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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. - PowerPoint PPT Presentation
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ELISA Immuno Explorer™ Kit
Instructors:Stan Hitomi
Director, Edward Teller Education CenterUC Davis / Lawrence Livermore NationalLaboratory, Livermore, CA
Kirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High School and Delta College, Tracy, CA
ELISA
Enzyme-Linked ImmunosorbantAssay
• Mammalian immune system
• Antibody specificity
• Biology’s “magic bullet”
• Evolved over millions of years
• Harness nature’s tool kit
• Imagine the applications!
Links to the Real World
• Mad Cow Disease, SARS, HIV
• GMO
• Drug and steroid testing
• Pregnancy / Reproduction
• Biodefense
• Cancer treatment
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
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!
Immune Response
A. Pathogen
C. Macrophage
D. Macrophage
E. MacrophageF. T cell
B. B cells
G. B cell
H. Memory B cellsI. Plasma cells
J. Antibodies attach to pathogen
ELISA Antibody Structure
Light chain
Heavy chain
Disulfide bonds
ELISA ANIMATION
ELISA Enzyme-Linked ImmunosorbantAssay
ELISA Kit Workstation Inventory Reagents:
Yellow tubes Test samples
2Violet tube (+) Positive control
1Blue tube (-) Negative control
1Green tube (PA) Primary antibody
1Orange tube (SA) Secondary antibody
1
Lab Equipment and Supplies:Microplate strips, pipettor, pipette tips, transfer pipette, wash buffer, paper towels, marking pen
ELISA Kit Quick Guide
ELISA Kit Results
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
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
ELISA Kit Protocol Issues
Controls:•Protocol II
Positive control = antigenNegative control = PBS
Triplicate samples: controls against cross contamination (2 out of 3 ain’t bad)
Washing: eliminates non-specific binding and reduces background
Ways The ELISA Kit Can Be Used
Protocol Type of ELISA Real-World Application
I Tracking outbreaks of disease
HIV, SARS, smallpox & anthrax
II Detecting antigensGMO, BSE, pregnancy, drugs, (and all the above)
III Detecting antibodies in serum
HIV, Lyme disease, smallpox and West Nile virus
ELISA test for Transmissible Spongiform Encephalopathies (TSEs)
PrPres
•Proteinase K resistant
•Aggregates in detergent
PrPsens
•Proteinase K sensitive
•Soluble in detergent
Prion Proteins (PrPres and PrPsens)
•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
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
Protocol II: Antigen Detection ELISA
Protocol - ELISA on simulated animal brain samples
Tube Description
Actual Tube Contents
Simulated Tube Contents
Student samples
Antigen or PBS Processed brain
Primary antibody
Primary antibody Antibody against prion protein
Secondary antibody
Secondary antibody
HRP-linked antibody against primary antibody
Positive control Antigen Synthesized peptide with prion sequence
Negative control
PBS Buffer
Real-World Application – TSE Test
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
Example: Pregnancy Test