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ELISA Immuno Explorer ™ Kit Instructors :

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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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Page 1: ELISA Immuno Explorer ™  Kit Instructors :
Page 2: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 3: ELISA Immuno Explorer ™  Kit Instructors :

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!

Page 4: ELISA Immuno Explorer ™  Kit Instructors :

Links to the Real World

• Mad Cow Disease, SARS, HIV

• GMO

• Drug and steroid testing

• Pregnancy / Reproduction

• Biodefense

• Cancer treatment

Page 5: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 6: ELISA Immuno Explorer ™  Kit Instructors :

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!

Page 7: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 8: ELISA Immuno Explorer ™  Kit Instructors :

ELISA Antibody Structure

Light chain

Heavy chain

Disulfide bonds

Page 9: ELISA Immuno Explorer ™  Kit Instructors :

ELISA ANIMATION

Page 10: ELISA Immuno Explorer ™  Kit Instructors :

ELISA Enzyme-Linked ImmunosorbantAssay

Page 11: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 12: ELISA Immuno Explorer ™  Kit Instructors :

ELISA Kit Quick Guide

Page 13: ELISA Immuno Explorer ™  Kit Instructors :

ELISA Kit Results

Page 14: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 15: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 16: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 17: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 18: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 19: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 20: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 21: ELISA Immuno Explorer ™  Kit Instructors :

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

Page 22: ELISA Immuno Explorer ™  Kit Instructors :

Example: Pregnancy Test