2. ELISA Immuno Explorer TM Influenza Diagnostic Tool

3. 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 ELISA Immuno Explorer TM Kit Influenza Diagnostic Tool Instructors

4. Why Teach 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

6. 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

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

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

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

10. Solution: Perform Rapid 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.

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

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

13. ELISA ANIMATION

14. Laboratory Quick Guide For Protocol II

15. Steps 1 – 2 Label wells of 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 1Sample 2

16. Steps 3 – 6 Add 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

17. 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

18. Influenza species (antigen types) 5 genera, but only 3 of interest to us Each 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

19. Steps 7 – 8 Wash 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

20. 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. Steps 9 – 10 Add primary antibody

21. 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

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

23. 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!

24. More about detergent terms Lipophilic portion is also referred to as “hydrophobic” tail Hydrophilic portion is also referred to as “polar” head Types: nonionic, anionic, cationic and zwitterionic

25. Detergents: Ionic vs non- ionic Denaturing vs non-denaturing Swords (denaturing): “pointy” hydrophobic ends, ionic polar ends Gloves (non- denaturing): bulky, non- penetrating hydrophobic ends, non-ionic or zwitterionic polar ends. SDS Triton X-100

26. Steps 11 – 13 Wash & 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

27. 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

28. 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

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

30. What are viruses? Small organisms that can replicate only within other organisms Millions of types (only a handful characterized) All have genetic material (either RNA or DNA) and a protein coat that protects the nucleic acids Viral genome can code for as few as 4 or as many as 100+ proteins

31. CDC guidelines for RIDTs (-) for Flu A & B (+) for Flu B (+) for Flu A 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

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

33. What do the results mean? All 3 RIDTs detect viral nucleoprotein antigen RIDTs can: – 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

34. 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)

35. 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

36. 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

37. 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) 9 NA subtypes (N1-N9)

38. Viral serotypes 16 different HA subtypes (H1 to H16) – Different subtypes differ by 30% in sequence 9 different NA subtypes (N1 to N9) All subtypes have been isolated from wild aquatic birds (but avian infections are normally asymptomatic) Gene segments reassort during infection with multiple strains

39. 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

40. Human Influenza Serotypes H1N1: 1918 pandemic, 2009 pandemic H2N2: 1957 Asian Flu H3N2: 1968 Hong Kong Flu H5N1: avian influenza Others: – H7N7 – H1N2 – H9N2 – H7N2 – H7N3 – H10N7

41. 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

42. 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.

43. Viral reassortment Eurasian swine influenza strain Classical swine H1N1 influenza strain Human H3N2 influenza strain Avian influenza strain North American H3N2 & H1N2 influenza strain Human 2009 H1N1 influenza strain

44. 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.

45. 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!

46. So, what happened in 2009? Since it was believed that the virus would be a major component of the 2009-10 flu season, it was decided to make an additional monovalent influenza vaccine H1N1/09 is forecast to be prominent in the 2010-11 flu season, and it will be included in the annual, trivalent influenza vaccine

47. 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

48. 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 Influenza, HIV, smallpox, West Nile and Flu viruses Uses for antibodies in research, medicine, and consumer goods III Detecting antibodies in serum HIV, Lyme disease, trichinosis, West Nile virus, and Flu virus Detecting exposure to disease causing agents

49. Two types of vaccines – “flu shot”- prepared from inactivated viruses – nasal spray- prepared from live, weakened (attenuated) flu viruses

50. References Garten, J. et al. 2009. Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans. Science 325, 197-201. Ghedin, E. et al. 2005. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature 437: 1162–1166. Index of Viruses - Orthomyxoviridae (2006). In: ICTVdB - The Universal Virus Database, version 4. Büchen-Osmond, C (Ed), Columbia University, New York, USA. http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_index.h tm http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_index.h tm Peiris, JSM. 2009. Avian influenza viruses in humans. Rev. sci. tech. Off. Int. Epiz. 28:161-174. Strauss, JH, & Strauss, EG. 2008. Viruses and human disease. Academic Press: Boston. Taubenberger et al. 2005. Characterization of the 1918 influenza virus polymerase genes. Nature 437: 889-893.

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