Presentation Transcript
Slide2: 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
ELISA Enzyme-Linked Immunosorbant Assay: ELISA Enzyme-Linked Immunosorbant Assay 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: 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: 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?: 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: 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
ELISA Antibody Structure: ELISA Antibody Structure
Slide9:
ELISA ANIMATION
ELISA Enzyme-Linked Immunosorbant Assay: ELISA Enzyme-Linked Immunosorbant Assay
ELISA Kit Workstation Inventory: 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
ELISA Kit Quick Guide: ELISA Kit Quick Guide
ELISA Kit Results: ELISA Kit Results
What Are The Reagents? And What Function Do They Perform?: 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
What Are The Reagents? And What Function Do They Perform?: 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
ELISA Kit Protocol Issues: 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
Ways The ELISA Kit Can Be Used: Ways The ELISA Kit Can Be Used
Slide18: ELISA test for Transmissible Spongiform Encephalopathies (TSEs) a PrPres
Proteinase K resistant
Aggregates in detergent
PrPsens
Proteinase K sensitive
Soluble in detergent
b 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
Slide19: Sample brain tissue
Homogenize brain tissue
Digest with Proteinase K (normal prions are digested, diseased prions are resistant)
Concentrate
Denature Proteinase K
Perform ELISA TSE test sample preparation
Slide20: Protocol II: Antigen Detection ELISA Protocol - ELISA on simulated animal brain samples Real-World Application – TSE Test
Real-world Applications of Antibodies: 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: Example: Pregnancy Test