Laboratory #2: ELISA Immuno Explorer Wheeler High School The Center for Advanced Studies in Science, Math & Technology Laboratory #2: ELISA Immuno Explorer Lab Timeline: Intro to ELISA H1N1 Genetics ELISA Lab Overview ELISA Assay Post-AP DNA/Genetics – Ms. Kelavkar

Day #1: Introduction to ELISA ELISA stands for enzyme-linked immunosorbent assay Also used to test for pregnancy, drugs and genetically modified organisms (GMO’s) Antibody based test to diagnose disease such as HIV, SARS, 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 disease. Molecules that cause your body to start the immune response are called antigens. Light chain Heavy chain Disulfide bonds 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… One Possibility 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.

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 Obtain a test-sample 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 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?