunogen to immunize 4~ 6 weeks old female BALB/c mice and screen secreting anti-dextran monoclonal antibody hybridoma cell lines by cell fusion. Expanding hybridoma cells grown in vitro, and injecting into BALB/c mice peritoneal cavity, anti-dextran solution containing monoclonal antibody ascites. Three secreting anti-dextran monoclonal antibody hybridoma cell lines were obtained and they were named with D9, D24 and D27. By intraperitoneal injection D24 hybridoma ascites and rprotein A affinity chromatography, the purified antibodies were abtained (antibody purity of about 95 %, and a concentration of about 2 mg/ml), indirect ELISA results showed that the titers was 2.5 10-5 or more, which indicates a higher affinity. III
Abstract Secondly, establishing competitive ELISA detection method to quantitatively detect dextran with anti-dextran monoclonal antibody. Determine the optimal system conditions: (1) Antigen concentration was 1.25 μg/ml, (2) Pack time is 4 overnight, (3) Blocking buffer was 2 % BSA, (4) Monoclonal antibody dilution of 1: 8000, (5) The competitive reaction time was 37,1 h, (6) Substrate time was 10 min.the optimization system for detecting the concentration of dextran 2000 standards 19.53 ng/ml ~1250 ng/ml range a good linear relationship, correlation coefficient R 2 = 0.9964, a minimum detection limit of 9.6 ng/ml, standard recoveries were 90.7 % ~ 115.6 %, among both intra-assay coefficient of variation less than 15 %, indicating that the method has good detection sensitivity and high accuracy. Finally, determination of clinical samples by quantitative detection method. results showed that 45 cases of human serum and urine samples in 29 parts of dextran were not detected (<9.6 ng/ ml), 16 parts of dextran can be detected (range: 10.0 ± 1.2 ng/ml ~ 22.2 ± 2.8 ng/ml), 2 parts of a urine sample which has a higher concentration appears dextran, dextran detection in urine than average (14.3 ng/ml) over 5 times higher, suggesting that these two samples dextran may be produced microbial infection (consistent with the clinical diagnosis of urinary tract infection). Competitive ELISA assay results were similar with the results of measurement of double antibody sandwich ELISA. The results further confirm the feasibility of ELISA determination of dextran content in competition in clinic. Key words: Dextran; Monoclonal antibody; Kit; Application IV
目录 目录 摘要... I Abstract... III 英文缩略词表... XIII 第一章绪论... 1 1.1 右旋糖酐的简介... 1 1.1.1 右旋糖酐的结构... 1 1.1.2 右旋糖酐的性质... 1 1.1.3 右旋糖酐的分类... 1 1.2 右旋糖酐的应用... 2 1.2.1 右旋糖酐在医学上的应用... 2 1.2.2 右旋糖酐在食品工业上的应用... 3 1.2.3 右旋糖酐在化工上的应用... 3 1.2.4 右旋糖酐在其他领域上的应用... 4 1.3 右旋糖酐检测技术概述... 4 1.3.1 医学上右旋糖酐测定技术... 4 1.3.1 制糖行业右旋糖酐测定技术... 5 1.4 右旋糖酐单克隆抗体及应用... 7 1.5 研究目的和内容... 7 第二章右旋糖酐单克隆抗体的制备 纯化及鉴定... 9 2.1 实验材料... 9 2.1.1 细胞株和实验动物... 9 2.1.2 主要试剂及耗材... 9 2.2 主要仪器及设备... 10 V
目录 2.3 主要溶液的配制... 11 2.4 实验方法... 14 2.4.1 Dextran 40 和 BSA OVA 交联物的制备... 14 2.4.2 杂交瘤细胞株的建立... 15 2.4.2.1 动物免疫... 15 2.4.2.2 血清抗体水平检测... 15 2.4.2.3 细胞融合 筛选及克隆化... 15 2.4.2.4 杂交瘤细胞株稳定性检测... 15 2.4.3 抗体亚型的鉴定... 15 2.4.4 抗体的生产... 16 2.4.5 抗体的纯化 透析和冻干... 16 2.4.6 抗体的效价测定... 16 2.4.7 抗体的特异性鉴定... 16 2.5 结果与分析... 17 2.5.1 Dextran 40-BSA 交联物的制备... 17 2.5.2 小鼠血清效价测定... 17 2.5.3 杂交瘤细胞株的建立... 18 2.5.4 抗体纯度测定... 19 2.5.5 抗体效价测定... 19 2.5.6 特异性鉴定... 20 2.6 本章小结... 21 第三章右旋糖酐竞争 ELISA 检测方法的建立... 22 3.1 供试材料... 22 3.2 实验方法... 22 3.2.1 血清和尿液样品的收集... 22 3.2.2 右旋糖酐标准品配制... 22 3.2.3 抗原包被浓度和抗体工作浓度的确定... 22 VI
目录 3.2.4 竞争 ELISA 方法... 22 3.2.5 竞争 ELISA 体系的优化... 23 3.2.5.1 抗原包被方式的选择... 23 3.2.5.2 封闭剂的选择... 23 3.2.5.3 竞争时间... 23 3.2.5.4 底物作用时间的选择... 24 3.2.6 竞争 ELISA 反应体系特性研究... 24 3.2.6.1 标准曲线的建立... 24 3.2.6.2 标准品回收率... 24 3.2.6.3 最低检测限... 24 3.2.6.4 变异系数... 24 3.2.6.5 稳定性... 25 3.2.6.6 检测体系干扰试验... 25 3.2.7 竞争 ELISA 法的初步应用... 25 3.3 结果与分析... 25 3.3.1 抗原包被浓度和单抗工作浓度... 25 3.3.2 竞争 ELISA 体系的优化... 26 3.3.2.1 抗原包被方式的选择... 26 3.3.2.2 封闭剂的选择... 26 3.3.2.3 竞争时间... 27 3.3.2.4 底物作用时间的选择... 27 3.3.2.5 竞争 ELISA 体系的建立... 28 3.3.3 竞争 ELISA 反应体系特性研究... 29 3.3.3.1 标准曲线的建立... 29 3.3.3.2 加标回收实验... 30 3.3.3.3 最低检测限... 30 3.3.3.4 变异系数... 31 3.3.3.5 稳定性... 32 3.3.3.6 检测体系干扰试验... 33 VII
目录 3.3.4 竞争 ELISA 法的初步应用... 33 3.4 本章小结... 35 第四章结论与展望... 36 4.1 结论... 36 4.2 本文存在的问题和今后开展的研究... 37 参考文献... 38 致谢... 43 VIII
Contents Contents Abstract (Chinese)... I Abstract... III Appendix... XIII Contents... 1 Chapter I Introduction... 1 1.1 The summary of dextran... 1 1.1.1 The structure of dextran... 1 1.1.2 The property of dextran... 1 1.1.3 The classification of dextran... 1 1.1.4 The application of dextran... 2 1.2 The Application of dextran... 2 1.2.1 Application in medicine... 2 1.2.2 Application in food industry... 3 1.2.3 Application in chemical industry... 3 1.2.4 Application in other fields... 4 1.3 Detection techniques of dextran... 4 1.3.1 Medical dextran measurement technology... 4 1.3.2 Dextran measurement technology in the sugar industry... 5 1.4 The mab of dextran and Application... 7 1.5 The purpose and content of the research... 7 Chapter II Production, purification and characterization of dextran mab... 9 2.1 Materials... 9 IX
Contents 2.1.1 Cell lines and experimental animal... 9 2.1.2 Main reagents and consumables... 9 2.2 Main instruments and equipment... 10 2.3 Main solution preparation... 11 2.4 Methods... 14 2.4.1 Preparation of dextran and BSA,OVA cross link... 14 2.4.2 Establishment of hybridoma cell lines... 15 2.4.3 Antibody isotype analysis... 15 2.4.4 Antibody production... 16 2.4.5 Purification, dialysis and freeze dryingof the antibody... 16 2.4.6 Identification the titer of antibody... 16 2.4.7 Identification the specificity of antibody... 16 2.5 Results and analyses... 17 2.5.1 Preparation of dextran and BSA,OVA cross link... 17 2.5.2 Determination of serum titer in mice... 17 2.5.3 Establishment of hybridoma cell lines... 18 2.5.4 Isotype analysis... 19 2.5.5 Purity analysis... 19 2.5.6 Specificity identification... 20 2.6 Conclusion of part II... 21 Chapter III Dextran mab based competitive ELISA for quantitative detection of dextran...22 3.1 Materials... 22 3.2 Methods... 22 3.2.1 Collection of serum and urine samples... 22 3.2.2 Preparation of standard test reagent for dextran... 22 X
Contents 3.2.3 Determination of the concentration of antigen and the antibody... 22 3.2.4 Competitive ELISA method... 22 3.2.5 System optimization of competitive ELISA method... 23 3.2.6 Research on system characteristics of competitive ELISA... 25 3.2.7 Preliminary application of competitive ELISA... 25 3.3 Results and analyses... 25 3.3.1 The concentration of antigen and the antibody... 25 3.3.2 System optimization of competitive ELISA method... 26 3.3.2.1 The choice of antigen package... 26 3.3.2.2 The choice of blocking Reagent... 26 3.3.2.3 The choice of competition time... 27 3.3.2.4 The choice of selection of substrate action time... 27 3.3.2.5 The establishment of competitive ELISA system... 28 3.3.3 Study on the characteristics of competitive ELISA reaction system... 29 3.3.3.1 Establishment of standard curve... 29 3.3.3.2 Experiment of adding standard recovery... 30 3.3.3.3 Lowest detectable limit... 30 3.3.3.4 Coefficient of variation... 31 3.3.3.5 Stabilization... 32 3.3.3.6 Test system interference test... 33 3.3.4 Preliminary application of competitive ELISA... 33 3.4 Conclusion of part III... 35 Chapter IV Conclusion...36 4.1 Conclusion... 36 4.2 Limitations of this thesis and its further studies in the future... 37 XI
Contents References...38 Acknowledgments...43 XII
英文缩略词表 英文缩略词表 英文缩写英文全称 中文全称 BCA Bicinchoninic acid 2,2- 联喹啉 -4,4- 二甲酸二钠 BSA Bovine serum albumin 牛血清白蛋白 DMSO Dimethyl sulphoxide 二甲基亚砜 ELISA Enzyme-linked immunosorbent assay 酶联免疫吸附试验 FDA Food and Drug Administration 食品药物管理局 HAT Hypoxathine aminopterin thymidine 次黄嘌呤氨基蝶呤胸腺嘧啶 HPLC High performance liquidchromatography 高效液相色谱法 HRP Horse radish peroxidase 辣根过氧化物酶 HT Hypoxathine thymidine 次黄嘌呤核苷 ICUMSA International Commission for Uniform Methods of Sugar Analysis 国际糖分析方法委员会 IgG Immunoglobulin G 免疫球蛋白 G mab Monoclonal antibody 单克隆抗体 OD Optical density 光密度 OPD O-Phenylenediamine 邻苯二胺 OVA Ovalbumin 鸡卵血清蛋白 PBS Phosphate buffer saline 磷酸盐缓冲液 SDS Sodium dodecyl sulfate 十二烷基磺酸钠 XIII
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