Analytical Proteins BIT 230. Clinical Chemistry Detection, Monitoring and Quantification of marker substances in biological samples –blood –urine –saliva.

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Presentation transcript:

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 NADHreduced (H) nicotinamide adenine dinucleotide dehydrogenase Substrate (Analyte) + NAD > Product + NADH + H+ How to measure: NADH absorbs at 340nm SPECTROPHOTOMETRY

Oxidase Reaction 1 oxidase Substrate + O 2 + H 2 O > Product + H 2 O 2 (hydrogen peroxide) Reaction 2 peroxidase H 2 O 2 + colorless substrate > Dye (colored) + H 2 O SPECTROPHOTOMETRY

Blood Glucose oxidase/peroxidase Glucose > quinoneimine + H 2 0 hexokinase/dehydrogenase Glucose > NADH Measure one point in time!

Blood Glucose Erythrocyte Hemoglobin Glycosylated Hemoglobin glucose binds to valine of Bets chain of hemoglobin This reflect glucose levels over several weeks

Cholesterol and Triglycerides 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

Biosensors Immobilized Biological Sensing element (analyte binds here) Transducer Electronics Read Out (Quantitative)

Antibodies as Reagents Immunoassays - assays which use Ab to detect and quantify substances Ab are extremely specific - ADVANTAGE Ab can not be detected, need a marker Radioactive labels (RIA) Enzymes(EIA) (ELIZA)

Disadvantages of RIA Safety issue for lab personnel - Radioactivity waste shelf life short expensive

Enzyme Immunoassay ELISA (antibody sandwich) Enzymes used horseradish peroxidase, alkaline phosphatase Alternates Fluorescent tag Chemiliminescent tags

ELISA for HIV Detection of Ab against HIV coat proteins Disadvantage: lapse between infection and Ab production most assays detect only HIV1 concentrations of Ab in saliva and urine too low Recombinant DNA approaches make this assay safer!

Pregnancy Tests Detection of hCG (human chorionic gonadotropin) Graves disease (Graves antibodies)