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
Sherri Andrews, Ph.D.
North Carolina School of the Arts
Winston-Salem, NC
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 HIV, Mad Cow, SARS
Biodefense
Cancer treatment
Drug and steroid testing
Pregnancy / Reproduction
GMO
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
Health sciences
Immunology
Immune response – antibody/antigen interactions
Disease – infection, detection, transmission
Biowarfare – defense against acts of aggression
Links to research, 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:
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
Ways The ELISA Kit Can Be Used: Ways The ELISA Kit Can Be Used
Slide18: Protocol I: Tracking Disease Outbreaks Step 1 - Share ‘body fluid’ samples to simulate disease transmission
Step 2 - ELISA to detect infection Results – Infection of about 50% of students Real-World Application - HIV Transmission & Detection
Slide19: Protocol II: Antigen Detection ELISA Protocol - ELISA on simulated urine samples Real-World Application – Pregnancy Test
Slide20: Protocol III: ELISA Antibody Test Protocol - ELISA to detect exposure to a disease
Do you have antibodies to the disease? Real-World Application – Lyme Disease
Real-world Applications of Antibodies: Real-world Applications of Antibodies Uses
– Disease diagnosis
– Basic Research
– Immunotherapy Applications
– Dipstick tests/ELISA
– Immunostaining
– Western blotting Bio-Rad’s HIV-2
ELISA Kit Bio-Rad’s HIV
Western Blot Kit
Example: Pregnancy Test: Example: Pregnancy Test