Published on October 24, 2007
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 ImmunosorbantAssay: 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 ImmunosorbantAssay: 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?AndWhat 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?AndWhat 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
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