ELISA Immuno ExplorerTM

Influenza Diagnostic Tool

ELISA Immuno
ExplorerTM Stan Hitomi
Kit Coordinator – Math & Science
Principal – Alamo School
Influenza San Ramon Valley Unified School District
Diagnostic Tool Danville, CA

Kirk Brown
Instructors Lead Instructor, Edward Teller Education Center
Science 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
Why Teach • Hands-on Immunology
ELISA?
• 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 • Lab completed in a 45 min period
Immuno
Explorer • Supplies for 48 students (12 workstations)
Kit Advantages
• Comprehensive and flexible curriculum

• Compelling real-world links

• Striking results

• Cost effective

• Classroom Safe
 • Introduction
Workshop
Time Line • 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: • RIDT is an ELISA that can be performed in the
doctor’s office in less than 30 minutes
Perform Rapid
Influenza • There are 3 RIDTs currently approved for use in
Diagnostic Test the U.S.
(RIDT)
ELISA Immunoglobulin (IgG) Structure

Enzyme-Linked
Immunosorbant
Assay
Heavy
chain

Disulfide Light
bonds chain

Influenza
Antigens
RIDT detects 1) Load samples &
viral antigens controls into wells

Wash

2) Add primary
antibody to all wells

Wash

3) Add enzyme-
linked secondary
antibody to all wells

Wash

4) Add enzyme
substrate to all wells
ELISA ANIMATION
Laboratory
Quick Guide

For Protocol II
Steps 1 – 2 • Obtain a microplate strip and “serum
samples”
Label wells • Label the 12-well strip
of microplate –First 3 wells: positive controls “+”
strip –Next 3 wells: negative controls “-”
–Remaining wells to identify test

samples

Sample 1 Sample 2
Steps 3 – 6 • 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
Add controls
and samples • 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 are made of polystyrene
Microplate Strips

• 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 • Type A
species –Natural host: wild aquatic birds
(antigen types)
–Has serotypes (based on antibody
response)
5 genera, but only 3 • Type B
of interest to us
–Infects mostly humans (ferrets & seals
can get it too)
Each genera has a –Less common than Type A
single species!
–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 • Remove sample from wells by firmly
tapping the strip on a paper towel
Wash plates
• 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

Steps 9 – 10 • Using a fresh pipet tip, add 50 µl of
Add primary primary antibody to each well of the
microplate strip
antibody
• Incubate for 5 minutes

• If any flu antigen bound to the well in

previous step primary antibody will bind
to antigen.
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:
…
I like fat!
are amphiphiles,
containing a
lipophilic portion
and a hydrophilic
portion.
lower the
interfacial energy
between unlike
phases. I like water!

emulsify or
solubilize
aggregated
particles.
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
Detergents:
Ionic vs non-
ionic
Denaturing vs
non-denaturing
SDS
Swords
(denaturing):
“pointy”
hydrophobic
ends, ionic polar
ends

Gloves (non-
denaturing): Triton X-100
bulky, non-
penetrating
hydrophobic
ends, non-ionic
or zwitterionic
polar ends.
Steps 11 – 13 • Wash unbound primary antibody from
Wash & add microplate wells as before
enzyme-linked • Wash twice
secondary • Add 50 µl of the enzyme-linked
antibody secondary antibody to each well
• Wait 5 minutes
Antibody • Secondary antibody (enzyme-linked
Specificity antibody) will only bind to the primary
antibody

• Secondary antibody specifically

recognizes the constant region of the
primary antibody
Steps 14 – 15 • Wash unbound enzyme-linked secondary
antibody from microplate wells as before
Add enzyme
• Wash THREE times
substrate
• 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 Flu B (+) for Flu A (-) for Flu A & B
for RIDTs

Detect and distinguish between Type A and Type B

influenza viruses
OR
Detect Type A and Type B influenza viruses, but not
tell them apart
OR
Detect Type A influenza virus
What about
H1N1?
• RIDT’s do not distinguish H1N1
specifically from other Type A Flu
viruses.
Lab tests for • The most sensitive & specific laboratory
tests are rRT-PCRs (real-time reverse
H1N1/09 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 • Roughly spherical virus, 80-120
nanometers
Type A
• 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 • Hemagglutinen (HA)- viral glycoprotein
that mediates binding of virus to target
viral proteins 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 • New human influenza viruses occur
through:
viruses
–Genetic reassortment within an
existing human virus

–Avian viruses developing capacity for

human-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 • Derived from several viruses circulating
H1N1 Origins 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: • Each seasonal influenza vaccine
contains 2 influenza A viruses and 1
What’s in influenza B virus.
them?
• 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 • Manufacturers grow the 3 strains in eggs
or in chicken kidney cells (3 strains 
production 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 Purified antigen: Chicken gamma globulin
reagents?
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 Real-World Application Objectives

ELISA
Tracking HIV, Bird Flu and West Nile Epidemiology,
outbreaks viruses, common cold, disease spread,
I of disease cholera, smallpox, anthrax, public health
and STDs
Detecting Pregnancy, drug, GMO and Uses for
antigens allergen tests antibodies in
II Air food and water testing research,
Influenza, HIV, smallpox, medicine, and
West Nile and Flu viruses consumer goods
Detecting HIV, Lyme disease, Detecting
antibodies trichinosis, West Nile exposure to
III in serum virus, and Flu virus disease causing
agents
Webinars • Enzyme Kinetics — A Biofuels Case Study

• Real-Time PCR — What You Need To Know

and Why You Should Teach It!

• Proteins — Where DNA Takes on Form and

Function

• From plants to sequence: a six week

college biology lab course

• From singleplex to multiplex: making the

most out of your realtime experiments

explorer.bio-rad.comSupportWebinars