Data Package. Multiplex Oncology I 96 96
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1 Data Package Multiplex Oncology I 96 96
2 Table of contents 1. Introduction Technology Data analysis 3 2. Performance characteristics Sample types Analytical Measurement 4 Detection limit 4 Measuring ranges 4 High dose hook effect 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
3 1. Introduction Proseek Multiplex Oncology I 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 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 Data Package 3
4 2. Performance characteristics 2.1 Sample types The ability to use different sample types was evaluated with the Proseek Multiplex Oncology I 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 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) LOD: pg/ml LLOQ: pg/ml ULOQ: pg/ml Hook: pg/ml Range: 2.4 log 0.1 LOD: 0.06 pg/ml LLOQ: 0.24 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 4. log 0.1 LOD: 46.6 pg/ml LLOQ: 61.0 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 3.0 log 0.1 LOD: 0.06 pg/ml LLOQ: 0.24 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 4.2 log 0.1 LLOQ LLOQ Carcinoembryonic antigen LLOQ Concentration (pg/ml) Adrenomedullin 0 Interleukin 0 LLOQ ULOQ 000 Concentration (pg/ml) 000 Concentration (pg/ml) Interleukin ULOQ 000 Concentration (pg/ml) ULOQ ULOQ LOD LOD LOD LOD Fig 2. Calibrator curves from 4 representative assays and their corresponding analytical measurement data. Signal ( Cq) Signal ( Cq) Proseek Multiplex Oncology I Data Package
5 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 % 0% 60% 40% 20% 0% 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 Data Package 5
6 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 P % 76% 56% % 26% 39% Amphiregulin P % 7% 111% % 22% 26% Angiopoietin-1 receptor Q % 74% 93% % 13% % B-cell activating factor Q9Y % 70% 96% % 14% 24% Betacellulin P NR NR 5% NR NR NR CA242 tumor marker % % 4% NR NR NR NR NR NR NR NR Carbonic Anhydrase IX Q % 69% 93% % % 27% Carcinoembryonic antigen P % 95% 115% % 27% % Caspase-3 P % 52% 35% % 1% 37% Cathepsin D P % 71% 114% % 23% 22% C-C motif chemokine 19 Q % 6% 1% % 15% 27% C-C motif chemokine 21 O % 4% 93% % 27% 31% C-C motif chemokine 24 O % 76% 4% % 17% 20% CD40 ligand P % 26% 93% % 22% 35% C-X-C motif chemokine P % 64% 1% % 17% 39% C-X-C motif chemokine 11 O % 2% 321% % 1% 35% C-X-C motif chemokine 13 O % 77% 3% % 1% % C-X-C motif chemokine 5 P % 13% 249% % 1% 17% C-X-C motif chemokine 9 Q % 75% 97% % 14% 23% Cystatin B P % 5% 95% % 20% 24% Early activation antigen CD69 Q % 9% 233% % 17% 25% Epidermal growth factor P % 19% 564% % 14% 37% Epidermal growth factor receptor P % 6% 96% % 15% 42% Epididymal secretory protein E4 Q % 77% 99% % 15% 29% Epiregulin O % 299% % 23% 40% Epithelial cell adhesion molecule P % 6% 3% % 17% 19% Erythropoietin P % 74% 11% % 27% 21% E-selectin P % 63% 96% % 17% 29% Estrogen receptor P NR NR 114% NR NR NR Extracellular matrix metalloproteinase inducer P % 77% 94% % 15% 31% Fas antigen ligand P % 91% 111% % 20% 30% Fatty acid binding protein 4 adipocyte P % 73% 2% % 19% 17% Fms-related tyrosine kinase 3 ligand P % 2% 111% % % 21% Folate receptor alpha P % 79% 9% % 23% 34% Follistatin P % 76% 3% % % 26% Galectin-3 P % 77% 1% % % 21% Granulocyte-macrophage colonystimulating factor P NR NR 6% NR NR NR Growth Hormone P % 77% 5% % 15% 23% Growth/differentiation factor 15 Q % 72% 4% % % 32% Heparin-binding EGF-like growth factor Q % 40% 221% % 14% 27% Hepatocyte growth factor P % 53% 119% % 14% 27% Hepatocyte growth factor receptor P % 3% 2% % 17% 22% Interferon gamma P % 6% 4% % 24% 32% Interleukin 1 receptor antagonist protein P % 67% 7% % % 24% Interleukin P % 72% 0% % 22% 21% Interleukin 17 receptor B Q9NRM % 77% 111% % 21% 23% Interleukin 2 P % 92% 2% NR NR NR Interleukin 2 receptor subunit alpha P % 0% 7% % 15% 23% Interleukin 4 P % 90% 3% NR NR NR Interleukin 6 P % 75% 113% % 14% 1% 6 Proseek Multiplex Oncology I Data Package
7 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 P % 2% 114% % % 29% Interleukin 7 P % 6% 223% % 1% 52% Interleukin P % 56% 1% % % 23% Kallikrein-11 Q9UBX % 5% 1% % 1% 1% Kallikrein-6 Q % 77% 111% % 15% 2% Macrophage colony-stimulating factor 1 P % 0% % % 23% 20% Matrix metalloproteinase-3 P % 97% 119% NR NR NR NR NR 5% 3% 46% Melanoma-derived growth regulatory protein Q % 7% 95% % 13% 2% MHC class I polypeptide-related sequence A Q % 1% % % 27% 2% Midkine P % 75% % % 21% 37% Monocyte chemotactic protein-1 P % 93% 140% % 13% 20% Myeloid differentiation primary response protein MyD Q % 65% 2% % 47% 55% Myeloperoxidase P % 64% 204% % 15% 24% Osteoprotegerin O % 71% 96% % 11% 20% Ovarian cancer-related tumor marker 5 QWXI % 0% 119% NR NR NR NR NR % 26% 34% Placenta Growth Factor P % 71% 6% % 14% 2% Platelet endothelial cell adhesion molecule P % 75% 3% % % 20% Platelet-derived growth factor subunit B P % % 203% % 21% 62% Prolactin P % 74% 4% % 1% 1% Prostasin Q % 4% 117% % 17% 30% Prostate-specific antigen P % 91% 9% % 31% 31% Receptor tyrosine-protein kinase ErbB-2 P % 73% 95% % 19% 1% Receptor tyrosine-protein kinase ErbB-3 P % 73% 7% % 17% 22% Receptor tyrosine-protein kinase ErbB-4 Q % 0% 113% % 22% 29% Regenerating islet-derived protein 4 Q9BYZ % 7% 3% % 23% 25% Stem cell factor P % 74% 0% % 17% 20% Tartrate-resistant acid phosphatase type 5 P % 60% 92% % 17% 27% Thrombopoietin P % 57% 15% % 19% 22% Tissue Factor P % 74% 7% % 14% 22% Transforming growth factor alpha P % 7% 379% % 20% 35% Transforming growth factor beta 1 P % 55% 202% % % 2% Tumor necrosis factor alpha P % 77% 92% NR NR NR Tumor necrosis factor ligand superfamily member 14 O % 69% 365% % 20% 24% Tumor necrosis factor ligand superfamily member P % 60% 7% % 27% 35% Tumor necrosis factor receptor 1 P % 0% 9% % 13% 23% Tumor necrosis factor receptor 2 P % 74% % % 14% 25% Tumor necrosis factor receptor superfamily member 4 P % 0% 115% % 2% 20% Tumor necrosis factor receptor superfamily member 6 P % 0% 11% % 14% 29% Urokinase plasminogen activator surface receptor Q % 76% % % % 19% Vascular endothelial growth factor A P % 70% 143% % % 26% Vascular endothelial growth factor D O % 77% 93% % 20% 31% Vascular endothelial growth factor receptor 2 P % 1% 2% % 17% 23% Proseek Multiplex Oncology I Data Package 7
8 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 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 Intra Inter β 2 14% Intra 1: 1% Intra 2: 9% Intra: % Inter: 19% 22% β 4 Intra 1: 1% Intra 2: 14% > 40 Inter: 19% Inter: % %CV Fig 4. Distribution of intra-assay and inter-assay variations of Proseek Multiplex Oncology I 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 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 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 Data Package
9 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 Hemolysate Lipids Bilirubin Adrenomedullin % Interleukin 2-115% 7% Amphiregulin - 1% 9% Interleukin 2 receptor subunit alpha Angiopoietin-1 receptor % Interleukin 4-79% B-cell activating factor - 115% 1% Interleukin 6-9% Betacellulin 130% Interleukin 6 receptor subunit alpha CA242 tumor marker Interleukin 7 3% Carbonic Anhydrase IX - 96% 99% Interleukin 136% Carcinoembryonic antigen - 3% 59% Kallikrein-11-97% 91% Caspase-3-137% 2% Kallikrein-6-3% 7% Cathepsin D % Macrophage colony-stimulating factor 1 - % C-C motif chemokine % Matrix metalloproteinase C-C motif chemokine 21 67% Melanoma-derived growth regulatory protein - 9% 7% C-C motif chemokine MHC class I polypeptide-related sequence A - 52% 69% CD40 ligand - 3% Midkine - - 5% C-X-C motif chemokine - 6% Monocyte chemotactic protein-1-5% C-X-C motif chemokine % Myeloid differentiation primary response protein MyD - 62% 51% C-X-C motif chemokine 13 % Myeloperoxidase % C-X-C motif chemokine 5 136% Osteoprotegerin - % C-X-C motif chemokine 9-4% 1% Ovarian cancer-related tumor marker Cystatin B % Placenta Growth Factor - 2% Early activation antigen Platelet endothelial cell - 130% 134% CD69 adhesion molecule % Epidermal growth factor 144% Platelet-derived growth factor subunit B - - 1% Epidermal growth factor receptor % 96% Prolactin % 91% Epididymal secretory protein E4-79% 6% Prostasin - 0% Epiregulin - 2% Prostate-specific antigen - 1% 92% Epithelial cell adhesion Receptor tyrosine-protein kinase - 0% molecule ErbB % Erythropoietin - 22% 6% Receptor tyrosine-protein kinase ErbB % E-selectin Receptor tyrosine-protein kinase ErbB-4-46% 67% Estrogen receptor - - 9% Regenerating islet-derived protein % Extracellular matrix metalloproteinase inducer - 61% Stem cell factor - 99% 1% Fas antigen ligand - 7% Tartrate-resistant acid phosphatase type % Fatty acid binding protein 4 adipocyte % Thrombopoietin - % 91% Fms-related tyrosine kinase 3 ligand 1% Tissue Factor - 7% Folate receptor alpha - 2% 91% Transforming growth factor alpha - 0% Follistatin % Transforming growth factor beta % Galectin Tumor necrosis factor alpha - 56% 50% Granulocyte-macrophage Tumor necrosis factor ligand - 3% 2% colony-stimulating factor superfamily member 14-11% Growth Hormone - 97% Tumor necrosis factor ligand superfamily member - 77% Growth/differentiation factor 15-0% 119% Tumor necrosis factor receptor % Heparin-binding EGF-like growth factor 14% Tumor necrosis factor receptor % Hepatocyte growth factor - 135% Tumor necrosis factor receptor superfamily member 4-2% Hepatocyte growth factor Tumor necrosis factor receptor receptor superfamily member % Interferon gamma - 150% 7% Urokinase plasminogen activator surface receptor - 62% Interleukin 1 receptor Vascular endothelial growth antagonist protein factor A % Interleukin 1% Vascular endothelial growth factor D - - 1% Interleukin 17 receptor B - 62% 7% Vascular endothelial growth factor receptor % ng/ml 5 ng/ml 50 ng/ml 200 ng/ml 1 Proseek Multiplex Oncology I Data Package 9
10 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 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) y = x R² = plex (dcq) y = 1.017x R² = 0.99 B) Lipids plex (dcq) Serum C) y = 1,025x - 0,427 R² = 0,9971 C) Bilirubin 24-plex (dcq) Serum plex (dcq) Fig 6. Endogenous interference. Levels tested for hemolysate were g/l hemoglobin, lipids and bilirubin 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 panel. The observed dcq (log2) values were plotted, and the correlation coefficient R2 value was generated by linear regression. Proseek Multiplex Oncology I Data Package
11 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 Data Package 11
12 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 Olink AB. All third party trademarks are the property of their respective owners. 0962, v1.3, Olink Bioscience Dag Hammarskjölds v. 52B SE Uppsala, Sweden
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