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Radioimmunoassay (RIA): A Remarkably Sensitive Bioassay

Radioimmunoassay (RIA): A Remarkably Sensitive Bioassay. Sarah Dekat NCSS 2006. Biochemistry Principles. Radioimmunoassay Procedure. Standard Curve & Unknown Sample. Characteristics of Binder and Ligand. Availability Synthetic Natural – Produced From Animals Monoclonal Antibodies

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Radioimmunoassay (RIA): A Remarkably Sensitive Bioassay

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  1. Radioimmunoassay (RIA):A Remarkably Sensitive Bioassay Sarah Dekat NCSS 2006

  2. Biochemistry Principles

  3. Radioimmunoassay Procedure

  4. Standard Curve & Unknown Sample

  5. Characteristics of Binder and Ligand • Availability • Synthetic • Natural – Produced From Animals • Monoclonal Antibodies • Purity – Competing Reactions with Impurities • Stability – Store in Albumin Serum • Specificity – Binding Constant

  6. Characteristics of Tracer • Must Have Similar Binding Properties as Unlabeled Ligand • Internally Labeled Ligand • 14C and 3H • Externally Labeled Ligand • 131I and 125I • By-Products or Incomplete Synthesis • Purification by chromatography (gel filtration)

  7. Separation of Bound and Free Ligand • Electrophoresis • Gel Filtration • Adsorption Chromatography • Fractional Precipitation • Centrifugation • Filtration • Partition Chromatography • Dialysis

  8. Increasing Assay Precision

  9. Sensitivity – Decrease [Ligand]

  10. Sensitivity – Decrease [Binder]

  11. Comparison of Assays • Enzymeimmunoassays (EIA) • millimolar • Fluoroimmunoassays (EIA) • micromolar • Radioimmunoassays (EIA) • nanomolar to picomolar

  12. Applications of Radioimmunoassays • Endocrinology • Insulin, HCG, Vasopressin • Detects Endocrine Disorders • Physiology of Endocrine Function • Pharmacology • Morphine • Detect Drug Abuse or Drug Poisoning • Study Drug Kinetics

  13. Applications of Radioimmunoassays • Epidemiology • Hepatitis B • Clinical Immunology • Antibodies for Inhalant Allergens • Allergy Diagnosis • Oncology • Carcinoembryonic Antigen • Early Cancer Detection and Diagnosis

  14. Summary • Based on Simple Biochemistry Principles • Establish Ideal Binder and Ligand • Synthesize Tracer Ligand • Separation of Bound and Free Parts • High Precision and Sensitivity • Powerful Applications to a Wide Range of Medical Fields

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