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Lecture 3A- Automated Drug Analysis and Systems.docx

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Biochemistry 3386B
L.Graham Smith

Lecture 3: Drug Analysis Systems/Instruments Part I – Automated Assays Automated Assays (done in the Core Labs) 1. Homogeneous Competitive Binding Immunoassays  Drugs/Drug Testing 2. Alcohol Dehydrogenase-Based  Ethanol/Ethanol Testing 3. Colourimetric  Lipids  triglycerides/cholesterol Homogeneous Competitive Protein Binding Immunoassays  All dissolved in solution  The most common automated assays for therapeutic drug monitoring and toxicology  A labeled drug corresponding to the one in the patient sample is added  Label: o Fluorescent compound (FPIA; fluorescence polarization immunoassay)  pass plane polarized light through a sample with the added antibody and fluorescent labeled drug (fluorescein)  concentration of the drug in the sample is inversely related to the amount of polarization retained in the fluorescent ligh then emitted by the sample  Best Method o Particle (PETIA; particle enhanced turbimetric inhibition immunoassay)  Antibody bound drug-linked particles form insoluble aggregates  Causes light scatter (turbidity)  Measured at 340nm  Tag= spherical latex particle (40nm dia- turbidity density) o Enzyme (EMIT; enzyme mediated immunoassay technique)  any drug-linked enzyme which becomes bound to the antibody is inactivated  Tag= G6P – Dehydrogenase  Residual amount (unbound) enzyme activity is proportional to the concentration of drug in the patient sample (observe NAD +  NADH conversion at 340nm) – because free has activity and bound does not →  The unlabeled drug in the sample and the labeled drug are added to the sample and compete for the binding site on the antibody (binding protein)  Note: antibody is against that drug (monoclonal) and is limited to the amount needed to bind the drug Alcohol Dehydrogenase-Based Automated Test for Ethanol  This is one of the most requested tests  This test specifically quantitates and detects ethanol →  NADH absorbance is measured at 340 nm and related to the ethanol concen
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