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ELISA Immuno Explorer ™ Kit Instructors :

ELISA Immuno Explorer ™ Kit Instructors :. Stan Hitomi Director, Edward Teller Education Center UC Davis / Lawrence Livermore National Laboratory, Livermore, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA.

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ELISA Immuno Explorer ™ Kit Instructors :

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  1. ELISA Immuno Explorer™ KitInstructors: Stan Hitomi Director, Edward Teller Education Center UC Davis / Lawrence Livermore National Laboratory, Livermore, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA

  2. ELISAEnzyme-LinkedImmunosorbantAssay • Mammalian immune system • Antibody specificity • Biology’s “magic bullet” • Evolved over millions of years • Harness nature’s tool kit • Imagine the applications!

  3. Links to the Real World • Mad Cow Disease, SARS, HIV • GMO • Drug and steroid testing • Pregnancy / Reproduction • Biodefense • Cancer treatment

  4. ELISA Immuno Explorer Kit Features • Lab completed in a 45 min period • Supplies for 48 students (12 workstations) • Comprehensive and flexible curriculum • Compelling real-world links • Cost effective • Classroom Safe • Striking results

  5. What Can You Teach With The ELISA Kit? • Hands-on Immunology • One lesson integrates multiple standards • Environmental Science – surveillance • Immunology – antibody/antigen interactions • Disease/Health Science – infection, detection, transmission • Biowarfare – defense against acts of aggression • Links to research, agriculture, medicine, and consumer products • Modern way to teach classic and contemporary science • Teach more with less!

  6. Immune Response C. Macrophage A. Pathogen D. Macrophage B. B cells F. T cell E. Macrophage G. B cell J. Antibodies attach to pathogen H. Memory B cells I. Plasma cells

  7. Heavy chain Disulfide bonds Light chain ELISA Antibody Structure

  8. ELISA ANIMATION

  9. ELISA Enzyme-LinkedImmunosorbantAssay

  10. ELISA Kit Workstation Inventory Reagents: Yellow tubes Test samples 2 Violet tube (+) Positive control 1 Blue tube (-) Negative control 1 Green tube (PA) Primary antibody 1 Orange tube (SA) Secondary antibody 1 Lab Equipment and Supplies: Microplate strips, pipettor, pipette tips, transfer pipette, wash buffer, paper towels, marking pen

  11. ELISA Kit Quick Guide

  12. ELISA Kit Results

  13. What Are The Reagents?AndWhat Function Do They Perform? PBS: Phosphate buffered saline – provides stable buffered environment to maintain antibody structure Tween 20: Nonionic detergent – removes non-specifically bound proteins to reduce background and blocks protein binding sites on the polystyrene Microplates: Polystyrene – proteins absorb (bind) by hydrophobic bonds to the polystyrene

  14. What Are The Reagents?AndWhat Function Do They Perform? Antigen: Chicken gamma globulin Primary antibody: Polyclonal anti-chicken antibody made by rabbits Secondary antibody: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP) Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue

  15. ELISA Kit Protocol Issues Controls: • Protocol II Positive control = antigen Negative control = PBS Triplicate samples: controls against cross contamination (2 out of 3 ain’t bad) Washing: eliminates non-specific binding and reduces background

  16. Ways The ELISA Kit Can Be Used

  17. Prion Proteins (PrPres and PrPsens) ELISA test for Transmissible Spongiform Encephalopathies (TSEs) a b • Uses differences in diseased prions vs. normal prions to prepare sample. • Proteinase K only digests normal, not diseased, prions . • ELISA tests for any prion protein • PrPsens • Proteinase K sensitive • Soluble in detergent • PrPres • Proteinase K resistant • Aggregates in detergent

  18. TSE test sample preparation • Sample brain tissue • Homogenize brain tissue • Digest with Proteinase K (normal prions are digested, diseased prions are resistant) • Concentrate • Denature Proteinase K • Perform ELISA

  19. Protocol II: Antigen Detection ELISA Real-World Application – TSE Test Protocol - ELISA on simulated animal brain samples

  20. Real-world Applications of Antibodies Agricultural Uses – Crop-specific disease diagnosis – Animal disease diagnosis – Detection of GM crops – Basic research Applications – Dipstick tests/ELISA – Immunostaining – Western blotting Bio-Rad’s TSE ELISA Kit

  21. Example: Pregnancy Test

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