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ELISA Immuno Explorer TM Influenza Diagnostic Tool

ELISA Immuno Explorer TM Influenza Diagnostic Tool. ELISA Immuno Explorer TM Kit Influenza Diagnostic Tool Instructors. Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown

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ELISA Immuno Explorer TM Influenza Diagnostic Tool

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  1. ELISA Immuno ExplorerTM Influenza Diagnostic Tool

  2. ELISA Immuno ExplorerTMKitInfluenza 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. sherri_andrews@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

  3. WhyTeach 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

  4. 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

  5. WorkshopTime Line • Introduction • Rapid Influenza Diagnostic Test (RIDT) • Viruses, influenza, and H1N1 • Ways the ELISA Immuno Explorer Kit can be used

  6. Lab Scenario • A room full of sick people (you guys!) • Various symptoms • Coughing • Sneezing • Temperature • Other nasties! (what are you doing here, anyway?)

  7. Question: • Is this the 2009-2010 pandemic H1N1? • Food poisoning? • Cholera? • Or lots of psychosomatic symptoms (because the person next to you is sick)?

  8. Solution:PerformRapid 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.

  9. Heavy chain Light chain Disulfide bonds Immunoglobulin (IgG) Structure ELISAEnzyme-LinkedImmunosorbantAssay Influenza Antigens

  10. RIDT detects viral antigens 1) Load samples & controls into wells Wash 2) Add primary antibody to all wells Wash 3) Add enzyme-linked secondary antibody to all wells Wash 4) Add enzyme substrate to all wells

  11. ELISA ANIMATION

  12. Laboratory Quick Guide For Protocol II

  13. Steps 1 – 2Label wellsof 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

  14. Steps 3 – 6Add 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

  15. 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

  16. Influenza species(antigen types)5 genera, but only 3 of interest to usEach 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

  17. Steps 7 – 8Wash 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

  18. 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.

  19. 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

  20. Chemistry in action….Or…Ask your friendly chemist…about detergents.

  21. 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!

  22. More about detergent termsLipophilic portion is also referred to as “hydrophobic” tailHydrophilic portion is also referred to as “polar” headTypes: nonionic, anionic, cationic and zwitterionic

  23. SDS Triton X-100 Detergents: Ionic vs non-ionicDenaturing vs non-denaturingSwords (denaturing): “pointy” hydrophobic ends, ionic polar endsGloves (non-denaturing): bulky, non-penetrating hydrophobic ends, non-ionic or zwitterionic polar ends.

  24. Steps 11 – 13Wash & 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

  25. 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

  26. 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

  27. Results • Some positive by RIDT • Some negative • Did the controls work?

  28. (+) for Flu B (+) for Flu A Interpretation: Influenza B infection likely Interpretation: Influenza A infection likely. Could be H1N1/09, seasonal H1N1, or H3N2 Interpretation: Cannot rule out influenza infection CDC guidelines for RIDTs (-) 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

  29. What about H1N1? • RIDT’s do not distinguish H1N1 specifically from other Type A Flu viruses.

  30. 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)

  31. 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

  32. 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

  33. 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) • 9NA subtypes (N1-N9)

  34. 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

  35. 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

  36. 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.

  37. 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.

  38. 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 vaccine- just separately!

  39. 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

  40. Ways The ELISA Kit Can Be Used

  41. Webinars • Enzyme Kinetics — A Biofuels Case Study • Real-Time PCR — What You Need To Know and Why You Should Teach It! • Proteins — Where DNA Takes on Form and Function • From plants to sequence: a six week college biology lab course • From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars

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