蛋白质是所有生命形式和生命活动的主要载体和功能执行者，蛋白质科学既是当代生命科学的前沿，也是生物技术与生物产业发展的重要源泉。“蛋白质组研究计划”作为四大基础研究计划之一，已被列入《国家中长期科学和技术发展规划纲要(2006-2020)》。蛋白质组学研究在过去的十年中取得了巨大的进步，主要表现在三个方面：一是技术发展快速，尤其是在定量蛋白质芯片技术、蛋白质相互作用及复合物分析技术，以及相关数据库和分析软件等方面尤为显著；二是大量研究数据呈几何级数增加；三是研究正在从蛋白表达种类与数量谱向功能谱研究的转移，如疾病标志蛋白的研究目前正得到空前广泛的关注与开展，同时许多有影响的实验室已从开发技术和积累数据，转向寻求对生命科学问题的解决。

MSD电化学发光免疫分析技术

当蛋白质组学研究人员开始采用高通量技术不断发现疾病生物标志物时，基础科学领域的科学家和仪器设备制造商却仍然致力于证明这一新型检测方法的可靠性。 许多研究生物标志物的研究人员都提到验证是该领域的最大难题，即如何从现有的大量候选生物标志物中缩小范围进一步筛选，最终获得少量几个真正有用的生物标志物的过程。多因子检测金标准 MSD（MesoScale Discovery）基于电化学发光免疫分析技术 的 V-PLEX Human 54-PLEX 经过了长期和多个不同实验基地的验证，确保了稳定性，重复性，批次间一致性。V-PLEX Human 54-PLEX 包括了54个关键检测指标( CRP,Eotaxin, Eotaxin-3, FGF (basic), Flt-1/VEGFR-1,GM-CSF, ICAM-1, IFN-γ,IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16,IL-17A, IL-17A/F, IL-17B,IL-17C, IL-17D, IL-1RA, IL-1α, IL-1β, IL-2, IL-21,IL-22, IL-23, IL-27, IL-3, IL-31, IL-4, IL-5, IL-6,IL-7, IL-8, IL-8 (HA),IL-9, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α,PlGF, SAA, TARC, Tie-2,TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C, VEGF-D ), 涉及到炎症反应，免疫系统调节，以及多种不同的生物学过程。

V-PLEX在血清，血浆，细胞上清，PBMC，尿液中验证了54种蛋白标志物。并可以在46种蛋白标志物中选择出特定标志物进行任意组合，选择其中1-54个指标均可。以下为54-plex前一代试剂盒40-PLEX的发表文献，覆盖了癌症治疗，神经内科，精神科，病理与免疫，妇产科，泌尿外科，细胞医学研究，内分泌等。  

以下案例涉及： 

1)  细胞治疗的细胞因子风暴 （案例：Kite CAR- T临床1期）

2) 筛选疾病相关蛋白标志物（案例：精神障碍，膀胱过度活动症等） 

3）药理研究（案例：急性脊髓炎，急性呼吸窘迫综合征） 

4）移植手术研究（案例：离体肺灌流） 

5）细胞评估（案例：成体多能祖细胞）。

 

  

CAR- T细胞治疗的细胞因子风暴

 

文章：Locke, Frederick L.,et al. "Phase 1 Results of ZUMA-1: A Multicenter Study of KTE-C19Anti-CD19 CAR T Cell Therapy in Refractory Aggressive Lymphoma." MolecularTherapy the Journal of the American Society of Gene Therapy 25.1(2017):285.

 

作者单位：Kite制药有限公司，美国MD安德森癌症中心，美国Moffitt癌症中心

 

检测样本：血清

 

细胞因子检测检测方法&相关发现：Samples were analyzed using thefollowing MSD kits: MSD V-PLEX Plus Angiogenesis Panel 1 (Human) Kit (bFGF,Flt-1/VEGFR-1, PlGF, Tie-2, VEGF-A, VEGF-C, VEGF-D),MSDV-PLEX Plus ChemokinePanel 1(Human)Kit (Eotaxin, Eotaxin-3, IL-8 [HA], IP-10, MCP-1, MCP-4, MDC, MIP-1a, MIP-1b,TARC),MSDV-PLEX Plus Cytokine Panel 1 (Human) Kit (GM-CSF, IL-12/IL-23p40,IL-15, IL-16, IL-17A, IL-1a, IL-5, IL-7, TNF-b), MSDV-PLEX Plus ProinflammatoryPanel 1 (Human) Kit (IFN-g, IL-10, IL-12p70, IL-13, IL-1b, IL-2, IL-4, IL-6,IL-8,TNF-a), MSD V-PLEX Plus Vascular Injury Panel 2 (Human) Kit (CRP, ICAM-1,SAA, VCAM-1), All MSD assays were read using a MESO QuickPlex SQ 120 andanalysis was performed using DISCOVERY WORKBENCH 4.0 (MSD).

 

The low-dose cyclophosphamide and fludarabine conditioning chemotherapyalone was capable of enhancing several homeostatic cytokines and chemokines,most notably IL-15, a critical T cell proliferative cytokine.24 Conditioning chemotherapyfollowed by KTE-C19 infusion resulted in a rapid and sequential induction, elevation,and general clearance of an array of cytokines, chemokines, and immune effectorproteins (Figure 3C). More specifically,markers, including CRP, IL-6, TNF-a, IFN-g, and IL-15, increased with peakvalues occurring 2–3 days post-infusion, and typically resolved to baselinewithin the first 28 days (Figure S3).  

 

 

Kinetics of Peripheral Blood CAR T Cells and Serum Biomarkers

Analysis of patient serum reveals a biomarker profile composed ofspecific cytokines, chemokines, and effector proteins associated with KTE-C19 treatment.Induction of IL-15 occurs during conditioning chemotherapy and levels continueto rise post-infusion, promoting anti-CD19 CAR T cell expansion. CRP levels parallelCRS and generally resolve within the first 28 days.

Heat map of serum biomarkers demonstrates sequential induction andgradual resolution within the first 2 weeks after KTE-C19 infusion of keycytokines, chemokines, and effector proteins. Patients 1–6 demonstrated similarbaseline levels and post-infusion kinetics for induction of IL-15

 

 

筛选疾病相关蛋白标志物： 精神障碍

 

文章：Föcking, Melanie, etal. "Differential expression of the inflammation marker IL12p40 in theat-risk mental state for psychosis: a predictor of transition to psychoticdisorder?." Bmc Psychiatry 16.1(2016):326.

 

作者单位：都柏林博蒙特医院（精神科），爱尔兰

 

检测样本：血浆

 

细胞因子检测检测方法&相关发现：Neuroinflammatory biomarkers were measured with the V-PLEX HumanBiomarker 40-Plex Kit (Meso Scale Discovery (MSD), Maryland, US) according tothe manufacturer’s instructions. In brief, 250 μl plasma (10–50 μl for each ofthe five plates) was diluted as recommended in buffer and 25 μl applied to thebottom of each well in duplicate, incubated for two hours, washed three timesand a solution containing detection antibodies was applied and incubated foranother two hours. Subsequently the plate was washed again three times beforethe read buffer was applied and the plate read in an MSD instrument. Theinstrument measures the intensity of emitted light to provide a quantitativemeasure of analytes in the sample. Forty markers were quantitatively measuredat the same time. This allowed simultaneous detection of cytokines and chemokineswith high precision and accuracy. These analytes were: bFGF, CRP, Eotaxin,Eotaxin-3, Flt-1, GM-CSF, ICAM-1, IFN-γ, IL-1α, IL-1β, IL-10, IL-12 p70,IL-12/IL-23p40, IL-13, IL-15, IL-16, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, IL-8 (high abundance), IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, PlGF,SAA, TARC, Tie-2, TNF-α, TNF-β, VCAM-1, VEGF, VEGF-C, VEGF-D. Each sample wastested in duplicate and the levels reported represent the mean of theduplicates. Sensitivity and coefficient of variation measures were within theirexpected ranges.

 

Our data provides preliminary evidence that elevations in thebaseline plasma levels of the inflammatory marker IL12/IL23p40 are associatedwith transition from ARMS to psychotic disorder. IL12/IL23p40 levels did not changefollowing 12 weeks administration of ω-3 PUFAs. These findings provide evidencethat elevated plasma IL12/IL23p40 is a potential biomarker of increased riskfor transition to psychotic disorder.

 

 

 

筛选疾病相关蛋白标志物：膀胱过度活动症

 

文章：Ma, E., et al. "Amultiplexed analysis approach identifies new association of inflammatoryproteins in patients with overactive bladder. " Am J Physiol Renal Physiol 311.1(2016):ajprenal.00580.2015.

 

作者单位：华盛顿大学医学院（妇产科，泌尿外科，病理与免疫学），美国

 

检测样本：血浆

 

细胞因子检测检测方法&相关发现：Plasmafrom patients was obtained from the KTRC, and specific markers forinflammation, chemokines, cytokines, angiogenesis factors, and vascular injurymarkers were assayed using a V-PLEX human biomarker 40-plex kit from Meso ScaleDiscovery (MSD; Rockville, MD) (Table 2). All 40 analytes were measured by an electrochemiluminescenceimmunoassay technique per the manufacturer’s protocol and analyzed on a MesoScale Discovery model 1250 Sector Imager 2400. TNF-a, MIP-1_, SAA, IL-4, andTie2 concentrations were measured according to the manufacturer’s instructions.Controls and plasma samples were diluted 2-fold, 4-fold, 1,000-fold, 2-fold,and 2-fold, respectively, in assay diluent for a final volume of 50 ul (TNF-a,MIP-1_, IL-4, Tie2), and a final volume of 25 ul (SAA). The units of concentrationfor all analytes shown are pictograms per milliliter.

 

Our findings demonstrate that five analytes, interleukin 4, tumor necrosis factoralpha, macrophage inflammatory protein-1, serum amyloid A, and Tie-2 canreliably differentiate OAB relative to controls and can be used to distinguish OAB from the other conditions. 

 

 

药理研究：急性脊髓炎

 

文章：Hsam, N.B. Ohnmar, etal. "Fulminant Acute Ascending Hemorrhagic Myelitis Treated withEculizumab." Frontiers in Neurology 8(2017):345.

 

作者单位：雷根斯堡大学（神经内科），德国

 

检测样本：血清，脑脊液

 

细胞因子检测检测方法&相关发现：Electrochemiluminescence(MesoScale Discovery., MD, USA) was performed with serum samples from healthycontrols (n = 11) and the patient (pretreatment and posttreatment with ivsteroids) and with a pretreatment CSF sample of the patient V-PLEX HumanBiomarker 40-Plex Kit (MesoScale Discovery.) was used according tomanufacturer’s instructions.

Multiplexanalysis revealed, relative to control, elevated serum interleukin-6 (IL-6),IL-8, IL-15, angiogenesis marker (Flt-1, vascular endothelial growth factor),and vascular injury marker (serum amyloid A, C-reactive protein, vascular celladhesion protein-1) levels, which returned to baseline after treatment with ivsteroids (Table 1). Serum IL-7, IL-17A, eotaxin, eotaxin-3, andphosphatidylinositol-glycan biosynthesis class F protein levels remainedincreased after steroids, whereas IL-12/-23p, Tie-2, and intercellular adhesionprotein-1 were higher only after steroid treatment. Accordingly, CSF IL-6(988.98 pg/ml) and IL-8 (308.14 pg/ml) seemed to be markedly higher. Earlier findingsrevealed that healthy patients or patients with diseases.

药理研究：急性呼吸窘迫综合征

 

作者单位：剑桥大学医学院，GSK呼吸道炎症药物研发，英国

 

检测样本：血清，脑脊液

 

文章：Juss, J. K., et al."Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotypeand Are Resistant to Phosphoinositide 3-Kinase Inhibition." Am J Respir Crit Care Med 194.8(2016):961-973.

 

细胞因子检测检测方法&相关发现：BALFand serum mediators were measured by Human Biomarker 40-Plex V-PLEX Kit andHuman MMP 3-Plex Ultra-Sensitive Kit ( Meso Scale Discovery).

Heat map of the inflammatory markers in the serum of healthy volunteers and patients with ARDS

Inflammatorymediators in serum were measured using an electrochemical luminescenceimmunoassay Meso Scale Discovery (MSD) multiplex. In the heatmap each row is a differentcytokine and each column is a different patient. The coloring represents theabundance of the inflammatory marker measured. The lowest abundance measuredare presented by bright green while the highest by bright red. To assess themean difference in abundance between the disease groups a linear mixed modelwas fitted to the data with disease as a fixed effect and the donor pairing as a random effect. The heat maps and dendrogram (variable tree) were obtained from a hierarchicalclustering of the cytokines using complete linkage. The variable tree to theleft of the heat map shows how the cytokines cluster together based on theirPearson’s correlation i.e., the more correlated two cytokines are the closerthey are in the branches of the dendrogram. The stars on the plot represent theFDR adjusted p-value of the disease effect in this model where NS=FDRp>0.05, *=FDR p<0.05, **=FDR p< 0.001 and ***=FDR p< 0.0001.

 

手术研究：离体肺灌流

 

文章：Asi, Andreasson, et al. "The role ofinterleukin-1β as a predictive biomarker and potential therapeutic targetduring clinical ex vivo lung perfusion." Journal of Heart & Lung Transplantation the Official Publication ofthe International Society for Heart Transplantation (2017).

 

单位：纽卡斯尔大学（细胞医学研究所），英国

 

样本：灌流液和组织裂解物

 

细胞因子检测检测方法&相关发现：Perfusate and tissue lysates wereanalyzed with a V-PLEX Human Biomarker 40-Plex Kit and a Human MMP 3-Plex Ultra-SensitiveKit (both Meso Scale Diagnostics, LLC, Rockville, MD). BALwas analyzed using a V-PLEXhuman pro-inflammatoryPanel 1 Kit and a Human MMP3-Plex Ultra-Sensitive Kit (both Meso ScaleDiagnostics, LLC).

 

细胞评估：成体多能祖细胞

 

文章：Cunha, João Paulo M. C. M., et al. "Human multipotentadult progenitor cells enhance islet function and revascularisation whenco-transplanted as a composite pellet in a mouse model of diabetes." Diabetologia60.1(2017):134-142.

 

单位：鲁汶大学临床和实验内分泌实验室，比利时

 

样本：细胞培养上清（成体多能祖细胞）

 

细胞因子检测检测方法&相关发现：Culture medium of human MAPCs was analysed with human biomarker40-Plex kit containing a pro-inflammatory panel, cytokine panel, chemokinepanel, angiogenesis panel and vascular inflammation panel. Data are means ±SEM. SAA, serum amyloid A; sVCAM-1, soluble vascular cell adhesion molecule-1;CRP, C-reactive protein. (c) Proangiogenic properties of 2.5 Å~ 105 human MAPCs(hMAPC) with or without 150 C57BL/6mouse islets in a CAMassay; 150 C57BL/6mouseislets alone and BSA were used as negative controls and VEGF-A as positivecontrol (mean ± SEM, n = 3–9 per group). **p < 0.01 and ***p < 0.001 forindicated comparisons

 

 

V-PLEX Human Biomarker 54-Plex Kit

V-PLEX Human Biomarker 54-Plex Overview
The V-PLEX Human Biomarker 54-Plex is optimized for long-term and multi-site biomarker studies that require reliable, reproducible results with lot-to-lot consistency. The V-PLEX Human Biomarker 54-Plex includes 54 key analytes that are important in inflammation response and immune system regulation as well as numerous other biological processes. To learn more about the advantages of V-PLEX assays click here. 

V-PLEX Human Biomarker 54-Plex Features

• Validated to measure 54 biomarkers in serum, plasma, cell culture supernatants, PBMCs, and urine

• Easily customized to create subset multiplexes via the V-PLEX Assay Designer

Typical Data
Calibration curve accuracy and precision for each analyte in the V-PLEX Human Biomarker 54-Plex were assessed and the representative data are presented below. 

Figure 1. V-PLEX Human Biomarker 54-Plex Overview Data

V-PLEX Human Biomarker 54-Plex

Description -The 54 assays in the V-PLEX Human Biomarker 54-Plex are provided in seven multiplex panels— Proinflammatory Panel 1 (human), Cytokine Panel 1 (human), Chemokine Panel 1 (human), Angiogenesis Panel 1 (human), TH17 Panel 1 (human), Cytokine Panel 2 (human), and Vascular Injury Panel 2 (human). The panels are optimized to achieve the highest level of performance from each assay, providing efficient biomarker screening and profiling without compromising performance. These panels are composed of assays against human cytokines and chemokines that are involved in many biological processes such as inflammation, the Th1/Th2 pathway, chemotaxis, the Th17 pathway, angiogenesis, and immune system regulation. As a result of their association with such a wide number of diseases, these assays are the subject of drug discovery projects, diagnostics development, and basic research. The 54-plex panel can be initially used as a screening panel to identify relevant biomarkers that can then be assembled into a custom panel for long-term studies.

Disease Research Focus - Wide ranging, including autoimmune conditions, cancer, intestinal inflammation, obesity, heart disease, asthma, cardiovascular disease, bacterial infections, and fungal infections.

Application Area - Inflammation, cytokines, and chemokines