Development of non-overlapping multiplex ELISA arrays for the quantitative measurement of 400 human and 200 mouse proteins in parallel Yingqing Mao1, Zhiqiang Lv1, Haw-Han Yen1, Yanni Sun1 and Ruo-Pan Huang1,2,3 1RayBiotech, Inc., 3607 Parkway Lane, Suite 100, Norcross, GA 30092, USA; 2RayBiotech Inc., Guangzhou, 510600 China; 3South China Biochip Research Center, Guangzhou, China 510630 Web: www.raybiotech.com Email: [email protected] Phone: 770-729-2992 Abstract Platform & Principal Sensitivity & X-reactivity Highlights Accurate detection of multiple cytokines in complex biological samples is essential to progress in immunological research. However, development of Human receptor array • Proven sandwich ELISA detection high-density multiplex sandwich-based ELISA panels is hampered by 60,000 50,000 Control Array sensitivity test nonspecific cross-reactivity between and among target-specific reagents 40,000 Std7 (Left): One set of 3x • High throughput multiplex array format (i.e., capture antibodies, detection antibodies, and/or antigens); this is 30,000 Std6 Std5 diluted standards were 20,000 particularly challenging for bead-based assays, in which all three are free to Std4 run on human receptor 10,000 • Quadruplicate data for each target per run interact in solution. To overcome this bottleneck, we tested for nonspecific Std3 0 Std2 array. 2 1 1 3 1 1 2 1 1 - - - - - - - - - 3L b1 Fas - Dtk - 1 RI 1 17R 21R 1BB DR6 2 Rg 2 1 R4 1 - B7 GITR - - - - Std1 - uPAR CD14 CD30 MICB MICA RAGE 10 Rb 10 Flt cross-reactivity among reagents used on our quantitative antibody arrays (in TIM 4 ErbB3 IL BCMA HVEM LIMPII - IL IL IL XEDAR IL LYVE CD40L ALCAM ICAM Selectin Selectin Fluor dye (cy3 equivalent) IL VCAM - PDGF Rb Endoglin - TRAIL R3 TRAIL NRG1 • ELISA sensitivity with wider dynamic range PECAM L Trappin E CEACAM which capture antibodies are affixed to a glass slide). Combinations of Lipocalin Biotin-Streptavidin complex reagents exhibiting nonspecific interactions were separated on discrete B Detect antibody • High specificity, accuracy & reproducibility arrays. Using this divide-and-conquer strategy, we developed and validated Cytokine 25,000 10 human and 5 mouse non-overlapping 40-plex arrays for parallel, Capture antibody Array X-reactivity test 20,000 • Less sample, time, and cost quantitative detection of 400 human and 200 mouse proteins respectively. To Glass Slide Support (Right): Individual DAB 15,000 our best knowledge, this is the highest density multiplex ELISA developed was tested in the 10,000 TRAIL R3 NRG-b1 • Suitable for diverse sample types 5,000 Lipocalin-2 to date. Such a system hold a great promise in determining key factors and Configuration of array platform and illustration of sandwich presence of mixed IL-10 Rb 0 GITR mechanisms in immune cell physiology and pathology, in identifying novel ELISA principal. One standard glass slide is spotted with 16 wells of antigens (10 ng/ml Endoglin identical cytokine antibody arrays. The sandwich ELISA detection CD30 • Software supports one step data computation each) with all CAB. 4-1BB drug targets, and in screening for putative biomarkers from patients' principal is depicted below after the consequent reaction of sample, samples. biotinylated detection antibody, and Streptavidin labeled fluor dye. Strategy Array vs. ELISA Accuracy & Precision Sample Test Step 1: ELISA confirmation of cytokine antibody IL-6 Detection Dynamic Range Example of Levey Jennings (pg/ml) LOD Normal Serum (n=31) pairs; System optimization in array platform. in Quantibody and ELISA Quadruplicate data IL-1a 5 6 ± 2 106 Chart for IL-6 in 14 runs Coefficient of Variation < IL-1b 1 1 ± 0 IL-1ra 59 222 ± 60 150.3 105 (n=8124) LOD IL-2 5 27 ± 5 IL-4 4 7 ± 2 Step 2: Print capture antibody(CAB) arrays, and 4 35 17% 10 140.3 UCL 139.53 IL-5 11 82 ± 15 test individual detection antibody (DAB) in the IL-6 7 42 ± 7 3 10 IL-6sR 7 755 ± 25 presence of mixed antigens at 10 ng/ml each. ELISA 30 < Quantibody 130.3 IL-7 11 50 ± 6 102 > 25 LOQ IL-8 1 13 ± 2 IL-10 3 7 ± 1 101 120.3 LOQ 16% IL-11 34 39 ± 11 Step 3: Do inter-array signal normalization, and 0 20 IL-12p40 18 30 ± 4 10 CL 109.87 67% create cross reactivity table (DAB vs. CAB). 110.3 IL-12p70 1 0 ± 0 10-1 15 IL-13 3 16 ± 3 IL-15 17 158 ± 33 Signal Intensity (Quantibody) or (ELISA) or OD (Quantibody) Intensity Signal 100.3 10-2 IL-16 16 69 ± 14 6 Concentration Concentration 6 (pg/ml) 0.1 1 10 100 1000 10000 - 10 IL-17 18 2 ± 0 IL Step 4: Divide cytokines into different subgroups IL-6 Concentration (pg/ml) 90.3 MCP-1 7 180 ± 23 Percentage of Assays of Percentage based upon the serum abundance, functionality 5 MCSF 3 1 ± 0 LCL 80.21 etc. and generate multiple 40-plex arrays. 80.3 Comparison of array and ELISA dynamic range and detection sensitivity. The 0 <5% 5-10% 10-15% 15-20% >20% dynamic range of IL-6 in array and ELISA (Left) and the correlation of IL-6 detection 70.3 Cytokine quantification in normal human sera (n=31). CV Range Step 5:Test individual array sensitivity, cross by both methods in 40 human culture media samples (Right). The same IL-6 pair of Limit of Detection (LOD): 3 standard deviation above negative control. reactivity, and reproducibility. Real sample test. antibodies together with the same antigen resource were used in both systems. Limit of Quantification (LOQ) = 10 standard deviation above negative control. Array Development Multiplex ELISA Arrays List of Target Proteins Conclusions Human targets Mouse targets Individual cross reactivity test Cross Reactivity Database 70000 IL-17 1. Here we developed and validated multiple non- # Cytokine Gene Symbol Uniprot Number HUGO Approved Name # Cytokine Gene Symbol Uniprot Number HUGO Approved Name # Cytokine Gene Symbol Uniprot Number HUGO Approved Name # Cytokine Gene Symbol Uniprot Number HUGO Approved Name # Cytokine In Array Gene Symbol # Cytokine In Array Gene Symbol 60000 1 2B4 CD244 Q9BZW8 CD244 molecule, natural killer cell receptor 2B4 101 Endostatin COL18A1 P39060 collagen, type XVIII, alpha 1 201 IL-1F6 IL36A Q9UHA7 interleukin 36, alpha 301 P-Cadherin CDH3 P22223 cadherin 3, type 1, P-cadherin (placental) 1 4-1BB mCYT6 TNFRSF9 101 IL-21 mCYT5 IL21 5PL Curve Signal Intensity 2 4-1BB TNFRSF9 Q07011 tumor necrosis factor receptor superfamily, member 9 102 Eotaxin-1 CCL11 P51671 chemokine (C-C motif) ligand 11 202 IL-1F7 IL37 Q9NZH6 interleukin 37 302 PD-1 PDCD1 Q15116 programmed cell death 1 2 6Ckine mCYT8 CCL21 102 IL-22 mCYT8 IL22 Array Cross Reactivity Check 3 6Ckine CCL21 O00585 chemokine (C-C motif) ligand 21 103 Eotaxin-2 Ccl24 O00175 chemokine (C-C motif) ligand 24 203 IL-1F8 IL36B Q9NZH7 interleukin 36, beta 303 PD-ECGF TYMP P19971 thymidine phosphorylase 3 ACE mCYT6 ACE 103 IL-23 mCYT4 IL23A CEA 4 A2M A2M P01023 alpha-2-macroglobulin 104 Eotaxin-3 CCL26 Q9Y258 chemokine (C-C motif) ligand 26 204 IL-1F9 IL36G Q9NZH8 interleukin 36, gamma 304 PDGF R alpha PDGFRA P16234 platelet-derived growth factor receptor, alpha polypeptide 4 Activin A mCYT8 INHBA 104 IL-28 mCYT4 IFNL2 50000 5 ACE-2 ACE2 Q9BYF1 angiotensin I converting enzyme 2 105 EpCAM EPCAM P16422 epithelial cell adhesion molecule 205 IL-1R3 IL1RAP Q9NPH3 interleukin 1 receptor accessory protein 305 PDGF Rb PDGFRB P09619 platelet-derived growth factor receptor, beta polypeptide 2,000 5 ADAMTS1 mCYT8 ADAMTS1 105 IL-3 mCYT5 IL3 3500 6 Activin A INHBA P08476 inhibin, beta A 106 Epo R EPOR P19235 erythropoietin receptor 206 IL-1R5 Il18r1 Q13478 interleukin 18 receptor 1 306 PDGF-AA PDGFA P04085 platelet-derived growth factor alpha polypeptide 6 Adiponectin mCYT8 ADIPOQ 106 IL-3 Rb mCYT6 Csf2rb 7 ADAM-9 ADAM9 Q13443 ADAM metallopeptidase domain 9 107 ErbB2 ErbB2 P04626 v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 207 IL-1ra IL1RN P18510 interleukin 1 receptor antagonist 307 PDGF-AB PDGFB P01127 platelet-derived growth factor beta polypeptide overlapping multiplex ELISA arrays for quantitative 7 ALK-1 mCYT6 SLPI 107 IL-33 mCYT7 IL33 8 Adiponectin ADIPOQ Q15848 adiponectin, C1Q and collagen domain containing 108 ErbB3 ErbB3 P21860 v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 208 IL-2 IL2 P60568 interleukin 2 308 PDGF-BB PDGFB P01127 platelet-derived growth factor beta polypeptide 8 ANG-3 mCYT8 ANGPTL1 108 IL-4 mCYT5 IL4 3000 40000 9 Adipsin CFD P00746 complement factor D (adipsin) 109 E-Selectin SELE P16581 selectin E 209 IL-2 Ra IL2RA P01589 interleukin 2 receptor, alpha 309 PECAM-1 PECAM1 P16284 platelet/endothelial cell adhesion molecule 1 9 ANGPTL3 mCYT8 ANGPTL3 109 IL-5 mCYT5 IL5 10 aFGF FGF1 P05230 fibroblast growth factor 1 (acidic) 110 FABP2 FABP2 P12104 fatty acid binding protein 2, intestinal 210 IL-2 Rb IL2RA P01589 interleukin 2 receptor, alpha 310 Pepsinogen I PGA3 P0DJD8 pepsinogen 3, group I (pepsinogen A) 10 AR mCYT4 AREG 110 IL-6 mCYT5 IL6 1,500 11 AFP AFP P02771 alpha-fetoprotein 111 FAP FAP Q12884 fibroblast activation protein, alpha 211 IL-2 Rg IL2RG P31785 interleukin 2 receptor, gamma 311 Pepsinogen II PGC P20142 progastricsin (pepsinogen C) 11 Artemin mCYT8 ARTN 111 IL-7 mCYT5 IL7 2500 12 AgRP AgRP O00253 agouti related protein homolog (mouse) 112 FAS Fas P25445 Fas cell surface death receptor 212 IL-20 IL20 Q9NYY1 interleukin 20 312 Periostin POSTN Q15063 periostin, osteoblast specific factor 12 Axl mCYT4 Axl 112 IL-7 Rα mCYT7 IL7R 30000 13 Albumin ALB P02768 albumin 113 FAS L FASLG P48023 Fas ligand (TNF superfamily, member 6) 213 IL-21 IL21 Q9HBE4 interleukin 21 313 Persephin PSPN O60542 persephin 13 B7-1 mCYT7 CD80 113 IL-9 mCYT6 IL9 14 ALCAM ALCAM Q13740 activated leukocyte cell adhesion molecule 114 Fcr RIIBC FCGR2B P31994 Fc fragment of IgG, low affinity IIb, receptor (CD32) 214 IL-21R IL21R Q9HBE5 interleukin 21 receptor 314 PF4 CXCL14 O95715 chemokine (C-X-C motif) ligand 14 14 BAFF R mCYT7 TNFRSF13C 114 I-TAC mCYT4 CXCL11 measurement of 400 human and 200 mouse proteins.