2. 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

3. ELISA E nzyme- L inked I mmuno s orbant A ssay Mammalian immune system Antibody specificity Biology’s “magic bullet” Evolved over millions of years Harness nature’s tool kit Imagine the applications!

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

5. 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

6. 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!

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

8. ELISA Antibody Structure Light chain Heavy chain Disulfide bonds

9. ELISA ANIMATION

10. ELISA E nzyme- L inked I mmuno s orbant A ssay

11. ELISA Kit Workstation Inventory Reagents: Yellow tubesTest samples2 Violet tube (+)Positive control1 Blue tube (-)Negative control1 Green tube (PA)Primary antibody1 Orange tube (SA)Secondary antibody1 Lab Equipment and Supplies: Microplate strips, pipettor, pipette tips, transfer pipette, wash buffer, paper towels, marking pen

12. ELISA Kit Quick Guide

13. ELISA Kit Results

14. What Are The Reagents? And What 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

15. What Are The Reagents? And What 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

16. 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

17. Ways The ELISA Kit Can Be Used ProtocolType of ELISAReal-World Application I Tracking outbreaks of disease HIV, SARS, smallpox & anthrax II Detecting antigens GMO, BSE, pregnancy, drugs, (and all the above) III Detecting antibodies in serum HIV, Lyme disease, smallpox and West Nile virus

18. ELISA test for T ransmissible S pongiform E ncephalopathies (TSEs)  PrP res Proteinase K resistant Aggregates in detergent PrP sens Proteinase K sensitive Soluble in detergent  Prion Proteins (PrP res and PrP sens ) 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

19. 1.Sample brain tissue 2.Homogenize brain tissue 3.Digest with Proteinase K (normal prions are digested, diseased prions are resistant) 4.Concentrate 5.Denature Proteinase K 6.Perform ELISA TSE test sample preparation

20. Protocol II: Antigen Detection ELISA Protocol - ELISA on simulated animal brain samples Tube Description Actual Tube Contents Simulated Tube Contents Student samples Antigen or PBSProcessed brain Primary antibody Antibody against prion protein Secondary antibody HRP-linked antibody against primary antibody Positive controlAntigenSynthesized peptide with prion sequence Negative control PBSBuffer Real-World Application – TSE Test

21. 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

22. Example: Pregnancy Test