Transcript Laboratory #2: ELISA Immuno Explorer

Wheeler High School
The Center for Advanced Studies in Science, Math & Technology
Laboratory #2: ELISA Immuno
Explorer
Lab Timeline:
1) Intro to ELISA
2) H1N1 Genetics ELISA Lab Overview
3) ELISA Assay
Post-AP DNA/Genetics – Ms. Kelavkar
Day #1: Introduction to ELISA

ELISA stands for enzyme-linked
immunosorbent assay
•Antibody based
test to diagnose
disease such as
HIV, SARS,
H1N1, STD’s,
anthrax
Post-AP DNA/Genetics – Ms. Kelavkar
•Also used to test
for pregnancy,
drugs and
genetically modified
organisms (GMO’s)
Introduction to ELISA


In this lab you will share simulated
‘body fluids’ with your classmates to
see how easily it is to pass on
H1N1.
Then you will perform an ELISA test
to determine if you have been
exposed to this contagious disease.
Post-AP DNA/Genetics – Ms. Kelavkar
Introduction to ELISA


Antibody Structure
ELISA tests use
antibodies to
detect the
presence of a
disease.
Heavy
chain
Disulfide
bonds
Light
chain
Molecules that
cause your body
to start the
immune response
are called
antigens.
Antigens
Introduction to ELISA

After becoming
infected, within days
your body will have
produced millions of
antibodies.

Recall from AP Bio
that antibodies are
proteins that
recognize the antigen
and bind to it.
Introduction to ELISA

In this lab we will use antibodies to
determine the presence of an
antigen (H1N1).
Immunologists inject
chickens, goats, rabbits or
sheep with the antigen
and then harvest the
antibodies in their blood to
use as a diagnostic tool in
the lab.
Introduction to ELISA

The antibodies used to recognize
antigens are called primary
antibodies.
•Secondary
antibodies, which
come from another
species, bind to
primary antibodies
when injected.
Let’s See How ELISA is used in
determining Pregnancy…

http://www.sumanasinc.com/webcontent/
animations/content/pregtest.html
Day #2: H1N1 Genetics & Fluid
Transfer

The ‘swine flu’ is actually a
combination of avian (bird) and
swine influenza genes.
Why H1N1?

Influenza and their subtypes are
named & classified based on their
surface proteins.
•Both Hemagglutinin
and Neuramindase are
surface proteins found
on the influenza virus.
Influenza Virus
Surface Proteins

Hemagglutinin – an
antigenic glycoprotein


Binds the virus to the
cell it’s trying to
infect
Neuramindase –
allows the virus to
inject it’s viral
genome into the host
and replicate
Did You Know…

The ‘swine flu’ has
never been isolated from
pigs!!!


The genes of human
‘swine flu’ VERY SIMILAR
to swine influenza thus the
media dubbing it ‘swine
flu’.
Therefore, it is unknown if
the swine flu is actually
zoonotic.
One Possibility
So if it didn’t come directly from
pigs…where the heck did it come from?


Genetic re-assortment
occurs when more
than 1 virus infects a
cell
Viral DNA/RNA gets
mixed & matched up
giving various genetic
combinations
ELISA Assay Overview: Step #1
1.
2.
Obtain a test-sample
Label the 12-well strip:
First 3 wells: positive controls “+”
Next 3 wells: negative controls “-”
Remaining wells to identify testsamples
Proteins in the
samples will bind
to the wells via
hydrophobic
interaction.
ELISA Assay Overview



Microplate strips are made
of polystyrene
Hydrophobic side chains in
amino acids bind to the
polystyrene wells
No special coating is needed
ELISA: Step #2

Remove samples from wells by firmly tapping them on
a paper towel

Discard the top paper towel

Using a disposable transfer pipette wash wells with
wash buffer

Remove wash buffer by firmly tapping the wells on a
paper towel

Discard the top paper towel

Repeat wash step
Step #3: Add controls to your samples

Add 50 ul of positive control to 1st 3 wells

Add 50 ul of negative control to 2nd 3 wells



Add 50ul of student sample A which represents
students serum sample to 3rd set of 3 wells
Add 50ul of other student sample B which
represents that student’s serum sample to last 3
wells
Samples are left in wells for 5 minutes.
Step #4: Wash antibody & add
enzyme-linked antibody




Wash the primary antibody from
polystyrene wells as before
WASH 2X
Add 50ul of the enzyme-linked secondary
antibody to each well
Wait 5 minutes
Step #4: Add enzyme substrate

Wash the enzyme-linked secondary antibody from
polystyrene wells as before

Using a disposable transfer pipette wash wells with
wash buffer

WASH 3X

Add 50ul of the enzyme substrate to each well

Wait 5 minutes

positive samples
will
begin to turn blue
What Reagents Are We Using?
Purified
Antigen: Chicken gamma globulin
Primary
antibody (Serum Samples): Polyclonal
anti-chicken antibody made by rabbits
Secondary
antibody (enzyme-linked): 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
Results?????
Any Questions?