Laboratory #2: ELISA Immuno Explorer by t8XI89QX

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									 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

                                      •Also used to test
    •Antibody based                   for pregnancy,
    test to diagnose                  drugs and
    disease such as                   genetically modified
    HIV, SARS,                        organisms (GMO’s)
    H1N1, STD’s,
    anthrax


Post-AP DNA/Genetics – Ms. Kelavkar
             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                            Heavy
    disease.                                 chain

                      Disulfide                      Light
   Molecules that    bonds
                                                     chain
    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               One Possibility
    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.
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.    Obtain a test-sample
2.    Label the 12-well strip:
     First 3 wells: positive controls “+”

     Next 3 wells: negative controls “-”

     Remaining wells to identify test-
     samples

                                              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?

								
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