蛋白质与抗体芯片在生物医学研究和转化医学中的应用ppt课件

蛋白质与抗体芯片在生物医学研究和转化医学中的应用 Arrays of Discovery: The application of Protein Antibody Arrays in Biomedical Research and Translational Medicine RayBio C Series Array: Mouse Cytokine Antibody Array III IL-6 IL-6 Knockout mouse IL-6 Wild type mouse n Overview: Protein Array Technology 蛋白 /抗体芯片技术 Major components of protein arrays Classification of protein arrays Benefits of protein and antibody arrays n Applications of protein and antibody arrays Biomarker discovery Drug discovery New insight on molecular mechanism 主要内容 什么是蛋白质芯片？ - 蛋白质芯片 （ Protein Array） : 将大量不同的蛋白质有序 地排列、固定于固相载体表面，形成微阵列。利用蛋白质分 子间 特异性结合 的原理，实现对生物蛋白质分子精准、快 速、高通量的检测。 蛋白质芯片制作 150 pl reagents added to 50 nl drop / Structured surface landing pad! 蛋白质芯片制作 化学发光检 测 荧光检测 l Interest fact: 0.35 nanoliter (nL) per spot l 全球第一个蛋白质芯片公司 ： RayBiotech, Inc. (2001, USA) NC膜 玻璃 多种检测指标高密度集成在一个固相载体上 固定在芯片上的生物材料 n Antibody 抗体 n Protein 蛋白质 n Polypeptide 多 肽 n Peptoid 拟 肽 n Glycan 糖类 n Lipid 脂类 典型的抗体芯片（夹心法） 工 作 原 理 基于生物素标记的抗体芯片 Discover novel ligands to known proteins Discover auto-antibody signatures 蛋白、多肽、拟肽、多糖芯片的工 作原理 screening protein-carbohydrate interactions. Identify the glycans binding partners in biological samples Identify whether target proteins are carbohydrate binding proteins Probe binding of viruses and whole cells to glycans Profile the substrate specificity of enzymes (glycosyltransferases, glycosidases, etc.) Profile the inflammatory immune response 蛋白质芯片的优势 l High-content (高密度 ) 每张芯片采用同一样品，可同时检测多达几百，甚至上千种目标蛋白 或多肽，是蛋白质组学研究的最佳工具。 l Miniaturization (微型化 ) 节省样品（ 10 50 L）、试剂、实验空间、仪器设备 l High-throughput (高通量 ) 每天能完成多达上千样品的检测 l High sensitivity, specificity, and reproducibility 高灵敏度（ ng pg/mL)，高特异性，高重复性 l Automation (自动化 ) - 可实现从加样到结果分析的全自动化 The system CV for IL-5 from a 60-slide (960 arrays) run is about 9% Sandwich array System CV 自动清洗系统 芯片的结果提取 激光扫描仪 GenePix 博奥生物有限公司的 LuxScan10K 玻片芯片结果 大多数基因芯片扫描仪都可以使用 只要具有 Cy3荧光剂（绿色）通道 20m的像素分辨率 能够扫描标准大小的玻片即可（ 25*75*1mm） 膜芯片提取结果 X-ray 胶片 比色法检测 TMB底物 化 学 发 光 系 统 UVP,Bio-Rad 红 外 荧 光 扫 描 LICOR Odyssey双色红外激光成像系统 1SLIDE 2SLIDE 3SLIDE 4SLIDE 1STD1 STD2 STD0 STD3 STD0 STD3 STD0 STD3 2STD3 STD4 STD4 2-5 STD4 2-12 STD4 refernce 3STD5 STD6 2-6 2-7 2-13 2-14 2-19 2-20 4STD7 STD0 2-8 2-9 2-15 2-16 2-21 2-22 5 2-1 2-2 2-10 2-11 2-17 2-18 2-23 2-24 6 2-3 2-4 1-5 1-6 1-11 1-12 1-17 1-18 7 1-1 1-2 1-7 1-8 1-13 1-14 1-19 1-20 8 1-3 1-4 1-9 1-10 1-15 1-16 1-21 1-22 5SLIDE 6SLIDE 7SLIDE 8SLIDE 1STD1 STD2 STD0 STD3 STD0 STD3 STD0 STD3 2STD3 STD4 STD4 2-29 STD4 2-36 STD4 2-44 3STD5 STD6 2-30 2-31 2-37 2-38 2-45 2-46 4STD7 STD0 2-32 2-33 2-39 2-40 2-47 2-48 5 2-25 2-26 2-34 2-35 2-41 2-42 2-49 2-50 6 2-27 2-28 1-27 1-28 2-43 1-33 2-51 1-38 7 1-23 1-24 1-29 1-30 1-34 1-35 1-39 1-40 8 1-25 1-26 1-31 1-32 1-36 1-37 1-41 Blockingbuffer An example 项目： Biomarker scanning of leukemia 样本： 51 physical examination serum samples, 41 leukemia patient serum samples 芯片： QAH-CAA-3000 (160 targets) Normalization with IC data hGFhCHE hREChINF3 Comparison data between US and GZ GZ Med SA 2-51 group 1 2 predicted group 1 36 2 2 5 49 correct rate of classification 92.39% hierarchical method sperman rank similarity metric Average 51 detected targets with P 1.5 信号通路预测蛋白相互作用预测 Applications of RayBio protein and antibody arrays RayBiotech生物高科技公司蛋白和抗体芯片的应用 1. 生物标志物 （ Biomarker）研究 发现新生物标志物 早期诊断 多指标、多疾病同时诊断 2. 新药研发 发现药物的靶标 筛选有前景的新药 研究药物作用、确定潜在副作用 选择临床试验、治疗的合适病人 3. 蛋白质表达谱 蛋白质 多肽 抗体 小分子 4. 蛋白功能谱 蛋白质间相互作用 DNA-蛋白质相互作用 RNA-蛋白质相互作用 小分子 -蛋白质相互作用 蛋白质修饰 5. 生物医学研究 发现研究模型的重要因素 阐明分子机制 建立数据库 S7 S14 H7 H14 S7-1 S7-2 S7-3 S14-1 S14-2 S14-3 H7-1 H7-2 H7-3 H14-1 H14-2H14-3 CCR4 2,784 2,161 2,495 1,447 996 1,777 1,457 1,558 1,579 1,642 719 1,657 CD106 2,735 1,838 2,414 1,418 1,269 1,700 1,254 1,916 2,117 1,543 926 1,737 IL-1 beta 2,481 1,511 1,851 1,434 835 1,523 1,421 1,530 1,179 1,403 585 1,323 IL-4 2,478 1,591 1,997 1,182 956 1,562 1,136 1,628 1,345 1,219 714 1,402 IL-6 2,450 1,520 1,719 1,235 857 1,334 1,189 1,354 1,302 1,175 658 1,258 IL-10 2,258 1,495 1,563 1,015 856 1,260 1,004 1,354 1,287 1,025 694 1,204 GM-CSF 1,975 1,302 1,426 926 659 1,031 861 1,107 1,019 961 546 1,041 MCP-1 1,910 1,307 2,035 1,153 794 1,402 1,191 1,465 1,430 1,165 578 1,350 GFR alpha-2 1,766 1,180 1,288 816 671 1,024 786 1,058 932 831 453 929 RALT/MIG-6 709 238 519 354 402 428 321 428 525 299 222 475 IP-10 606 323 481 224 205 203 227 337 140 231 178 267 MDC 487 148 171 234 166 306 242 205 254 242 121 171 IL-1 R6/IL-1 R rp2 475 171 293 198 140 164 220 218 145 232 116 218 Fas Ligand/TNFSF6 450 142 1 246 68 70 36 103 72 44 56 126 ADFP 448 182 138 235 227 183 126 95 144 145 154 175 MIF 411 220 183 179 232 116 110 155 171 127 139 196 CINC-2 alpha/beta 401 197 113 137 133 180 51 252 217 90 155 199 EG-VEGF/PK1 392 483 193 295 151 245 756 135 341 501 36 327 Growth Hormone R 365 129 30 69 48 62 2 108 1 55 41 94 BDNF 319 176 166 145 126 80 65 129 1 100 64 135 CNTF 313 109 1 99 100 6 26 38 1 76 79 105 IL-5 288 329 164 127 305 289 35 294 304 125 212 331 MIP-2 280 142 115 352 447 508 57 647 571 92 258 494 Growth Hormone 251 57 1 49 52 57 1 25 1 43 42 92 Adiponectin/Acrp30 236 75 1 79 126 1 1 119 1 33 97 66 Integrin alpha M beta 2 234 218 13 402 711 232 85 251 142 496 218 82 ICK 229 4 1 100 16 1 30 1 1 15 5 1 Cancer Cell 25, 605620, May 12, 2014 (IF: 23.893) 本实验所用产品 ： RayBiotech人 AAH-CYT-5抗体芯片 研究背景 : n 乳腺癌是女性死亡的主要恶性肿瘤之一，乳腺癌转移是导 致乳腺癌患者死亡的主要原因 n 上皮 -间质转化（ EMT）是乳腺癌肿瘤转移的关键步骤 n 乳腺癌是巨噬细胞浸润程度最高的恶性肿瘤之一，部分病 例的巨噬细胞甚至占了肿瘤成分 50%以上。 n 肿瘤相关巨噬细胞浸润程度与肿瘤转移成正相关 n 肿瘤相关巨噬细胞（ TAM）与乳腺癌转移的机制未明 A Positive Feedback Loop between Mesenchymal-like Cancer Cells and Macrophages Is Essential to Breast Cancer Metastasis Highlights Cancer cells with EMT activate macrophages to a TAM-like phenotype by GM-CSF TAMs induce EMT of breast cancer cells via CCL18 to form a positive feedback loop The interaction between cancer cells with EMT and TAMs is essential to metastasis High GM-CSF or CCL18 in cancer samples is associated with EMT and poor prognosis 研究内容 n 思路： 表型观察 表型变化相关蛋白筛查 机制探讨 间质型乳腺癌 细胞上清可以 诱导巨噬细胞 为肿瘤相关巨 噬细胞（ TAM ） 乳腺癌细胞被 诱导发生 EMT时 可以激活巨噬细 胞分化为肿瘤相 关性样的巨噬细 胞 采用 RayBiotech AAH-CYT-5的 细胞因子抗体芯片筛查表皮 型乳腺癌细胞细胞上清和间 质型乳腺癌细胞上清 发现间质性乳腺癌细胞分泌 的 GM-CSF是刺激巨噬细胞发 生表型变化，表现肿瘤相关 巨噬细胞特征（ TAM）的关 键因子 A Positive Feedback Loop between GM-CSF from Cancer Cells and CCL18 from Macrophages In Vitro Annals of Neurology, Volume 57, Issue 1, pages 6781, January 2005 Is Autism an Inflammatory（ 炎症） Disease? Autism(自闭症 ) is a neuro-developmental disorder. Etiology and pathophysiology （病因和病理生理学） is largely unknown Inflammatory component was suspected but not yet confirmed.Can cytokine levels in brain tissues and CSF（脑脊液 ） reveal an immunopathology associated with autism? Vargas, et al. Ann Neurol 2005;57: 67-81. Vargas, et al. Ann Neurol 2005;57: 67-81. MCP1, IL 6, TGF1, IGFBP-1 cerebellum (Cbl), 小脑 anterior cingulate gyrus (Acg) 前扣带回 middle frontal gyrus (Mfg). 额中回 Cytokine profile in brain tissues obtained using cytokine protein array methods (A) Cytokine protein arrays in CSF samples from an autistic patient and a control. The spots for macrophage chemoattractant protein (MCP)1, interferon-, TGF-2, interleukin-8, and IGFBP-1 showed a marked density increase as compared with the CSF control. (BI) Profile of expression (fold increase) of cytokines that were found markedly increased in autistic patients as compared with controls. p 0.05, MannWhitney U test. Cytokine profiles in cerebrospinal fluid (CSF) from autistic and control patients Inflammatory Component of Autism n Neuroinflammatory pathogenesis of autism（自闭症神 经炎性发病机制） is mediated by glial cell activation of innate immune pathways.（由神经胶质细胞活化自身免 疫路径调控） n TGF-beta 1 and MCP-1 represent important clinical biomarkers or therapeutic targets for autism. n Seminal paper gave first evidence of neuroinflammation in autism论文首次揭示自闭症的神经炎性 n This paper been cited over 700 times. Vargas, et al. Ann Neurol 2005;57: 67-81. In a seminal study (Cell. 2013 Jan 17;152(1-2):51-67) by Anura Rambukkana and colleagues at the Medical Research Council Center for Regenerative Medicine in London and the Rockefeller Institute in New York, the RayBio Mouse Chemokine/Cytokine Antibody Array C series 1000 was utilized to test for the presence of biologically active proteins in media conditioned by adult Schwann cell reprogrammed progenitor/stem-like cells. The application of this array allowed for the identification of immunomodulatory factors secreted by these cells that promote bacteria-laden macrophage survival and migration, thus exacerbating the effect of leprosy bacterium infection. Please click here for a link to the publication. Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw , Karen Burr , Ryan Raaum , Anura Rambukkana Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection Cell Volume 152, Issues 1?2 2013 51 - 67 The proposed model: Schwann cells in the adult PNS infected with ML undergo a reprogramming process that coverts Schwann cells to pSLC that promote bacterial dissemination. Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw , Karen Burr , Ryan Raaum , Anura Rambukkana Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection Cell Volume 152, Issues 1?2 2013 51 - 67 Reprogrammed pSLC secrete Immune/growth factors that promote macrophage survival and migration The RayBio Mouse Chemokine/Cytokine Antibody Array C series 1000 (96) Axl, bFGF, BLC, CD30 Ligand, CD30, CD40, CRG-2, CTACK, CXCL16, DPPIV/CD26, Dtk, Eotaxin-1, Eotaxin-2, E -Selectin, Fas Ligand, Fc gamma RIIB, Flt-3 Ligand, Fractalkine, G-CSF, GITR, GM-CSF, HGF R, ICAM-1, IFN- gamma, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IGF-1, IGF-2, IL-1 beta, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-17, IL-17B R, IL-1 alpha, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-6, IL-7, IL-9, I-TAC, KC/CXCL1, Leptin/OB, Leptin R, LIX, L-Selectin, Lungkine, Lymphotactin, MCP-1, MCP-5, M-CSF, MDC, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2, MIP-3 beta, MIP-3 alpha, MMP-2, MMP-3, Osteopontin, Osteoporotegerin, PF-4, Pro- MMP-9, P-Selectin, RANTES, Resistin, SCF, SDF-1 alpha, Shh-N, sTNFRI, sTNFRII, TARC, TCA-3, TECK, Thymus CK-1, TIMP-1, TIMP-2, TNF alpha, Thrombopoietin, TRANCE, TROY, TSLP, VCAM-1, VEGF-A, VEGFR1, VEGFR2, VEGFR3, VEGF-D Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw , Karen Burr , Ryan Raaum , Anura Rambukkana Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection Cell Volume 152, Issues 1?2 2013 51 - 67 Reprogrammed pSLC secrete Immune/growth factors that promote macrophage survival and migration The RayBio Mouse Chemokine/Cytokine Antibody Array C series 1000 (96) Axl, bFGF, BLC, CD30 Ligand, CD30, CD40, CRG-2, CTACK, CXCL16, DPPIV/CD26, Dtk, Eotaxin-1, Eotaxin-2, E -Selectin, Fas Ligand, Fc gamma RIIB, Flt-3 Ligand, Fractalkine, G-CSF, GITR, GM-CSF, HGF R, ICAM-1, IFN- gamma, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IGF-1, IGF-2, IL-1 beta, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-17, IL-17B R, IL-1 alpha, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-6, IL-7, IL-9, I-TAC, KC/CXCL1, Leptin/OB, Leptin R, LIX, L-Selectin, Lungkine, Lymphotactin, MCP-1, MCP-5, M-CSF, MDC, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2, MIP-3 beta, MIP-3 alpha, MMP-2, MMP-3, Osteopontin, Osteoporotegerin, PF-4, Pro- MMP-9, P-Selectin, RANTES, Resistin, SCF, SDF-1 alpha, Shh-N, sTNFRI, sTNFRII, TARC, TCA-3, TECK, Thymus CK-1, TIMP-1, TIMP-2, TNF alpha, Thrombopoietin, TRANCE, TROY, TSLP, VCAM-1, VEGF-A, VEGFR1, VEGFR2, VEGFR3, VEGF-D RayBiotech抗体芯片在心力衰竭研究中的应用 心血管疾病生物标志物筛选 样品临床信息 RayBiotech人 AAH-BLM-1抗体膜芯片 血清样品分组： Healthy control (1) (n = 3) Hypertension (2) (n = 3) HFPEF* (3) (n = 3) 芯片筛选结果： HFPEF vs Healthy controls， Hypertensive patients 14个上调因子 3个下调因子 芯片筛选出 17个因子做聚类分析，将 Non-HFPEF 和 HFPEF 很好的区分； 17个新发现的潜在标志物对 HFPEF的诊断有重要意义 Nature Medicine. 2007 . 13(11) 1359-1362. Stanford University School of Medicine 老年痴呆症临床诊断： 基于血浆信号蛋白质的分类和预后 Alzheimers Disease the search for biomarkers n Alzheimers is a progressive, fatal neurodegenerative brain disorder the most common form of dementia n Diagnosis is made by a process of elimination using a combination of imaging, cognitive and neuropsychological exams n No chemistry-based diagnostic currently exists 259 patients AD: Alzheimers patients （ 老年痴呆症患者） OD: Other dementias （其他痴呆症） MCI: Mild cognitive impairment （ 轻度认知障碍 ) NDC: Non-demented control （ 非痴呆症者 ) 假设 ： Does Alzheimer patient have a distinct cytokine signature detectable in plasma? 老年痴呆症患者的血浆中是否有明显的细胞因子标签（ biomarker)？ Nature Medicine. 2007 . 13(11) 1359-1362. Training set 43 AD, 40 NDC 18 cytokines associated with Alzheimers Ray S, et al. Classification and prediction of clinical Alzheimers diagnosis based on plasma signaling proteins (2007) Nature Med. Statistical analysis RayBio Cytokine Array 1000 Search for Alzheimers Biomarkers DISCOVERY Blinded test set 42 AD, 39 NDC, 11 OD PAM algorithm: 18 cytokines Overall accuracy 89% VALIDATION RayBio Cytokine Array 1000 Classification of Alz vs. other dementias Blinded test set 47 MCI Overall accuracy 81%PAM algorithm: 18 cytokines RayBio Cytokine Array 1000 Prediction of Alz Ray S, et al. Classification and prediction of clinical Alzheimers diagnosis based on plasma signaling proteins Nature Med. Training set 43 AD, 40 NDC 18个细胞因子 与 Alzheimers相 关 Blinded test set 42 AD, 39 NDC, 11 OD Alz vs. 其他老年痴呆症 准确度 89% Blinded test set 47 MCI 预测 Alz 准确度 81% 人细胞因子抗体芯片 1000 (AAH-CYT-1000) 18个蛋白质的生物网络功能分析： 确定了在网络中最为显著的蛋白的生物学功能 指出在阿尔茨海默氏病 发生症状前，造血功能、免疫反应、细胞凋亡和 神经支持都会全身失调、紊乱； Paracrine networks and the corresponding receptor pathways in CSC and CAF crosstalk Clinical significance of IGF-II in CAFs and IGF1R as well as Nanog in cancer cells for stage I NSCLC patients. A schematic illustration of the crosstalk between CAFs and lung CSCs 抗体芯片的国际刊物引用数 ： -用 “ 蛋白质芯片 ” 或 “ 抗体芯片 ” 查询，发表的论文统计分析 Before 1999 2004200320021999-2001 发表论文数 0 50 145 437 700 862 1494 2005 2006 5671 2007 New England Journal of Medicine Cell Nature Nature Medicine Science PNAS Lancet 上千篇 高水平研究论文 采用了本公司开发的技术和产品 Figure 1 Wnt heterogeneity in primary tumours and in CSC culture. Figure 5 Myofibroblasts support stem-cell properties and regulate Wnt signalling. Three different groups were established: (1) 12 healthy volunteers as the control group. (2) 14 patients with PDAC who had not received any treatment (denoted as pretreated). (3) 13 patients with PDAC under 2 weeks of gemcitabine + erlotinib combined therapy (denoted as posttreated). Soluble proteins in the sera of the patients with PDAC. a biotin label-based human antibody array(Human Antibody L-Series 507 Array; RayBiotech, Norcross,Ga) in accordance with the recommended protocols. Briefly, all samples had to be biotinylated. The antibodies were immobilized in specific spot locations on glass slides. The incubation of array membranes with biological samples resulted in the binding of cytokines to corresponding antibodies. Signals were visualized using streptavidin-hor