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ELISA Immuno Explorer ™ Kit Pr esenter:

ELISA Immuno Explorer ™ Kit Pr esenter:. Dr. Sherri Andrews Science Department Chair North Carolina School of the Arts Instructor – General Studies. ELISA E nzyme- L inked I mmuno s orbant A ssay. Mammalian Immune System Antibody Specificity- Biology’s magic bullet

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ELISA Immuno Explorer ™ Kit Pr esenter:

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  1. ELISA Immuno Explorer™ KitPresenter: Dr. Sherri Andrews Science Department Chair North Carolina School of the Arts Instructor – General Studies

  2. ELISAEnzyme-LinkedImmunosorbantAssay • Mammalian Immune System • Antibody Specificity- Biology’s magic bullet • Evolved over millions of years • Harness and manipulate tools of nature • Imagine the applications!

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

  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 • Striking results • Cost effective • Classroom Safe

  5. What Can You Teach With The ELISA Kit? • Hands-on Immunology • One lesson integrates multiple standards • Immunology • Immune response – antibody/antigen interactions • Disease – infection, detection, transmission • Biowarfare – natural or acts of aggression • Links to research, medicine, and consumer products • Contemporary way to teach classic and compelling classroom science • Teach more with less

  6. ELISA Kit Step-By-Step Summary

  7. 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

  8. Heavy chain Disulfide bonds Light chain ELISA Antibody Structure

  9. Four Step ELISA Procedure: Step 1 Proteins from body fluids (including antigens) stick or absorb to plastic wells.

  10. Four Step ELISA Procedure: Step 2 Primary antibody binds to its specific antigen.

  11. Four Step ELISA Procedure: Step 3 Enzyme-linked secondary antibody binds to primary antibody.

  12. Four Step ELISA Procedure: Step 4 Enzyme linked to secondary antibody catalyzes substrate turning it blue.

  13. ELISA Enzyme-LinkedImmunosorbantAssay

  14. 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

  15. ELISA Kit Quick Guide

  16. ELISA Kit Results

  17. 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

  18. 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’ – tetraethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue

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

  20. Ways The ELISA Kit Can Be Used

  21. What is the Antigen in Each Protocol? Protocol 1: Protein in shared body fluids. Protocol 2: Protein in urine (e.g. hCG) Protocol 3: Purified HIV surface protein

  22. What is the Primary Antibody in Each Protocol? Protocol 1: Antibody to disease antigen Protocol 2: Antibody to disease antigen (e.g. anti-hCG) Protocol 3: Human serum antibody to HIV in blood sample

  23. Applications – Dipstick tests/ELISA – Immunostaining – Western blotting Real-world Applications of Antibodies Uses – Disease diagnosis – Basic Research – Immunotherapy Bio-Rad’s HIV-2 ELISA Kit Bio-Rad’s HIV Western Blot Kit

  24. Example: Pregnancy Test

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