Analytical Proteins分析蛋白质.pptVIP

Analytical Proteins BIT 230 Clinical Chemistry Detection, Monitoring and Quantification of marker substances in biological samples blood urine saliva Where do proteins come in? Analyte Part of Assay enzyme antibody Enzymes $100 million market Enzyme-Substrate interaction Specificity Stability pH temperature storage Source recombinant vs natural (Fig 9.2) Enzyme-Based Diagnostic tests Dehydrogenases Oxidases Dehydrogenase Oxidase Blood Glucose Blood Glucose Cholesterol and Triglycerides Biosensors Antibodies as Reagents Disadvantages of RIA Enzyme Immunoassay ELISA for HIV Pregnancy Tests * NADH reduced (H) nicotinamide adenine dinucleotide dehydrogenase Substrate (Analyte) + NAD+ ------------------------ Product + NADH + H+ How to measure: NADH absorbs at 340nm SPECTROPHOTOMETRY Reaction 1 oxidase Substrate + O2 + H2O ------------------------------ Product + H2O2 (hydrogen peroxide) Reaction 2 peroxidase H2O2 + colorless substrate --------------------------- Dye (colored) + H2O SPECTROPHOTOMETRY oxidase/peroxidase Glucose --------------------------------- quinoneimine + H20 hexokinase/dehydrogenase Glucose ---------------------------------- NADH Measure one point in time! Erythrocyte Hemoglobin Glycosylated Hemoglobin glucose binds to valine of Bets chain of hemoglobin This reflect glucose levels over several weeks Hydrophobic Transported in complexes called LIPOPROTEINS Low Density Lipoproteins -BAD atheroschlerosis fatty deposits on walls of arteries High Density Lipoproteins -Good transports cholesterol from tissue to liver bile salts oxidase/peroxidase Cholesterol ------------------------------------ quinoneimine Immobilized Biological Sensing element (analyte binds here) Transducer Electronics Read Out (Quantitative) Immunoassays - assays which use Ab to detect and quantify substances Ab are extremely specific - ADVANTAGE Ab can not be detected, need a