Transcript ELISA as a Diagnostic Tool — Influenza - Bio-Rad

ELISA Immuno ExplorerTM
Influenza Diagnostic Tool
ELISA Immuno
ExplorerTM
Kit
Influenza
Diagnostic Tool
Instructors
Stan Hitomi
Coordinator – Math & Science
Principal – Alamo School
San Ramon Valley Unified School District
Danville, CA
Kirk Brown
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.
[email protected]
Leigh Brown, M.A.
[email protected]
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
Workshop
Time 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
Immunoglobulin (IgG) Structure
Enzyme-Linked
Immunosorbant
Assay
Heavy
chain
Light
chain
Disulfide
bonds
Influenza
Antigens
RIDT detects
viral antigens
1) Load samples &
controls into wells
Wash
2) Add primary
antibody to all wells
Wash
3) Add enzymelinked secondary
antibody to all wells
Wash
4) Add enzyme
substrate to all wells
ELISA ANIMATION
Laboratory
Quick Guide
For Protocol II
Steps 1 – 2
Label wells
of 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
Steps 9 – 10
Add primary
antibody
•
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.
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
Detergents:
Ionic vs nonionic
Denaturing vs
non-denaturing
Swords
(denaturing):
“pointy”
hydrophobic
ends, ionic polar
ends
Gloves (nondenaturing):
bulky, nonpenetrating
hydrophobic
ends, non-ionic
or zwitterionic
polar ends.
SDS
Triton X-100
Steps 11 – 13
Wash & 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 B
(+) for Flu A
(-) for Flu A & B
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
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 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
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 vaccinejust 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
I
Tracking
outbreaks
of disease
Detecting
antigens
II
III
Detecting
antibodies
in serum
Real-World Application
HIV, Bird Flu and West Nile
viruses, common cold,
cholera, smallpox, anthrax,
and STDs
Pregnancy, drug, GMO and
allergen tests
Air food and water testing
Influenza, HIV, smallpox,
West Nile and Flu viruses
HIV, Lyme disease,
trichinosis, West Nile
virus, and Flu virus
Objectives
Epidemiology,
disease spread,
public health
Uses for
antibodies in
research,
medicine, and
consumer goods
Detecting
exposure to
disease causing
agents
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