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1、Application of Multiplexing in IVD Manufactory Grace Yuan Instrument Field Application Scientist Merck Millipore To explore bio-marker and in vitro diagnostic Outline Luminex xMAP technology Muse Cell Analyzer Direct Detect Spectrometer Outline Luminex xMAP technology Muse Cell Analyzer Direct Detec
2、t Spectrometer Ab conjugated to microbeads as “suspension arrays” Antibodies in a sandwich immunoassay (ELISA) Ab immobilized on chips as “planar arrays” Milliplex: high throughput ELISA tubes 96 well plate microarray Multiplexing The history of protein analysis technology Solution-phase kinetics So
3、lid-phase kinetics Single Plex Assays Multi Plex Assays Insulin Leptin GLP-1(A) Add samples, stds, QCs and matrix Add Biotinylated Secondary cocktail Add strep-PE Wash Wash Luminex 200 Millipore Endocrine: Insulin, Leptin and GLP-1(active) xMAP Biomakers screening Detection principle Identification
4、of microshperes Quantification Red laser (635 nm) Green laser (532 nm) 鞘液 532 nm APD APD APD 635 nm PMT 6 6.5 um 磁珠磁珠 Core 3.5% Fe3O4 Coating Layer COOH Same chemicals used in magnetic bead conjugation to bio-molecules xMAP: Color coding of beads xMAP technology- color coding xMAP technology- color
5、coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding xMAP technology- color coding 18 100 specific microspheres Dyeing process creates u
6、nique “spectral address” Dye ratio 100 bead colors (regions) 1 100 10 IL-6 36 IL-1b 92 20 xMAP Application The application of xMAP technology covers immunology, nuclear acid, enzymology, reaction of receptor and the ligand. Milliplex Portfolio Offerings: Inflammation/Immunology Cytokine, Soluble Cyt
7、okine Receptors, Th17, CD8+, MMP, TIMP Immune Response Immunoglobulin Isotyping, Sepsis Metabolism Adipokine/Adipocyte, Metabolic Hormone, Liver Protein, Gut Hormone, Pituitary, Stress Hormone, Species Steroid/Thyroid Hormone, IGF Binding Protein Cardiovascular CVD, Apolipoproteins, Acute Phase Bone
8、 Metabolism Skin Panel Cancer Biomarker Circulating Cancer Biomarker, Human Angiogenesis/Growth Factor, Cancer/Metastasis Biomarker Neuroscience Neurodegenerative Disease, Neurological Disorders, Neuropeptide Toxicity Kidney Toxicity, Vascular Injury Cellular Metabolism Assays Pyruvate Dehydrogenase
9、, Fatty Acid Oxidation, Oxidative Phosphorylation, Oxidative Stress Stem Cell Assays Cell Signaling/Epiquant 21 SA-PE Pre-wet filter plate Add beads Add sample & controls (sample prepared in cell lysis buffer, serum diluent) Add detection antibody Add streptavidin- PE Read plate Instant Data Analysi
10、s! 30 min-O/N 30 min 10 min 40 min The experimental process xMAP Technology Multiplex： Multiple testing solutions with a small amount of sample. High speed：10000 test/hour. Low cost： Save cost when detecting of more than 3 factors. High sensitivity：Lowest limitation is 0.04pg/ml。 Reproducibility：acc
11、urate and stable results。 Broad linear range：Detection range covers 10e5 concentration range, providing accurate results of different samples at the same time. Number of plates required Total time to result Results per plate Total sample used per panel Internal controls possible? Dynamic Range Lower
12、 limit of detection xMAP Technology ELISA 3 hours 800 10 hours 80 YES 50 m ml 4 ml 1-10,000 pg/ml 1 pg/ml NO 10-2500 pg/ml 1 pg/ml 10 proteins 40 samples Comparison between xMAP technology & ELISA 25 xMAP technology applicationImmunology Multiplex detection serves as the most authentic and accurate
13、tools for animal model research. Results：After treated with LPS, the expression of cytokines fluctuates along with the time. DIAGNOSTIC MARKERS FOR EARLY DETECTION OF OVARIAN CANCER Prof. Gil Mor, Yale University and collaborators including Millipore Advantages: Early detection of malignant tumor Mu
14、ltiplex detection benefits for tissue specificity Non-invasive sampling Multiple tissue screening xMAP technology applicationcancer Kits Instruments Software Services Merck Millipore providing integrated application solution 28 Instruments K I S S Multiplex Assays Human Panels Adipokine Apoptosis Bo
15、ne Cancer Biomarker CVD Cytokine Endocrine Gut Hormone Pituitary Skin Sol. Cyto Receptor Rat Panels Adipokine Bone CVD Cytokine Endocrine Gut Hormone Pituitary Stress Thyroid Mouse Panels Adipokine Bone CVD Cytokine Endocrine Gut Hormone Soluble Cytokine Receptor Canine Panels Adipokine Cytokine End
16、ocrine 29 Non-Human Primate Panel Cytokine Milliplex detection for 7 species NEW：pig、cat Sample Types Culture supernatant Serum Plasma Tissue lysates Urine Blood spots Gingival Crevicular Fluid Nasal Lavage Fluid Tears Cerebral Spinal Fluid Broncho-Alveolar Fluid Saliva Vaginal Secretions Sample Volumes Routine protocol = 25-50ml/well Have mod
