ELISA Immuno ExplorerTM

Influenza Diagnostic Tool

Stan HitomiCoordinator – Math & SciencePrincipal – Alamo SchoolSan Ramon Valley Unified School DistrictDanville, CA

Kirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High School and Delta College, Tracy, CA

Bio-Rad Curriculum and Training Specialists:Sherri Andrews, Ph.D.

sherri_andrews@bio-rad.com

Leigh Brown, M.A. leigh_brown@bio-rad.com

ELISA Immuno ExplorerTM

Kit

Influenza Diagnostic Tool

Instructors

Why Teach ELISA?

• Hands-on Immunology

• Tangible results

• Laboratory extensions

• Real-world connections

• Link to careers and industry

• Standards-based: One lesson integrates multiple standards

–Health sciences –Immunology–Immune response – antibody/antigen interactions–Disease – infection, detection, transmission

ELISA Immuno Explorer

Kit Advantages

• Lab completed in a 45 min period

• Supplies for 48 students (12 workstations)

• Comprehensive and flexible curriculum

• Compelling real-world links

• Striking results

• Cost effective

• Classroom Safe

WorkshopTime Line

• Introduction

• Rapid Influenza Diagnostic Test (RIDT)

• Viruses, influenza, and H1N1

• Ways the ELISA Immuno Explorer Kit can be used

Lab Scenario • A room full of sick people (you guys!)

• Various symptoms

–Coughing

–Sneezing

–Temperature

–Other nasties! (what are you doing here,

anyway?)

Question: • Is this the 2009-2010 pandemic H1N1?

• Food poisoning?

• Cholera?

• Or lots of psychosomatic symptoms (because the person next to you is sick)?

Solution:

Perform Rapid Influenza Diagnostic Test(RIDT)

• RIDT is an ELISA that can be performed in the doctor’s office in less than 30 minutes

• There are 3 RIDTs currently approved for use in the

U.S.

ELISA

Enzyme-Linked ImmunosorbantAssay

Light chain

Heavy chain

Disulfide bonds

InfluenzaAntigens

Immunoglobulin (IgG) Structure

RIDT detects viral antigens

1) Load samples & controls into wells1) Load samples & controls into wells

2) Add primary antibody to all wells

2) Add primary antibody to all wells

3) Add enzyme-linked secondary antibody to all wells

3) Add enzyme-linked secondary antibody to all wells

4) Add enzyme substrate to all wells

4) Add enzyme substrate to all wells

WashWash

WashWash

WashWash

ELISA ANIMATION

LaboratoryQuick Guide

For Protocol II

Steps 1 – 2

Label wellsof microplate strip

• Obtain a microplate strip and “serum samples”

• Label the 12-well strip

–First 3 wells: positive controls “+”

–Next 3 wells: negative controls “-”

–Remaining wells to identify test

samples

Sample 1 Sample 2

Steps 3 – 6

Add controls and samples

• Add 50 µl of positive control to the 3 (+) wells

• Using a fresh pipet tip, add 50 µl of negative control to the 3 (−) wells

• Using a fresh pipet tip, add 50 µl of sample 1 to the next 3 wells

• Using a fresh pipet tip, add 50 µl of sample 2 to the final 3 wells

• Incubate for 5 minutes

Microplate Strips • Microplate strips are made of polystyrene

• Hydrophobic side chains of amino acids bind to the polystyrene wells

• If flu antigen is present it will bind to the polystyrene, (+) control, and possibly in the unknown sample

Influenza species(antigen types)

5 genera, but only 3 of interest to us

Each genera has a single species!

• Type A

–Natural host: wild aquatic birds

–Has serotypes (based on antibody response)

• Type B

–Infects mostly humans (ferrets & seals can get it too)–Less common than Type A

–Mutation rate 2-3x slower than type A, so less genetic diversity and more acquired immunity

• Type C

–Infects humans, dogs, & pigs, but less common–Causes only mild disease

Steps 7 – 8

Wash plates

• Remove sample from wells by firmly tapping the strip on a paper towel

• Discard the top paper towel

• Using a disposable transfer pipet, wash wells with wash buffer

• Remove wash buffer from wells by firmly tapping the strip on a paper towel

• Discard the top paper towel

• Repeat wash step

• Using a fresh pipet tip, add 50 µl of primary antibody to each well of the microplate strip

• Incubate for 5 minutes

• If any flu antigen bound to the well in previous step primary antibody will bind to antigen.

Steps 9 – 10Add primary antibody

Wash Buffer • Wash buffer contains phosphate buffer saline (PBS) to keep antibodies in a stable environment that helps keep their structure

• Also contains Tween 20: a nonionic detergent that removes non-specifically bound proteins and coats wells to act as a blocking agent to reduce background

• Antibody will bind only to influenza antigens

Chemistry in action….

Or…

Ask your friendly chemist…about detergents.

DETERGENTS:…

are amphiphiles, containing a lipophilic portion and a hydrophilic portion.

lower the interfacial energy between unlike phases.

emulsify or solubilize aggregated particles.

I like fat!

I like water!

More about detergent terms

Lipophilic portion is also referred to as “hydrophobic” tail

Hydrophilic portion is also referred to as “polar” head

Types: nonionic, anionic, cationic and zwitterionic

A generic detergent

polar headhydrophobic tail:hydrocarbon chain

Detergents: Ionic vs non-ionicDenaturing vs non-denaturing

Swords (denaturing): “pointy” hydrophobic ends, ionic polar ends

Gloves (non-denaturing): bulky, non-penetrating hydrophobic ends, non-ionic or zwitterionic polar ends.

S

O

OO

O- Na+

SDS

Triton X-100O

O

H

7

Steps 11 – 13Wash & add enzyme-linked secondary antibody

• Wash unbound primary antibody from microplate wells as before

• Wash twice

• Add 50 µl of the enzyme-linked secondary antibody to each well

• Wait 5 minutes

Antibody Specificity

• Secondary antibody (enzyme-linked antibody) will only bind to the primary antibody

• Secondary antibody specifically recognizes the constant region of the primary antibody

Steps 14 – 15 Add enzyme substrate

• Wash unbound enzyme-linked secondary antibody from microplate wells as before

• Wash THREE times

• Add 50 µl of the enzyme-linked substrate to each well

• Wait 5 minutes

• The positive samples will begin to turn blue

Results

• Some positive by RIDT

• Some negative

• Did the controls work?

CDC guidelines for RIDTs

(-) for Flu A & B

(-) for Flu A & B

(+) for Flu B

(+) for Flu B

(+) for Flu A

(+) for Flu A

Detect and distinguish between Type A and Type B influenza viruses

ORDetect Type A and Type B influenza viruses, but not tell them apart

ORDetect Type A influenza virus

What about H1N1?

• RIDT’s do not distinguish H1N1 specifically from other Type A Flu viruses.

Lab tests for H1N1/09

• The most sensitive & specific laboratory tests are rRT-PCRs (real-time reverse transcriptase PCR)

• rRT-PCRs detect viral RNA (very specific)

• Cannot be performed in doctor’s office; 2-4 days to get results (test takes 6-8 hours)

The flu! • Influenza viruses are single-stranded RNA viruses

• Family Orthomyxoviridae

• Affect birds and mammals

• 3 types A, B, and C

• 2009 H1N1 is Type A

Influenza Type A

• Roughly spherical virus, 80-120 nanometers

• Viral envelope with 2 types of glycoprotein wrapped around central core

• Core contains RNA genome and viral packaging proteins

• Single-stranded (-)RNA virus; 8 RNA molecules encode 11 proteins

Influenza A viral proteins

• Hemagglutinen (HA)- viral glycoprotein that mediates binding of virus to target cell and entry of viral genome into that cell

• Neuraminidase (NA)- viral glycoprotein that allows release of progeny virus from infected cells

–H & N? Sound familiar? (think H1N1)

• 16 HA subtypes – (H1-H16)

• 9 NA subtypes (N1-N9)

New human viruses

• New human influenza viruses occur through:–Genetic reassortment within anexisting human virus

–Avian viruses developing capacity forhuman-to-human transmission

• New influenza viruses may have novel HA proteins, with or without a novel NA proteins

• Called antigenic shift

• Novel antigens means that humans have no prior immunity

2009 Pandemic H1N1 Origins

• Derived from several viruses circulating in swine

• New strain is probably a result of the reassortment of two swine influenza viruses, one from North America and one from Europe

• North American virus already carried an avian and a human gene.

• The new H1N1 virus has genes from swine, avian, and human influenzas

Reassortments resulting in the current gene complement in the pandemic 2009 H1N1 virus. Figure from Garten, RJ, et al. 2009. Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans. Science 325, 197-201.

Flu vaccines: What’s in them?

• Each seasonal influenza vaccine contains 2 influenza A viruses and 1 influenza B virus.

• Data is gathered from 94 countries and analyzed by 4 WHO centers (USA, UK, Australia, & Japan). WHO makes recommendations in February for vaccines for Northern Hemisphere.

• Strains are selected based on forecasts about which are most likely to cause disease in the coming flu season.

Vaccine production

• Manufacturers grow the 3 strains in eggs or in chicken kidney cells (3 strains trivalent vaccine)

• It takes 6 months to grow sufficient quantities of virus for vaccine preparation

• Novel H1N1 strain (H1N1/09) developed too late to be included in the annual influenza vaccine

• H1N1 vaccine was prepared in the same way as the seasonal influenza vaccine- just separately!

What are the reagents?

Purified antigen: Chicken gamma globulin

Primary antibody: Polyclonal anti-chicken antibody made in rabbits

Enzyme-linked secondary antibody: Polyclonal anti-rabbit antibody (made in goats) linked to horseradish peroxidase (HRP)

Enzyme substrate: TMB(3,3’,5,5’-tetramethylbenzidine) - a colorless solution that turns blue when oxidized by HRP

Ways The ELISA Kit Can Be Used

Protocol Type of ELISA Real-World Application Objectives

ITracking outbreaks of disease

HIV, Bird Flu and West Nile viruses, common cold, cholera, smallpox, anthrax, and STDs

Epidemiology, disease spread, public health

II

Detecting antigens

Pregnancy, drug, GMO and allergen testsAir food and water testingInfluenza, HIV, smallpox, West Nile and Flu viruses

Uses for antibodies in research, medicine, and consumer goods

III

Detecting antibodies in serum

HIV, Lyme disease, trichinosis, West Nile virus, and Flu virus

Detecting exposure to disease causing agents

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