Protein  and Antibody Array Technology in  biomarker discovery and development

Scott Luo, Ph.D. 

R & D Director

shimly@biotnt.com or biotnt@biotnt.com

Cell: 13601690874 (China)shimly

217‐721‐9160 (US)

BioTNT

美国raybiotech研究中心

Tel/Fax:021-51692391Website:www.raybiotech.com.cn Or www.biotnt.com

RayBio® C Series Array: Mouse Cytokine Antibody Array III

IL-6

IL-6 Knockout mouse

IL-6

Wild type mouse

High-content (高密度)– 每张芯片同一样品可同时检测多达数百种指标。

Omics Tool (蛋白质组学的工具)– 可用于分析反应的模式。

High Throughput (高通量)– 每天完成多至上千样品的测试。

Miniaturization 微型化

– 节省样品和试剂, 实验空间,仪器设备

High sensitivity, specificity and reproducibility– 高灵敏，高特异性，重复性

蛋白芯片是通过芯片矩阵平台同时检测多种蛋白质表达及功能的技术

Benefits of protein and antibody arraysBenefits of protein and antibody arrays

蛋白和抗体芯片的优点

抗体芯片的载体

膜芯片 玻璃芯片

SandwichMethod

Direct LabelMethod

CompetitiveMethod

Label‐freeMethod

A. B. C. D.

Signal detection in most antibody arrays relies on common labels (biotin, fluors, or enzymes) attached to the antibody or antigen. The sandwich method (A) requires an immobilized capture antibody and a labeled, in-solution detection antibody. This method has the advantages of excellent specificity and sensitivity. The competitive method (B) relies on competitive binding between the analyte and a labeled antigen. In the direct-label method (C), the label is attached to the analyte itself, while the label-free method (D) relies on an inherent property of the analyte (such as mass per unit area or surface plasmon resonance) to produce a signal.

Processing Quantibody® Data

0

10000

20000

30000

40000

50000

60000

70000

0 100 200 300 400 500 600 700 800 900

Cytokine Concentration (pg/ml)

Inte

nsity

(Sig

nal -

BK

G)

IL-8

Negative IL-10

IL-2

IL-4

IL-5

IL-6

IL-13

GM-CSF

IFNγ

TNFα

Standard Curve

Positive

Sample Name IL-2 IL-4 IL-5 IL-6 IL-8 IL-10 IL-13 GM-CSF IFNg TNFa

Media control 0.1 0.0 0.2 0.1 0.0 0.1 0.1 0.1 0.3 0.1

Cell culture, 24hr 0.1 0.9 0.0 0.1 435.0 0.2 0.1 349.7 0.1 0.1

Add drug A, 24hr 0.1 2.4 0.4 0.4 1112.7 0.5 0.0 2399.7 0.1 1.0

Add drug B, 24hr 0.0 1.6 0.1 0.2 576.7 0.2 0.0 493.0 0.1 0.2

Add drug A and B, 24hr 0.1 4.6 0.4 0.8 934.9 1.2 0.2 3283.0 0.0 2.4

Cell culture, 72hr 0.1 0.5 0.0 0.2 453.7 0.8 0.3 563.8 0.1 0.6

Add drug A, 72hr 0.2 34.3 0.3 2.4 915.7 6.7 0.4 3304.8 0.3 20.8

Add drug B, 72hr 0.2 1.1 0.1 0.3 454.0 0.7 0.3 721.4 0.1 0.3

Add drug A and B, 72hr 0.1 60.5 0.5 4.1 810.6 8.1 0.7 3665.1 1.0 44.1

Data Extraction(ArrayVision, GenePix)

Standard Curve(Raybio Q-Analyzer)

Quantitative Data Export

全自动化蛋白质芯片检测仪：整合自动加样，清除，孵化，检测， 结果分析为一体的仪器， 已完成整体设计和样机制作。Automatic protein chip analyzer: integration of sample handling, wash, incubation, detection and data analysis into a single device.

全自动化蛋白质芯片检测仪 (Automatic protein chip analyzer)

Alzheimer’s Disease the search for biomarkers

• Alzheimer’s is a progressive, fatal neurodegenerative brain disorder – the most common form of dementia

• Diagnosis is made by a process of elimination using a combination of imaging, cognitive and neuropsychological exams

• No chemistry-based diagnostic currently exists

259 patients

AD: Alzheimer’s diseaseOD: Other dementiasMCI: Mild cognitive impairmentNDC: Control (non-demented)

Does Alzheimer’s have a distinct cytokine signature detectable in plasma?

Training set43 AD, 40 NDC

18 cytokines associatedwith Alzheimer’s

Ray S, et al. Classification and prediction of clinical Alzheimer’s diagnosis based on plasma signaling proteins (2007) Nature Med.

Statistical analysis

RayBio® Cytokine Array 1000

Search for Alzheimer’s Biomarkers

DISCOVERY

Blinded test set42 AD, 39 NDC, 11 OD

PAM algorithm: 18 cytokinesOverall accuracy 89%

VALIDATION

RayBio® Cytokine Array 1000 Classification of Alz vs. other dementias

Blinded test set47 MCI Overall accuracy 81%PAM algorithm: 18 cytokines

RayBio® Cytokine Array 1000Prediction of Alz

Ray S, et al. Classification and prediction of clinical Alzheimer’s diagnosis based on plasma

signaling proteins Nature Med.

Training set43 AD, 40

NDC

18个细胞因子与 Alzheimer’s相

关

Blinded test set42 AD, 39 NDC, 11 OD

Alz vs. 其他老年痴呆症

准确度> 89%

Blinded test set47 MCI

预测 Alz准确度> 81%

人细胞因子抗体芯片1000 (AAH-CYT-1000)

Annals of Neurology, Volume 57, Issue 1, pages 67–81, 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 unknownInflammatory 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.

MCP–1, IL– 6, TGF–1, 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 inCSF samples from an autistic patient and a control. The spotsfor macrophage chemoattractantprotein (MCP)–1,interferon-, TGF-2, interleukin-8, and IGFBP-1 showed amarked density increase as compared with the CSF control.(B–I) Profile of expression (fold increase) of cytokines thatwere found markedly increased in autistic patients as comparedwith controls. p 0.05, Mann–Whitney U test.

Cytokine profiles in cerebrospinal fluid (CSF) fromautistic and control patients

Inflammatory Component of Autism

• 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）

• TGF‐beta 1 and MCP‐1 represent important clinical biomarkers or therapeutic targets for autism.

• Seminal paper gave first evidence of neuroinflammation in autism论文首次揭示自闭症的神经炎性

• This paper been cited over 700 times.

Vargas, et al. Ann Neurol 2005;57: 67-81.

2012年7月最新Nature

肿瘤微环境通过分泌 HGF引起对RAF抑制剂的先天抵抗

癌细胞不死之谜

Nature. 2012 Jul 26;487(7408):500-4.

实验思路

• 1  提出假设‐‐肿瘤微环境与肿瘤的抗药性有关

• 2  设计实验

筛选上清因子（AAH-CYT-G4000(274个因子)（AAH-BLG-1 (507个因子)

肿瘤细胞 (45)和stromal cell line or primary cells共培养 (23)

HGF responsible for rescue.

鉴定HGF的作用

Nature. 2012 Jul 26;487(7408):500-4.

PLX4720

AAH-CYT-G4000(274个因子)AAH-BLG-1 (507个因子)

Nature. 2012 Jul 26;487(7408):500-4.

Stromal cell line Rescue score

LL 86 * 0.35Wi-38 * 0.33PC60163A1* 0.31Hs888Lu * 0.24CCD-13Lu * 0.23PC87985B1 * 0.21CCD-8Lu 0.07HS-5 0.06CCD-1068Sk 0.06HDF 0.05HUVEC-CS 0.05PC87399A1 0.05PC87322A1 0.05CCD-1090Sk 0.04CCD-1117Sk 0.04CCD-1065Sk 0.04HMF 0.03CCD-1069Sk 0.0

Correlations between proteins secreted by stromalcells and stromal average melanoma rescue scores

鉴定HGF的作用

其他RTK配体是否也会与抗药性相关

In vitro 鉴定

WB 鉴定 HGF/MET通路机制

In vivo 鉴定

BRAFiPLX4720

肿瘤微环境通过分泌 HGF引起先天抵抗 RAF抑制剂 （AAH-CYT-G4000(274个因子)

（AAH-BLG-1 (507个因子)

Without our technology, such breakthrough discovery will not be possible.

2010年8月:

皮质类固醇抑制婴幼儿血管瘤源性干细胞内的VEGF-A分泌

uPAR, IL‐6, MCP1, and MMP1 are down‐regulated in HemSC3 treated with dexamethasone

人血管生成抗体玻璃芯片-1: AAH-ANG-G1000；（检测43种不同的细胞因子）

uPAR, IL‐6, MCP1, and MMP1 are down‐regulated in HemSC3 treated with dexamethasone

人血管生成抗体玻璃芯片-1: AAH-ANG-G1000；

动脉粥样硬化的趋化因子/细胞因子性别差异研究

目的：由冠状动脉钙化（CAC）确定动脉粥

样硬化冠状动脉疾病程度的中年男性和女性的血清中血小板分泌的炎症趋化因子/细胞因子的性别差异。

女性体内的趋化因子RANTES比男性的要超出100.98%，女性体内的PDGF-BB比男性的超出55.30%。

细胞增活素/细胞活素类RANTES 和 PDGF-BB呈性别差异性分布

女性 男性

PDGF-BBRANTES

Fig. 1. Macrophage infiltration and increased endothelial cell number in human omental adipose tissue (OAT) of obese subjects

Fig. 2. Cytokine antibody array membranes were used to detect adipokinessecreted by each cellular fraction of omental adipose tissue. Raybiotechmembranes were probed with 24-h conditioned medium. The relative cytokine level was determined by chemiluminescence detection and autoradiography. Among the 120 cytokines tested, 16 were secreted into the medium.

Fig. 3. Secretion of adipokines already characterized in human obesity by adipocytesand SV cells isolated from OAT.

Fig. 4. Secretion of adipokines newly identified as oversecreted by OAT in human obesity by adipocytes (A) and SV cells (B) isolated from OAT of lean and obese subjects

肾移植监测

同时检测人体血和尿中20种蛋白标记物

新药毒性的监测

人体血和尿

监测治疗性药物,及时调整用药

早期发现肾脏损伤性疾病

Kidney transplantationDrug treatment (cisplatin)Kidney disease

Different stage

AKI arrays

Early diagnosisPredictive markers

AKI

Normal

肿瘤标志物检测抗体芯片‐定量检测十六种常见肿瘤血清标志物 (Cancer detection antibody arrays: simultaneous detection of 16 common cancer biomarkers)

AFP

CA

125

CA

15-3

CA

19-9

CE

A

HC

Gb

NS

E

PS

A-total

PS

A-free

Thyroglobulin

Procalcitonin

Ferritin

Prolactin

B2M

Pepsinogen 1

Pepsinogen 2

Lung x x x x xBreast x x x xOvary x x x x x

Uterine x x xProstate x x xTesticle x x

Colorectal x x x xPancreas x x

Liver x xStomach x xThyroid x x x x

Heptocellular xMultiple Myeloma x

Renal xGastric x x

Alph-FPCA125CEAPSABeta-HCGCA-15-3

CA19-9LHFerritinNSEProcalcitoninS-100b

采用肿瘤标志物检测抗体芯片同时检判断一次同时检测十六种常见肿瘤血清标志物，省时，省钱，有利于在广大基层开展长期监测十六种常见肿瘤血清标志物的动态变化，有利于检测出早期肿 瘤 。

一滴血

One drop of blood

16 common cancer biomarkers can be measured simultaneously using cancer biomarker antibody arrays. It is cost effective and convenient. The dynamic change of those cancer biomarkers can be monitored longitudinally. It will benefit for cancer screening and early detection

上千篇New England Journal of Medicine 、Nature、Science, Nature Medicine、Cell、Lancet、PNAS等高水平论文均使用了其所开发的技术和产品 (thousand of papers using our technologies and products have been published in the literatures)；

抗体芯片的国际刊物引用数

Publications on “Protein array” or “Antibody Array”用”蛋白芯片”或”抗体芯片”查询的发表论文统计分析

Before 1999

2004200320021999-2001

发表

论文

数

0

50 145

437

700862

1494

2005 2006

5671

2007

期刊名称 发表时间 链接 题目

Nature 4-Jul-12 http://www.nature.com/nature/journal/v487/n7408/full/nature11183.html

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Nature 15-Jul-09 http://www.nature.com/nature/journal/v460/n7253/full/460415a.html Systems biology: Untangling the protein web

Nature 22-Jun-06 http://www.nature.com/nature/journal/v441/n7096/full/nature04846.html

Smad4 signalling in T cells is required for suppression of gastrointestinal cancer

Nature 11-Oct-06 http://www.nature.com/nature/journal/v443/n7113/full/nature05143.html

Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism

Nature 14-Dec-11 http://www.nature.com/nature/journal/v481/n7380/full/nature10661.html

A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells

Nature 13-Dec-05 http://www.nature.com/nature/journal/v444/n7121/full/444963a.html Protein arrays: Table of suppliers

Nature 15-Jul-09 http://www.nature.com/nature/journal/v460/n7253/full/460419a.html Systems biology: Table of suppliers

Nature 30-Nov-05 http://www.nature.com/nature/journal/v438/n7068/full/438703a.html Cell biology: Table of suppliers

Nature 13-Aug-08 http://www.nature.com/nature/journal/v455/n7211/full/nature07209.html

Platelet-derived growth factor-α receptor activation is required for human cytomegalovirus infection

Nature 15-Jun-08 http://www.nature.com/nature/journal/v454/n7203/full/nature07034.html

Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells

期刊名称 发表时间 链接 题目

Nature Immunology 4-Mar-12 http://www.nature.com/ni/journal/v13/n4/full/ni.2238.html

NF-κB-mediated degradation of the coactivator RIP140 regulates inflammatory responses and contributes to endotoxin tolerance

Nature Medicine 7-May-07 http://www.nature.com/nm/journal/v13/n6/full/nm1578.html

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Nature Medicine 3-Feb-13 http://www.nature.com/nm/journal/v19/n3/full/nm.3070.html

Epidermal growth factor regulates hematopoieticregeneration after radiation injury

Nature Medicine 7-May-07 http://www.nature.com/nm/journal/v13/n6/full/nm1578.html

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Nature Medicine 20-Sep-09 http://www.nature.com/nm/journal/v15/n10/full/nm.2026.html

The obesity susceptibility gene Cpe links FoxO1 signaling in hypothalamic pro-opiomelanocortin neurons with regulation of food intake

Nature Cell Biology 22-Nov-09 http://www.nature.com/ncb/journal/v11/n12/full/ncb1993.html

YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway

Nature Biotechnology 9-Jan-11 http://www.nature.com/nbt/journal/v29/n2/abs/nbt.1733.html

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice

Nature Cell Biology 25-Apr-10 http://www.nature.com/ncb/journal/v12/n5/full/ncb2048.html

Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

Nature Cell Biology 22-Nov-09 http://www.nature.com/ncb/journal/v11/n12/abs/ncb1993.html

YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway

期刊名称 发表时间 链接 题目

Cell 17-Jan-13 http://www.sciencedirect.com/science/article/pii/S0092867412015012

Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection

Cell 11-May-12 http://www.sciencedirect.com/science/article/pii/S0092867412004679

DICER1 Loss and Alu RNA Induce Age-Related Macular Degeneration via the NLRP3 Inflammasome and MyD88

Cell 14-Oct-11 http://www.sciencedirect.com/science/article/pii/S0092867411010051

Inducible NOS Inhibition Reverses Tobacco-Smoke-Induced Emphysema and Pulmonary Hypertension in Mice

Cell 10-Jun-11 http://www.sciencedirect.com/science/article/pii/S0092867411005253

Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast

Cell 24-Dec-09 http://www.sciencedirect.com/science/article/pii/S0092867409014378 Tumor Self-Seeding by Circulating Cancer Cells

Cell Stem Cell 6-Jan-12 http://www.sciencedirect.com/science/article/pii/S1934590911005807

Rejuvenation of Regeneration in the Aging Central Nervous System

Cell Stem Cell 3-Apr-09 http://www.sciencedirect.com/science/article/pii/S1934590909000186

Migratory Chondrogenic Progenitor Cells from Repair Tissue during the Later Stages of Human Osteoarthritis

Molecular Cell 24-Aug-12 http://www.sciencedirect.com/science/article/pii/S1097276512005096

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by

Expanding the Cancer Stem Cell Population

Molecular Cell 13-Jan-12 http://www.sciencedirect.com/science/article/pii/S1097276511009051

Hydrogen Sulfide-Linked Sulfhydration of NF-κB Mediates Its Antiapoptotic Actions

Cancer Cell 1-Oct-05 http://www.sciencedirect.com/science/article/pii/S1535610805002989

Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors

Hospitals ： 麻省总医院、波士顿儿童医院

美国红十字会、纽约医疗中心······Universities 大学： 哈佛大学、普林斯顿大学、

耶鲁大学、 麻省理工学院······Biotech R&D 生物技术公司： Amgen, Genetech ······Pharmaceutical R&D 制药公司 ： 默克、辉瑞、强生······Government Labs 政府实验室： CDC, NIH, FDA ······Research Institutions 研究所： Sloan-Kettering, MD Anderson,

Scripps······

Cancer 癌症Heart disease 心血管病Diabetes 糖尿病Obesity 肥胖症Arthritis 关节病Asthma 哮喘Infectious diseases 传染病Inflammation 炎症Nutrition 营养(不良)Drug targeting 药物靶标Basic research 基础科研Biomarker 生物标记

Cytokines 细胞因子Chemokines 趋化因子Growth factors 生长因子Angiogenic factors 血管生成因子Proteases 蛋白酶Phosphorylation 磷 酸化Adipokine 肥 胖 因子Apoptosis 凋亡Signal transduction 信号传导Others 其他

主要客户

应用领域主要产品

RayBiotech芯片产品和技术的客户

Examples of Diseases have been applied using RayBiotech Technologies and products

• AIDS

• Allergy

• Alzheimer's disease

• Asthma

• Atherosclerosis

• Autism

• Autoimmune diabetes

• Cancer

• Colitis

• Crohn’s disease

• Glaucoma

• Graft rejection

• Hypertension

• Infection

• Ischemia-reperfusion

• Mastocytosis

• Multiple sclerosis

• Obstructive pulmonary disease

• Psoriasis

• Rheumatoid arthritis

• Sepsis

• Systemic lupus erythematosus

Wide range of samples

Conditioned mediaPlasmaSerumUrineAmniotic fluidSputum Cerebrospinal fluid Synovial fluid Breath condensatesBronchoalveolar lavageGingival crevicular fluidLaser capture-microdissected tissueSalivaBreast interstitial fluidLiver cyst fluidsTearCell lysatesTissue lysatesOther

抗体抗体芯片芯片

蛋白芯片

抗体抗体

重组重组蛋白蛋白

ELISA/EI

A定制产品

多肽多肽

根据客户需求

灵活多变

产品定制

多种多样

•RayBiotech公司抗体抗原库的资源

•客户拥有的原材料

•市场上可购买到

定制服务

RayBiotechRayBiotech Custom ArrayCustom Array

联络我们

上海冠泰生物科技有限公司（biotnt）电 话:       021 – 5169 2391（免费:  400‐880‐1880）

传 真:       021 – 5169 2391 

销 售 部:       shimly@biotnt.com

网 址： http://www.biotnt.cn/

地 址:上海市龙吴路1500号交通大学海科科技园区A座515室

Questions  & Answer

Quantibody® vs. Luminex xMAP

1) No dedicated equipment needed

2) More Flexibility

3) Higher density arrays

4) Larger choice of targets

5) Better reproducibility

6) Better suited for screening and validation of biomarkers

Antibody Arrays vs. Bead Assays（luminex）

• Fewer unwanted Ab–Ab interactions– Bead‐conjugated antibodies can interact freely in solution

– Capture antibodies affixed to array solid support

– Easier to integrate more antibodies per assay

• More flexibility and more options– Multiple detection formats: chemiluminescent,fluorescent or 

chromogenic

– No dedicated equipment to purchase

– Can screen for hundreds of proteins, or focus on a few

– Can switch to quantitative multiplex after preliminary screening for relative expression

Stem Cell Array Coming soon, not finished yet

Figure 1 Wnt heterogeneity in primary tumours and in CSC culture.

Figure 5 Myofibroblasts support stem-cell properties and regulate Wnt signalling.