ELISA Immuno Explorer CCTCA February 12, 2016 Inspire your students – tomorrow’s researchers

ELISA Immuno Exlorer TM HIV/AIDS Diagnostic Tool Bob Malyk BSc., MEd. Biology Teacher (retired) Ridley College St. Catharines, ON Canada Bio-Rad (Canada) Life Science Education – Biotechnology Explorer Consultant

Why Teach ELISA and HIV? 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 – Biodefense – 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 $ ($3.77 / student) Classroom Safe

Workshop Time Line Introduction Human Immunodeficiency Virus ELISA-HIV Test Ways the ELISA-Immuno Explorer Kit can be used

Scenario I ELISA Tracking disease outbreaks Step 1. Classroom exchange of simulated bodily fluids Step 2. Antigen detection ELISA protocol Step 3. Track disease transmission Real World Application: HIV West Nile Common cold Cholera Smallpox Anthrax Influenza STDs

Scenario II ELISA Antigen Detection Test ELISA for detecting specific antigens in a sample. Real World Application: Pregnancy Drug abuse GMO Allergen tests Air, food and water testing HIV, smallpox, West Nile and SARS viruses

Scenario III ELISA Antibody Test ELISA for diagnosing exposure to a disease by testing for the presence of antibodies to the disease in a blood sample Real World Application: HIV Lyme disease West Nile SARS

ELISA Procedures Overview Add the purified antigen to all the wells. Incubate for 5 min. Rinse Add serum antibodies (student samples) to the appropriate wells. Incubate for 5 min. Rinse Add the enzyme-linked antibody to all wells. Incubate for 5 min. Rinse Add enzyme substrate to all wells. Incubate for 5 min.

ELISA ANIMATION Click here

Laboratory Quick Guide

Volume Measurement

TV or real life? Feb 11, 2014 Atlanta The Walking Dead show

Zombiefication Method of infection – Bodily fluid contact – Airborne – Foodborne – Other? Symptoms – Undead – Hungry for flesh Treatment – Under investigation

An Overview of the protocol Introduction to Immunology, Antibodies and ELISA Fluid swapping and disease spreading! Indirect ELISA to narrow down who were the initial carriers of the disease

Crash Course in Immunology and ELISA

Light chain Heavy chain Disulfide bridges Antigens Antibody Structure ELISA tests are based on immune system antibody molecules

ELISA E nzyme- L inked I mmuno- s orbant A ssay Add the purified antigen to all the wells. Incubate for 5 min. Rinse Add serum antibodies (student samples) to the appropriate wells. Incubate for 5 min. Rinse Add the enzyme-linked antibody to all wells. Incubate for 5 min. Rinse Add enzyme substrate to all wells. Incubate for 5 min.

Time to Swap Fluids and disease!

Swapping Fluids and Disease Part I Swap with 1 person at your table (record who) Add your 750 µl together, mix and each take 750 µl back

Swapping Fluids and Disease Part II Swap with 1 person at the closest table (record who) Add your 750 µl together, mix and each take 750 µl back

Swapping Fluids and Disease Part III Swap with 1 person at the another table (record who) Add your 750 µl together, mix and each take 750 µl back

ELISA Time! Testing for who is infected

ELISA Time! Testing for who is infected Your ID #Partner ID # Label Wells

ELISA Time! Testing for who is infected Add 50 µl + Control Add 50ul – Control Add 50ul of Bodily Fluid (you + partner)

RR HH Protein (Antigen) Hydrophobic interactions Concept Corner ELISA Time! Testing for who is infected Antigen = chicken gamma globulin

RR HH Polystyrene (plate) Protein (Antigen) Hydrophobic interactions Concept Corner Hydrophobic side chains in amino acids bind to the polystyrene wells

Incubate: 5 minutes ELISA Time! Testing for who is infected

Tap out liquid ELISA Time! Testing for who is infected

Wash with Buffer (contains Tween20)

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 removes non-specifically bound proteins and coats wells that acts as a blocking agent to reduce background Antibody will only bind to the simulated HIV antigen

ELISA Time! Testing for who is infected Tap out liquid Repeat 1x

ELISA Time! Testing for who is infected Add 50 µl “PA” (Primary Antibody) Primary Antibody = anti-chicken polyclonal antibody

Incubate: 5 minutes ELISA Time! Testing for who is infected

Tap out liquid

ELISA Time! Testing for who is infected Wash with Buffer (contains Tween20)

ELISA Time! Testing for who is infected Tap out liquid Repeat 2x

ELISA Time! Testing for who is infected Add 50µl “SA” Secondary Antibody = goat anti-rabbit conjugate to HRP HRP (Horseradish Peroxidase) for colormetric detection

Incubate: 5 minutes ELISA Time! Testing for who is infected

Tap out liquid

ELISA Time! Testing for who is infected Wash with Buffer (contains Tween20)

ELISA Time! Testing for who is infected Tap out liquid Repeat 3x

Antibody Specificity Secondary antibody (enzyme-linked antibody) will only bind to the primary antibody (serum antibody) Secondary antibody specifically recognizes the constant region of the primary antibody In which wells do you predict this is happening?

ELISA Time! Testing for who is infected Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) a colorless solution that when oxidized by HRP turns blue Add 50 µl enzyme “sub” Amplification of signal = +

Incubate: 5 minutes Watch for blue colour change = positive ELISA Time! Testing for who is infected

Deducing who was originally infected Everyone Stand UP! If your ELISA was negative sit down

Deducing who was originally infected  Round 1 – If someone you shared with just sat down, also sit down  Round 2 – If someone you shared with just sat down, also sit down  Round 3 – If someone you shared with just sat down, also sit down

Who is left? Deducing who was originally infected

Using Zombies to Model the Spread of an Infection!

The Zombie Autopsies by Dr. Steven Schlozman, MD

The Scenario A new virus has been discovered that affects the brains of humans, turning them into zombies! Epidemiologists are trying to figure out what to do to help stop the outbreak. The virus is airborne, being transported in saliva droplets of affected individuals. The zombies are attacking healthy humans as their primary food source and new zombies are showing up at an alarming rate.

Connections to Solid Science Practices Asking questions and defining problems – What happens to the brain to cause “zombification”? – How does a disease spread through a population? Using mathematical and computational thinking – Graphically and numerically evaluate the spread of a disease in a population Developing and using models – Brain models – Graphical representations of data Analyzing and interpreting data – Using mathematics and graphical literacy to evaluate data

Symptoms of the Infection Affected Cerebellum: Zombies clumsily shuffle forward. Affected Hypothalamus: Zombies have insatiable appetites. Affected Frontal Lobe: Zombies are poor problem solvers. Affected Amygdala: Zombies aren't nice. They are full of rage.

Normal Dude Pre- Infection

Stage 1 – Infected Dude Early Infection

Stage 2 – Sick Looking Dude

Stage 3 - Creepy Looking Dude

Stage 4 – Trying-to- Eat-You Dude Full-Blown Infection!!

Graphing the Infection Spread

Zooming In For a Better Look

Fill the Blocks With Zombies!!

Stocking Up For the “Z” Apocalypse!

Hmmm….Why are the Graphs Curvy???

Human Immunodeficiency Virus (HIV) First diagnosed in 1981 Over 20 million deaths worldwide, over a half million in the United States Over 40 million currently infected, over a million in the United States Half of all new infections are in people younger than 25 Education has been effective in limiting the spread of HIV/AIDS

HIV Biology What do we know? HIV is an RNA Retrovirus Transmitted by exchange of body fluids, sharing needles, or blood transfusion Infects T-Cells in the immune system and thus destroys the immune system Flu-like symptoms within 1-2 months followed by latent period of up to 10 years HIV may have spread from an animal host to humans Treated but not cured by drugs which inhibit the action of HIV enzymes High error rate of replication (1/2000 nucleotides)

ELISA E nzyme- L inked I mmuno s orbant A ssay Light chain Heavy chain Disulfide bonds Influenza Antigens Immunoglobulin (IgG) Structure

Immune Response A. Pathogen C. Macrophage D. Macrophage E. Macrophage F. T cell B. B cells G. B cell H. Memory B cells I. Plasma cells J. Antibodies attach to pathogen HIV Antibodies are ineffective Against HIV

ELISA-HIV Test Detecting Antibodies in Serum Protocol III After 4-8 weeks of exposure to the HIV virus, the body will have produced a detectable level antibodies (immune response) against HIV ELISA (HIV-Test) detects the presence of serum antibodies against HIV protein antigens This is how HIV is detected in clinical laboratories Most common AIDS test

Bio-Rad HIV Clinical Diagnostic Kits HIV can be detected by ELISA or western blot technology. (Both of which are developed using the basis of the mammalian immune system) ELISA tests are very quick. Western Blot tests are slower and more expensive and are used for confirmatory tests. Bio-Rad’s HIV-2 ELISA Kit Bio-Rad’s HIV Western Blot Kit

Ways The ELISA Kit Can Be Used Protocol Type of ELISA Real-World ApplicationObjectives I Tracking outbreaks of disease HIV, Bird Flu and West Nile viruses, common cold, cholera, smallpox, anthrax, and STDs Epidemiology, disease spread, public health II Detecting antigens Pregnancy, drug, GMO and allergen tests Air food and water testing HIV, smallpox, West Nile and Bird Flu viruses Uses for antibodies in research, medicine, and consumer goods III Detecting antibodies in serum HIV, Lyme disease, trichinosis, West Nile virus, and Bird Flu virus Detecting exposure to disease causing agents