ELISA Immuno Exlorer TM : Using Antibodies for Diagnosis and Detection

2. ELISA ImmunoExlorerTM: Using Antibodies for Diagnosis and Detection

3. ELISA Immuno ExplorerTMInstructors Sherri Andrews, Ph.D. Curriculum and Training Specialist Bio-Rad Laboratories Damon Tighe, Ph.D. Curriculum and Training Specialist Bio-Rad Laboratories Bill Woodruff Department Head, Biotechnology Alamance Community College

4. WhyTeach ELISA? • Hands-on Immunology • Tangible, visual 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

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. WorkshopTime Line • Introduction • Antigen Detection by ELISA • Antibody detection if time allows • Ways the ELISA-Immuno Explorer Kit can be used • Real-World Examples

8. ELISAEnzyme-LinkedImmunosorbantAssay • Mammalian immune system • Antibody specificity • Biology’s “magic bullet” • Evolved over millions of years • Harness nature’s tool kit • Imagine the applications!

9. Links to the Real World • Mad Cow Disease, SARS, HIV • GMO • Drug and steroid testing • Pregnancy / Reproduction • Biodefense • Cancer treatment

10. Overview • The Immune System • Those mechanisms by which the body protects itself from often damaging (allergy, death) environmental contaminants foreign to the body (antigen – Ag) • Without the immune system we could not survive on earth • Mechanisms • Innate immunity • Born with these elements • Non-specific cellular and molecular • Equally active against all types of foreign molecules • “First line of defense”

11. Acquired immunity • Specific attack against an invader • Requires activation through contact, hence, acquired • Present only in vertebrates • Includes cells and proteins • Basis of vaccination and immunity • Resistant to subsequent attack • As a child get chicken pox, as a parent care for our own children without a repeat episode • Immunity to one disease does not impart immunity to other, unrelated diseases • Cowpox vs smallpox

12. Characteristics of acquired immunity • Self/non-self discrimination • Major aspect to recognize and attack foreigness • Recognize and not attack self (autoimmunity) • Based on Ag-specific receptors • Memory • Anamnestic response • Ability to quickly respond to a foreign molecule that has been seen before • Faster, stronger, longer • Protects from pathogenic (often lethal) organisms • Also, basis of allergic response

13. Specificity • Based on ability to recognize each foreign molecule as a unique structure • Allows to respond only to the appropriate Ag without initiating a generic attack that activates the entire immune response • Immunity to one foreign molecule does not impart immunity to another, unrelated molecule • Involves cell surface molecules (markers) • Bind to opposing molecules in a highly specific fashion • Enzyme – substrate • Ligand (hormone) – receptor • Antigen – antibody

14. Cells of the Acquired Immune Response • Lymphocytes – cells that exhibit specificity • T cells • Develop in the thymus • B cells • Develop in the bone marrow • When B cells proliferate they differentiate into plasma cells that produce and secrete large amounts of Ag specific antibodies (Ab), an immune protein of high specificity

15. FIGURE 1.1. Clonal selection theory of B cells leading to antibody productionInc.

16. Each B cell expresses ~ 1 X 105 surface Ab of the exact same specificity • Surface Ab binds specific Ag => B cell activation => proliferation => plasma cell => secrete Ab to attack and destroy specific triggering Ag • Called an Immune Response (IR) • The antibody • All Abs share two common elements • specific Ag binding sites • Class specific biological functions • Structure • 2 identical light chains • 2 identical heavy chains

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

19. Heavy chain Disulfide bonds Light chain ELISA AntibodyStructure

20. ELISA-HIV TestDetecting Antibodies in SerumProtocol I & III • After 4-8 weeks of exposure to the antigen (virus, bacteria, etc) the body will have produced a detectable level of antibodies (immune response) against it • ELISA detects the presence of serum antibodies against the protein antigens • This is how HIV and other diseases are detected in clinical laboratories • Most common AIDS test

21. ELISA Animation http://www.sumanasinc.com/webcontent/animations/molecularbiology.html

22. ELISA ProceduresOverview

23. ELISA KitWorkstation Inventory Reagents: Yellow tubes Test samples 2 Violet tube (+) Positive control 1 Blue tube (-) Negative control 1 Green tube (PA) Primary antibody 1 Orange tube (SA) Secondary antibody 1 Lab Equipment and Supplies: Microplate strips, pipettor, pipette tips, transfer pipette, wash buffer, paper towels, marking pen

24. LaboratoryQuick Guide

25. Step OneLabel and add controls • 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 • Add 50 ul of positive control to 1st 3 wells • Add 50 ul of negative control to 2nd 3 wells • Add 50ul of the student samples to the appropriately labeled wells • Wait 5 minutes for the antigen to bind

26. Microplate Strips • Microplate strips are made of polystyrene • Hydrophobic side chains in amino acids bind to the polystyrene wells • No coating is needed

27. Step TwoWASH • 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

28. Step Three Add (PA) Primary Antibody • Add 50 ul of the primary antibody (PA) to all 12 wells • Samples are left in wells for 5 minutes • After 5 minutes WASH 2X

29. 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 antigen

30. Step FourWash antibody and add enzyme-linked secondary antibody (SA) • 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

31. 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?

32. Step FiveAdd enzyme substrate(SUB) • 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

33. What are the reagents? Antigen: Chicken gamma globulin Primary antibody (PA): Polyclonal anti-chicken antibody made by rabbits Secondary antibody (enzyme-linked) SA: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP) Enzyme substrate (SUB): 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue

34. ELISA KitResults

35. Ways The ELISA Kit Can Be Used

36. ELISA test forTransmissible Spongiform Encephalopathies (TSEs) Prion Proteins (PrPres and PrPsens) a b • Uses differences in diseased prions vs. normal prions to prepare sample. • Proteinase K only digests normal, not diseased, prions . • ELISA tests for any prion protein • PrPsens • Proteinase K sensitive • Soluble in detergent • PrPres • Proteinase K resistant • Aggregates in detergent

37. TSE test sample preparation • Sample brain tissue • Homogenize brain tissue • Digest with Proteinase K (normal prions are digested, diseased prions are resistant) • Concentrate • Denature Proteinase K • Perform ELISA

38. Protocol II: Antigen Detection ELISA Real-World Application – TSE Test Protocol - ELISA on simulated animal brain samples

39. Real-world Applications of Antibodies Agricultural Uses – Crop-specific disease diagnosis – Animal disease diagnosis – Detection of GM crops – Basic research • Applications • – Dipstick tests/ELISA • – Immunostaining • – Western blotting Bio-Rad’s TSE ELISA Kit

40. DNA RNA Protein ELISA to test for GMOs “Genetically Modified Organism (GMO)" an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination • ELISA can help farmers separate their GMO grain lots from non-GMO grain lots. • ELISA tests are used to identify specific proteins • - Delta-endotoxin Cry1Ab from Bt11 • - glyphosate from Round-up (RR)

41. How to test for GMOs ELISA: Test for presence of proteins expressed from genetic modifications Pro: Quick, inexpensive, low tech Con: Crop specific, protein stability PCR: Test for presence of inserted foreign DNA Pro: ID different GM crops, DNA stability Con: Expensive, timely

42. Example: Pregnancy Test