Data Package Multiplex Oncology I 96 96
Table of contents 1. Introduction 3 1.1 Technology 3 1.2 Data analysis 3 2. Performance characteristics 4 2.1 Sample types 4 2.2 Analytical Measurement 4 Detection limit 4 Measuring ranges 4 High dose hook effect 4 2.3 Precision Repeatability Reproducibility 2.4 Analytical Specificity Recovery by addition Endogenous interference 2.5 Scalability 3. References 11 Technical support For technical support, please contact us at support@olink.com or +46 1 444 3970
1. Introduction Proseek Multiplex Oncology I 96 96 is a reagent kit measuring 92 cancer related human protein biomarkers simultaneously in serum, plasma or other biological samples. The analytical performance of the product has been carefully validated and the results are presented below. 1.1 Technology The Proseek reagents are based on PEA, a Proximity Extension Assay technology 1, where 92 oligonucleotide labeled antibody probe pairs are allowed to bind to their respective target present in the sample. A PCR reporter sequence is formed by a proximity dependent DNA polymerization event and is subsequently detected and quantified using real-time PCR. The assay is performed in a homogeneous 96-well format without any need for washing steps, see Figure 1. 1.2 Data analysis Data analysis was performed by employing a preprocessing normalization procedure. For each data point, delta Cq (dcq) values were obtained by subtracting the value for the internal run control (Extension control), thus normalizing for technical variation. To generate delta delta Cq (ddcq) values, the normalized dcq was set relative to the background by subtracting the obtained value in the first step from the normalized background value. All calculations up to this point are based on log2 data. Linearization of data was employed by the mathematical operation 2 dcq or 2 dd Cq. Statistical analyses, e.g. coefficient of variation (CV) calculations were performed on linearized values. Incubation Extension Detection Allow the 92 antibody probe pairs to bind to their respective proteins in your samples. Create and pre-amplify 92 unique DNA reporter sequences by proximity extension. Quantify each biomarker s DNA reporter using high throughput real-time qpcr. Fig 1. Proseek Multiplex assay procedure employs three core steps: Incubation, Extension and Detection. High throughput real-time qpcr is performed by using the Fluidigm BiomarkTM HD system. Proseek Multiplex Oncology I 96 96 Data Package 3
2. Performance characteristics 2.1 Sample types The ability to use different sample types was evaluated with the Proseek Multiplex Oncology I 96 96 by collecting matched serum, acid citrate dextrose (ACD), ethylenediaminetetraacetic acid (EDTA) and heparin plasma samples from 5 individuals. Table 1 shows linearized signal-to-background values for each sample type and assay, as well as relative percentage differences of serum, citrate and heparin plasma to EDTA plasma. The results indicated that serum, as well as citrate and EDTA plasma are suitable sample types for most analytes. Highest number of assays below limit of detection was observed for heparin plasma, although many of the assays function without major limitation in that sample type as well. 2.2 Analytical Measurement Detection limit Limit of detection (LOD) was defined as 3 standard deviations of ddcq above background, and reported in pg/ml for 9 proteins out of 92, for which recombinant antigen was available, see Figure 2 and Table 1. Measuring ranges The analytical measuring range was defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) and reported in pg/ml. Quantification limits of LLOQ and ULOQ were calculated with the following trueness and precision criteria; relative error 30% and CV 30%, of backcalculated values, respectively. Measuring ranges were reported in order of log. See Figure 2 and Table 1. Calibrator curves were determined for 9 out of 90 protein biomarkers for which recombinant antigen was available. Representative assays with their analytical measurement data are exemplified in Figure 2 and the distribution of their corresponding measuring range per assay is shown in Figure 3. Separate calibrator curves established for each assay may be viewed at www.olink.com/products/proseekmultiplex/proseek-multiplex-oncology-i High dose hook effect A high dose hook effect is a state of antigen excess relative to the reagent antibodies resulting in falsely lower values. If undetected, a significantly lower value will be reported which can lead to misinterpretation of results. Therefore, the high dose hook effect was determined for each analyte, here reported in pg/ml. See Figure 2 and Table 1. A) C) B) D) Signal ( Cq) Signal ( Cq) Signal ( Cq) Signal ( Cq) 14 14 6 4 2 0 14 6 4 2 0 6 4 2 0 14 6 4 2 0 0.01 0.01 0.01 0.01 LOD: 227.1 pg/ml LLOQ: 976.6 pg/ml ULOQ: 250 000 pg/ml Hook: 1 000 000 pg/ml Range: 2.4 log 0.1 LOD: 0.06 pg/ml LLOQ: 0.24 pg/ml ULOQ: 15 625 pg/ml Hook: 15 625 pg/ml Range: 4. log 0.1 LOD: 46.6 pg/ml LLOQ: 61.0 pg/ml ULOQ: 62 500 pg/ml Hook: 250 000 pg/ml Range: 3.0 log 0.1 LOD: 0.06 pg/ml LLOQ: 0.24 pg/ml ULOQ: 3 906 pg/ml Hook: 3 906 pg/ml Range: 4.2 log 0.1 LLOQ LLOQ 1 1 1 1 Carcinoembryonic antigen LLOQ 0 00 000 Concentration (pg/ml) Adrenomedullin 0 Interleukin 0 LLOQ 00 00 ULOQ 000 Concentration (pg/ml) 000 Concentration (pg/ml) Interleukin 6 0 00 ULOQ 000 Concentration (pg/ml) 0 000 0 000 ULOQ 0 000 0 000 ULOQ LOD 00 000 LOD 00 000 LOD 00 000 LOD 00 000 Fig 2. Calibrator curves from 4 representative assays and their corresponding analytical measurement data. Signal ( Cq) Signal ( Cq) 14 6 4 2 0 14 6 4 2 0 0.01 0.01 4 Proseek Multiplex Oncology I 96 96 Data Package
CTSD Gal-3 ER CD62E REG-4 PRL MK PECAM-1 FABP4 EGFR MPO TNF-R2 IL6RA TIE-2 CPI-B ADM CCL21 hk11 IL-1ra THPO MIA PSA ErbB2/Her2 EPO GDF-15 FS TNF TR-AP MYD FAS BAFF IL-2 GM-CSF PDGF subunit B TGF-beta-1 MIC-A BTC CD30-L EPR CAIX CXCL11 IFN-gamma FR-alpha CEA HGF receptor HE4 VEGF-D CASP-3 VEGFR-2 ErbB4/Her4 ErbB3/Her3 CXCL9 SCF KLK6 CXCL13 PRSS AR TNFRSF4 CXCL5 HGF CXCL CCL19 FasL TNFRSF14 IL17RB CD69 HB-EGF CCL24 IL- IL-4 TGF-alpha U-PAR Ep-CAM EMMPRIN IL-7 TNF-RI TF CD40-L Flt3L IL2RA MCP-1 EGF GH CSF-1 PlGF VEGF-A IL- IL-6 OPG -1 0 1 0% 0% 60% 40% 20% 0% 2 3 4 5 6 7 Concentration (pg/ml) Fig 3. Distribution of analytical measuring range, defined by the limits of quantification LLOQ-ULOQ, for 9 out of 92 analytes. Proseek Multiplex Oncology I 96 96 Data Package 5
Table 1. Sample Types, Analytical Measurement; Limit of Detection, LOD, Lower Limit of Quantification, LLOQ, Upper Limit of Quantification, ULOQ, High Dose Effect, Hook, and Precision indicative of assay performance are shown for 92 analytes. Values below limit of detection were not reported (NR). Sample types Analytical measurment Precision Signal-to-background (2 ddcq ) Relative 2 ddcq to EDTA plasma pg/ml log Target UniProt No ACD EDTA Heparin Serum ACD Heparin Serum LOD LLOQ ULOQ Hook Range Intra-assay Inter-assay Inter-site Adrenomedullin P3531 30 40 30 22 74% 76% 56% 227.1 976.6 250000 00000 2.4 % 26% 39% Amphiregulin P15514 6 7 5 7 5% 7% 111% 1.5 15.3 15625 15625 3.0 % 22% 26% Angiopoietin-1 receptor Q02763 19 20 15 19 92% 74% 93% 46.3 976.6 250000 00000 2.4 7% 13% % B-cell activating factor Q9Y275 23 25 1 24 91% 70% 96% 56.0 244.1 250000 250000 3.0 9% 14% 24% Betacellulin P35070 1 1 1 1 NR NR 5% 7.9 61.0 3906 3906 1. NR NR NR CA242 tumor marker 1 1 1 1 % % 4% NR NR NR NR NR NR NR NR Carbonic Anhydrase IX Q790 2 3 2 3 79% 69% 93% 9.9 61.0 15625 62500 2.4 % % 27% Carcinoembryonic antigen P06731 2 2 2 2 93% 95% 115% 46.6 61.0 62500 250000 3.0 % 27% % Caspase-3 P42574 11 341 176 11 53% 52% 35% 1.7 61.0 62500 62500 3.0 % 1% 37% Cathepsin D P07339 6 5 9 5% 71% 114% 664.7 15625.0 00000 00000 1. 9% 23% 22% C-C motif chemokine 19 Q99731 523 624 535 699 4% 6% 1% 5.7 15.3 15625 15625 3.0 9% 15% 27% C-C motif chemokine 21 O0055 229 296 247 275 77% 4% 93% 54.5 244.1 3906 15625 1.2 6% 27% 31% C-C motif chemokine 24 O00175 9 143 9 177 90% 76% 4% 0.44 3. 3906 3906 3.0 7% 17% 20% CD40 ligand P29965 347 150 40 1405 231% 26% 93% 0.66 3. 15625 15625 3.6 % 22% 35% C-X-C motif chemokine P0277 62 56 97 70% 64% 1% 4.6 15.3 15625 15625 3.0 % 17% 39% C-X-C motif chemokine 11 O14625 64 33 9 7 192% 2% 321% 14.0 61.0 15625 15625 2.4 % 1% 35% C-X-C motif chemokine 13 O43927 14 214 5 263 69% 77% 3% 1. 15.3 3906 3906 2.4 % 1% % C-X-C motif chemokine 5 P4230 2 4 21 409 176% 13% 249% 3.0 15.3 15625 15625 3.0 7% 1% 17% C-X-C motif chemokine 9 Q07325 24 27 20 26 90% 75% 97% 4.2 61.0 62500 62500 3.0 7% 14% 23% Cystatin B P0400 31 43 25 41 73% 5% 95% 207.7 976.6 250000 250000 2.4 9% 20% 24% Early activation antigen CD69 Q07 119 56 73 132 2% 9% 233% 1.15 15.3 62500 62500 3.6 7% 17% 25% Epidermal growth factor P01133 4 90 17 506 13% 19% 564% 0.17 0.95 3906 3906 3.6 6% 14% 37% Epidermal growth factor receptor P00533 15 1 17 1% 6% 96% 149.6 976.6 250000 250000 2.4 11% 15% 42% Epididymal secretory protein E4 Q1450 20 22 17 22 93% 77% 99% 1. 61.0 62500 250000 3.0 7% 15% 29% Epiregulin O14944 1 1 1 3 % 299% 5.1 61.0 15625 15625 2.4 % 23% 40% Epithelial cell adhesion molecule P422 97 115 997 11 95% 6% 3% 0.44 3. 15625 15625 3.6 6% 17% 19% Erythropoietin P015 3 3 2 3 92% 74% 11% 1.6 244.1 62500 250000 2.4 % 27% 21% E-selectin P51 30 3 24 36 79% 63% 96% 50.5 3906.3 250000 250000 1. 11% 17% 29% Estrogen receptor P03372 1 1 1 1 NR NR 114% 375.0 3906.3 250000 00000 1. NR NR NR Extracellular matrix metalloproteinase inducer P35613 240 243 17 229 99% 77% 94% 0. 3. 15625 15625 3.6 6% 15% 31% Fas antigen ligand P4023 2 3 2 3 73% 91% 111% 2.6 15.3 15625 15625 3.0 7% 20% 30% Fatty acid binding protein 4 adipocyte P15090 22 26 19 27 2% 73% 2% 635.6 976.6 250000 250000 2.4 9% 19% 17% Fms-related tyrosine kinase 3 ligand P49771 9 9 0 91% 2% 111% 0.1 0.95 3906 3906 3.6 % % 21% Folate receptor alpha P1532 3 4 3 4 79% 79% 9%.7 61.0 62500 62500 3.0 7% 23% 34% Follistatin P193 4 23 1 24 17% 76% 3% 31.5 244.1 250000 250000 3.0 % % 26% Galectin-3 P17931 17 19 15 19 91% 77% 1% 354.5 15625.0 00000 00000 1. % % 21% Granulocyte-macrophage colonystimulating factor P04141 1 1 1 1 NR NR 6% 42.0 244.1 250000 00000 3.0 NR NR NR Growth Hormone P041 214 255 196 26 4% 77% 5% 0.34 0.95 15625 15625 4.2 7% 15% 23% Growth/differentiation factor 15 Q999 7 1% 72% 4% 21.2 244.1 62500 62500 2.4 % % 32% Heparin-binding EGF-like growth factor Q99075 5 17 7 3 29% 40% 221% 0. 3. 3906 3906 3.0 7% 14% 27% Hepatocyte growth factor P142 7 17 9 21 41% 53% 119% 1.5 15.3 15625 15625 3.0 7% 14% 27% Hepatocyte growth factor receptor P051 19 211 174 215 94% 3% 2% 20.5 61.0 62500 62500 3.0 5% 17% 22% Interferon gamma P01579 1 1 1 2 74% 6% 4% 14.2 61.0 15625 15625 2.4 11% 24% 32% Interleukin 1 receptor antagonist protein P15 6 7 4 97% 67% 7% 5.1 244.1 15625 15625 1. % % 24% Interleukin P29460 41 51 37 51 2% 72% 0% 0.77 3. 3906 15625 3.0 % 22% 21% Interleukin 17 receptor B Q9NRM6 4 4 3 5 % 77% 111% 3.9 15.3 62500 62500 3.6 % 21% 23% Interleukin 2 P6056 1 1 1 1 67% 92% 2% 55. 244.1 250000 250000 3.0 NR NR NR Interleukin 2 receptor subunit alpha P0159 655 693 557 741 95% 0% 7% 0.19 0.95 3906 3906 3.6 5% 15% 23% Interleukin 4 P051 1 1 1 1 3% 90% 3% 0.63 3. 15625 15625 3.6 NR NR NR Interleukin 6 P05231 79 66 99 90% 75% 113% 0.06 0.24 15625 15625 4. 9% 14% 1% 6 Proseek Multiplex Oncology I 96 96 Data Package
Sample types Analytical measurment Precision Signal-to-background (2 ddcq ) Relative 2 ddcq to EDTA plasma pg/ml log Target UniProt No ACD EDTA Heparin Serum ACD Heparin Serum LOD LLOQ ULOQ Hook Range Intra-assay Inter-assay Inter-site Interleukin 6 receptor subunit alpha P07 56 61 50 69 92% 2% 114% 1.2 976.6 250000 250000 2.4 9% % 29% Interleukin 7 P13232 1 2 1 4 71% 6% 223% 1.09 3. 15625 15625 3.6 % 1% 52% Interleukin P145 43 41 23 77 6% 56% 1% 0.06 0.24 3906 3906 4.2 % % 23% Kallikrein-11 Q9UBX7 7 7 6 9 97% 5% 1% 145.0 244.1 15625 15625 1. % 1% 1% Kallikrein-6 Q9276 62 6 52 76 91% 77% 111% 11.0 61.0 62500 62500 3.0 9% 15% 2% Macrophage colony-stimulating factor 1 P09603 64 66 53 71 97% 0% % 0.09 0.95 15625 15625 4.2 7% 23% 20% Matrix metalloproteinase-3 P0254 1 1 1 2 3% 97% 119% NR NR NR NR NR 5% 3% 46% Melanoma-derived growth regulatory protein Q674 11 15 11 14 74% 7% 95% 97.4 244.1 62500 62500 2.4 % 13% 2% MHC class I polypeptide-related sequence A Q2993 3 4 3 5 77% 1% % 17.1 61.0 3906 62500 1. % 27% 2% Midkine P21741 6 2 61 72 3% 75% % 232.9 976.6 62500 62500 1. 9% 21% 37% Monocyte chemotactic protein-1 P13500 774 761 709 6 2% 93% 140% 0.11 0.95 3906 3906 3.6 % 13% 20% Myeloid differentiation primary response protein MyD Q9936 1 2 1 2 66% 65% 2% 69.7 244.1 250000 250000 3.0 % 47% 55% Myeloperoxidase P054 33 45 29 91 74% 64% 204% 137. 976.6 250000 00000 2.4 % 15% 24% Osteoprotegerin O00300 1111 1443 31 137 77% 71% 96% 0.14 0.24 15625 15625 4. % 11% 20% Ovarian cancer-related tumor marker 5 QWXI7 2 2 2 3 76% 0% 119% NR NR NR NR NR % 26% 34% Placenta Growth Factor P49763 37 45 32 4 2% 71% 6% 0.20 0.95 15625 62500 4.2 9% 14% 2% Platelet endothelial cell adhesion molecule P24 9 13 1% 75% 3% 463.4 976.6 250000 250000 2.4 % % 20% Platelet-derived growth factor subunit B P017 67 5 13 2 64% % 203% 92.7 244.1 250000 250000 3.0 % 21% 62% Prolactin P036 13 17 17 76% 74% 4% 1925.4 3906.3 00000 00000 2.4 % 1% 1% Prostasin Q651 69 72 61 4 96% 4% 117% 9.5 15.3 15625 62500 3.0 6% 17% 30% Prostate-specific antigen P072 2 3 3 3 9% 91% 9% 4.6 244.1 62500 62500 2.4 6% 31% 31% Receptor tyrosine-protein kinase ErbB-2 P04626 57 62 45 59 93% 73% 95% 6.1 244.1 62500 62500 2.4 7% 19% 1% Receptor tyrosine-protein kinase ErbB-3 P2160 144 170 4 12 5% 73% 7%. 61.0 62500 62500 3.0 7% 17% 22% Receptor tyrosine-protein kinase ErbB-4 Q15303 6 75 60 5 91% 0% 113% 11.1 61.0 62500 62500 3.0 % 22% 29% Regenerating islet-derived protein 4 Q9BYZ 4 5 4 5 77% 7% 3% 259.9 3906.3 00000 00000 2.4 9% 23% 25% Stem cell factor P2153 53 60 44 59 9% 74% 0% 6.2 61.0 62500 62500 3.0 5% 17% 20% Tartrate-resistant acid phosphatase type 5 P1366 25 15 22 65% 60% 92% 5. 244.1 250000 250000 3.0 11% 17% 27% Thrombopoietin P40225 7 9 5 13 6% 57% 15% 146.6 244.1 62500 62500 2.4 13% 19% 22% Tissue Factor P13726 1 19 14 93% 74% 7% 0.1 3. 15625 62500 3.6 7% 14% 22% Transforming growth factor alpha P01135 4 4 4 9% 7% 379% 0.0 3. 15625 62500 3.6 % 20% 35% Transforming growth factor beta 1 P01137 29 27 15 55 9% 55% 202% 56.1 244.1 250000 250000 3.0 7% % 2% Tumor necrosis factor alpha P01375 2 2 1 2 90% 77% 92% 77.4 244.1 250000 250000 3.0 NR NR NR Tumor necrosis factor ligand superfamily member 14 O43557 5 4 3 4% 69% 365% 4.5 15.3 15625 15625 3.0 % 20% 24% Tumor necrosis factor ligand superfamily member P32971 4 3 2 4 0% 60% 7% 9.7 61.0 15625 15625 2.4 5% 27% 35% Tumor necrosis factor receptor 1 P1943 51 66 17 39 99% 0% 9% 0. 3. 15625 15625 3.6 7% 13% 23% Tumor necrosis factor receptor 2 P20333 11 13 9 14 7% 74% % 247.4 976.6 250000 250000 2.4 % 14% 25% Tumor necrosis factor receptor superfamily member 4 P4349 3 4 3 4 93% 0% 115% 4.7 15.3 15625 15625 3.0 % 2% 20% Tumor necrosis factor receptor superfamily member 6 P25445 3 6 5 5 97% 0% 11%.5 244.1 250000 250000 3.0 % 14% 29% Urokinase plasminogen activator surface receptor Q03405 223 229 175 294 97% 76% % 0.1 3. 15625 15625 3.6 5% % 19% Vascular endothelial growth factor A P15692 271 331 233 472 2% 70% 143% 0.32 0.95 15625 15625 4.2 % % 26% Vascular endothelial growth factor D O43915 1 130 1 1 93% 77% 93% 22.7 61.0 62500 62500 3.0 7% 20% 31% Vascular endothelial growth factor receptor 2 P3596 7 6 90% 1% 2%.2 61.0 62500 62500 3.0 7% 17% 23% Proseek Multiplex Oncology I 96 96 Data Package 7
2.3 Precision Repeatability Within-run variation (intra-assay) was calculated as the mean coefficient of variation (% CV) for individual serum samples, within each of 11 separate runs during the validation studies. Between-run variation (inter-assay) was calculated as the mean coefficient of variation (% CV), for the same individual serum samples, between the 11 separate runs during the validation studies. Variation calculations were assessed on linearized values for 5 out of 92 analytes. Assays with values below limit of detection were not reported. See Table 1. Across 5 assays, the mean CV within-run and between-run variations were observed to be % and 19%, respectively. The distribution of both within-run and between-run variations per assay is shown in Figure 4. Between-run variations (Inter-assay) ranged from 25% to % while the between-site variation (Intersite) ranged from 22% to 14%, here shown in direct comparison to Olink Bioscience in Figure 5. Overall, the Proseek Multiplex Oncology I 96 96 showed very good reproducibility and repeatability with average between-site variation of 2%, in particular considering that, all external sites were first time users. β 1 Intra 1: 15% Intra 2: % Inter: 25% 21% β 3 Intra 1: NR Intra 2: % Inter: NR 1% No. of protein biomarkers 35 30 25 20 15 5 Intra Inter β 2 14% Intra 1: 1% Intra 2: 9% Intra: % Inter: 19% 22% β 4 Intra 1: 1% Intra 2: 14% 0 2 2 4 4 6 6 14 14 1 1 20 20 22 22 24 24 26 26 2 2 30 30 32 32 34 34 36 36 3 3 40 > 40 Inter: 19% Inter: % %CV Fig 4. Distribution of intra-assay and inter-assay variations of Proseek Multiplex Oncology I 96 96 Reproducibility Between-site variation was also investigated during the validation in a β-site study, to estimate the expected variations in values between different laboratories, with different operators and using different equipment. Eight individual serum samples were distributed to each site together with Proseek Multiplex Oncology I 96 96 reagent kits. Each site was instructed to perform the analysis of the individual serum samples according to the same run design. Each site was also asked to perform two independent runs. The overall design of the β-site study enabled the estimation of both the within-run and between-run variations for 3 sites and the between-site variation for each site, here shown in Figure 5. Within-run variations (Intra-assay) ranged from 1% to 15% in the first analysis and 14% to % in the second analysis. Due to technical error of analysis number 1 at β-site 3, the between-run estimation could not be determined. Fig 5. Validation of the Proseek Multiplex Oncology I 96 96 at 4 (β1-β4) different laboratories. Larger boxes shows within-run and between-run variations for each site and small boxes represent the between-site run variations in direct comparison to Olink Bioscience. NR; data not reported. 2.4 Analytical Specificity Recovery by addition Recovery was calculated as the relative difference in signal between a complex (EDTA plasma) and a noncomplex matrix (buffer). Two concentrations, 5 ng/ml and 50 ng/ml, of target antigens were spiked in. Twentyfive assays out of 92 reached high dose hook effect values or had endogenous protein levels exceeding spike-in antigen concentrations and were withdrawn from the analysis. Recovery was calculated as the ratio of observed results to expected results, expressed as % recovery. The average recovery at 50 ng/ml spike-in antigen concentrations was determined to be 96% for 44 assays and at 5 ng/ml spike-in antigen concentrations to be 99% for 43 assays. For 23 assays results were obtained for both spike-in concentrations. Recovery value of 0% to 0% is considered acceptable and the results are summarized in Table 2. Proseek Multiplex Oncology I 96 96 Data Package
Table 2. Performance characteristics. Representative recovery values (%) for at least one spike-in concentration is presented for 67 out of 92 assays Endogenous interference was performed by addition of hemolysate, lipids and bilirubin in serum matrix. Reported are the highest tested concentrations without impact on assay performance. Recovery Endogenous interference Recovery Endogenous interference 1 ng/ml 5 ng/ml 50 ng/ml 200 ng/ml 1 g/l g/l Targets 1-46 Hemolysate Lipids Bilirubin Targets 47-92 Hemolysate Lipids Bilirubin Adrenomedullin - - 142% - - 15 20 630 Interleukin 2-115% 7% - - 15 630 Amphiregulin - 1% 9% - - 15 20 630 Interleukin 2 receptor subunit alpha - - - - - 15 20 630 Angiopoietin-1 receptor - - 69% - - 15 20 630 Interleukin 4-79% - - - 15 20 630 B-cell activating factor - 115% 1% - - 15 20 630 Interleukin 6-9% - - - 15 20 630 Betacellulin 130% - - - - 15 630 Interleukin 6 receptor subunit alpha - - - - - 15 20 630 CA242 tumor marker - - - - - 15 20 630 Interleukin 7 3% - - - - 15 20 630 Carbonic Anhydrase IX - 96% 99% - - 15 20 630 Interleukin 136% - - - - 7.5 20 630 Carcinoembryonic antigen - 3% 59% - - 15 20 630 Kallikrein-11-97% 91% - - 15 20 630 Caspase-3-137% 2% - - 0.469 20 630 Kallikrein-6-3% 7% - - 15 20 630 Cathepsin D - - - - 97% 15 20 630 Macrophage colony-stimulating factor 1 - % - - - 15 20 630 C-C motif chemokine 19 - - 74% - - 15 20 630 Matrix metalloproteinase-3 - - - - - 15 20 630 C-C motif chemokine 21 67% - - - - 15 20 630 Melanoma-derived growth regulatory protein - 9% 7% - - 15 20 630 C-C motif chemokine 24 - - - - - 15 20 630 MHC class I polypeptide-related sequence A - 52% 69% - - 15 20 630 CD40 ligand - 3% - - - 15 20 630 Midkine - - 5% - - 15 20 630 C-X-C motif chemokine - 6% - - - 15 20 630 Monocyte chemotactic protein-1-5% - - - 15 20 630 C-X-C motif chemokine 11 204% - - - - 0.93 20 630 Myeloid differentiation primary response protein MyD - 62% 51% - - 1. 20 630 C-X-C motif chemokine 13 % - - - - 15 20 630 Myeloperoxidase - - 45% - - 7.5 20 630 C-X-C motif chemokine 5 136% - - - - 15 20 630 Osteoprotegerin - % - - - 15 20 630 C-X-C motif chemokine 9-4% 1% - - 15 20 630 Ovarian cancer-related tumor marker 5 - - - - - 15 20 630 Cystatin B - - 117% - - 0.234 20 630 Placenta Growth Factor - 2% - - - 15 20 630 Early activation antigen Platelet endothelial cell - 130% 134% - - 15 20 630 CD69 adhesion molecule - - 140% - - 15 20 630 Epidermal growth factor 144% - - - - 15 20 630 Platelet-derived growth factor subunit B - - 1% - - 15 20 630 Epidermal growth factor receptor - - - 111% 96% 15 20 630 Prolactin - - - 14% 91% 15 20 630 Epididymal secretory protein E4-79% 6% - - 15 20 630 Prostasin - 0% - - - 15 20 630 Epiregulin - 2% - - - 15 20 630 Prostate-specific antigen - 1% 92% - - 15 20 630 Epithelial cell adhesion Receptor tyrosine-protein kinase - 0% - - - 15 20 630 molecule ErbB-2 - - 69% - - 15 20 630 Erythropoietin - 22% 6% - - 15 20 630 Receptor tyrosine-protein kinase ErbB-3 - - 65% - - 15 20 630 E-selectin - - - - - 15 20 630 Receptor tyrosine-protein kinase ErbB-4-46% 67% - - 15 20 630 Estrogen receptor - - 9% - - 15 20 630 Regenerating islet-derived protein 4 - - - - 4% 15 20 630 Extracellular matrix metalloproteinase inducer - 61% - - - 15 20 630 Stem cell factor - 99% 1% - - 15 20 630 Fas antigen ligand - 7% - - - 15 20 630 Tartrate-resistant acid phosphatase type 5 - - 9% - - 15 20 630 Fatty acid binding protein 4 adipocyte - - 117% - - 15 20 630 Thrombopoietin - % 91% - - 15 20 630 Fms-related tyrosine kinase 3 ligand 1% - - - - 15 20 630 Tissue Factor - 7% - - - 15 20 630 Folate receptor alpha - 2% 91% - - 15 20 630 Transforming growth factor alpha - 0% - - - 15 20 630 Follistatin - - 13% - - 15 20 630 Transforming growth factor beta 1 - - 137% - - 15 20 630 Galectin-3 - - - - - 1. 20 630 Tumor necrosis factor alpha - 56% 50% - - 15 20 630 Granulocyte-macrophage Tumor necrosis factor ligand - 3% 2% - - 15 20 630 colony-stimulating factor superfamily member 14-11% - - - 15 20 630 Growth Hormone - 97% - - - 15 20 630 Tumor necrosis factor ligand superfamily member - 77% - - - 1. 0.625 630 Growth/differentiation factor 15-0% 119% - - 15 20 630 Tumor necrosis factor receptor 1 - - 90% - - 15 20 630 Heparin-binding EGF-like growth factor 14% - - - - 15 20 630 Tumor necrosis factor receptor 2 - - 4% - - 15 20 630 Hepatocyte growth factor - 135% - - - 15 20 630 Tumor necrosis factor receptor superfamily member 4-2% - - - 15 20 630 Hepatocyte growth factor Tumor necrosis factor receptor - - - - - 15 20 630 receptor superfamily member 6 - - 4% - - 15 20 630 Interferon gamma - 150% 7% - - 0.234 20 630 Urokinase plasminogen activator surface receptor - 62% - - - 15 20 630 Interleukin 1 receptor Vascular endothelial growth - - - - - 15 20 630 antagonist protein factor A - - 99% - - 15 20 630 Interleukin 1% - - - - 15 20 630 Vascular endothelial growth factor D - - 1% - - 15 20 630 Interleukin 17 receptor B - 62% 7% - - 15 20 630 Vascular endothelial growth factor receptor 2 - - 52% - - 15 20 630 1 ng/ml 5 ng/ml 50 ng/ml 200 ng/ml 1 Proseek Multiplex Oncology I 96 96 Data Package 9
Endogenous interference Endogenous interference from heterophilic antibodies, e.g. HAMA, is known to cause problems in immunoassays. To evaluate the potential impact of this specific interference, a special mismatch system was designed. The only way to generate a signal here is by antibody probe pairs being brought into proximity, by cross-binding substances other than antigens, e.g. heterophilic antibodies and similarly acting rheumatoid factor. Six different mismatched probe pairs of varying antibody host species origin were designed and evaluated with a Heterophilic Assessment Panel from Scantibodies Laboratory Inc. (part no. 3KG027). No interference could be detected for any of the panel samples, indicating a sufficient blocking ability in all assays in the Proseek Multiplex Oncology I 96 96. The potential impact of certain known interfering serum and plasma components was evaluated by using serial dilutions of hemolysate, lipids and bilirubin, respectively in serum, as shown in Figure 6. These additions represent different patient health conditions and/or sample collection irregularities. No interference was detected by addition of bilirubin while 1 assay was observed to be affected by lipids and assays out 92 were altered by hemolysate. The latter is probably due to actual analyte leaking out from the disrupted blood cells rather than disturbance of the assay mechanism. Table 2 shows the highest concentrations without impact on assay performance for each component. A) Hemolysate 15 g/l 7.5 g/l 3.75 g/l 1. g/l 0.94 g/l 0.47 g/l 0.23 g/l Serum 2.5 Scalability Assay performance was further evaluated with regard to scalability, meaning the capability of the Proseek Multiplex technology to maintain the same quality of performance irrespective of multiplex grade. A stepwise increase of multiplex grade (24, 4, 72 and 96) was performed and the observed dcq values for the 24-plex were plotted against the 4-plex, 72-plex and 96-plex for each analyte. The correlation coefficient R 2 value generated by linear regression analysis reflects the correlation between the multiplex assays. The R 2 values were >0.99 for the different multiplex blocks, as shown in Figure 7, demonstrating the scalability of the system. A) B) 24-plex (dcq) 24-plex (dcq) 24 20 24 20 y = 1.0064x - 0.1351 R² = 0.997 20 24 4-plex (dcq) y = 1.017x - 0.399 R² = 0.99 B) Lipids 20 24 72-plex (dcq) 20 5 2.5 1.25 0.63 0.31 Serum C) 24 20 y = 1,025x - 0,427 R² = 0,9971 C) Bilirubin 24-plex (dcq) 630 315 15 79 39 20 Serum 20 24 96-plex (dcq) Fig 6. Endogenous interference. Levels tested for hemolysate were 0.23-15 g/l hemoglobin, lipids 0.3-20 and bilirubin -630. The highest hemolysate concentration translates to about % hemolysis. Fig 7. Scalability of the Proseek Mutliplex technology platform. Human serum samples were analyzed with a 24-plex, 4- plex and 72-plex assay and the complete Proseek Mutliplex Oncology I 96 96 panel. The observed dcq (log2) values were plotted, and the correlation coefficient R2 value was generated by linear regression. Proseek Multiplex Oncology I 96 96 Data Package
3. References 1. Lundberg M, Eriksson A, Tran B, Assarsson E and Fredriksson S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of owabundant proteins in human blood. Nucleic Acid Res 6 June (2011). doi:.93/nar/gkr424 Proseek Multiplex Oncology I 96 96 Data Package 11
This product is for research use only. Not for use in human diagnostic or therapeutic procedures. This product includes a license for non-commercial use of Proseek products. Commercial users may require additional licenses. Please contact Olink AB for details. There are no warranties, expressed or implied, which extend beyond this description. Olink AB is not liable for property damage, personal injury, or economic loss caused by this product. The following trademarks are owned by Olink AB: Olink, Olink Bioscience, Proseek, Duolink and PLA. This product is covered by several patents and patent applications including US 6,511,09, US 7,306,904, and related US and foreign patents. This product is sold under license from PHRI Properties, Inc. and may be used under PHRI Properties patent rights outside the field of human in vitro diagnostics. Components in the Proseek Multiplex Probe Kit utilise Lightning-Link technology and are provided under license from Innova Biosciences. 2013 Olink AB. All third party trademarks are the property of their respective owners. 0962, v1.3, 2013-09-06 Olink Bioscience Dag Hammarskjölds v. 52B SE-752 37 Uppsala, Sweden www.olink.com