Official ELISA powerpoint Summer 2005

Information about Official ELISA powerpoint Summer 2005

Published on October 24, 2007

Author: Felipe

Source: authorstream.com

Content

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

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