1. In Search of the Body’s Antibodies: Investigate Antibodies Using Enzyme Linked Immunosorbent Assay (ELISA) Module developed at Boston University School of Medicine Presented by Dr. Dan Murray

2. The human body has developed intricate means of defense against infections, tissue damage, and abnormal body cells.

3. Outline Non-specific immunity Specific immunity Immunoglobulin Structure and Function Enzyme Linked Immunosorbent Assay

4. Non-specific Immunity

5. Two Lines of Defense Non-specific (innate) Immunity –Body’s response is effective against a variety of “attackers” –Involves antimicrobial cells and proteins Specific (acquired) Immunity –Body’s response is tailored for a specific “attacker” –Involves antibodies

6. Non-specific (innate) Defenses Mediated by host cells –Phagocytosis (by phagocytes) –Non-phagocytic cells Mediated by host proteins –Complement system –Interferons Each of these play a role following a microbial infection and/or a wound to tissue.

7. Phagocytosis Ingestion of infecting microbes by phagocytic white blood cells (i.e., leukocytes) Neutrophils – short-lived; 60-70% of leukocytes Macrophages – long-lived; develop from monocytes

8. Non-phagocytic Cells Killing is by means other than phagocytosis Eosinophils – effective against larger parasites; attach to parasite and discharge destructive enzymes Natural Killer Cells – destroy infected cells or precancerous cells by destroying the cell membrane http://www.cat.cc.md.us/courses/ bio141/lecguide/unit3/nknomhc.html http://www.som.tulane.edu/classware /pathology/Krause/Blood/BL11a.html

9. Complement System Made up of about 20 serum proteins Form pores in microbial cells that cause them to lyse Also functions in Specific Immunity

10. Interferons Proteins secreted by virus-infected cells Inhibit virus reproduction in neighboring cells

11. Specific Immunity

12. Antibody-Antigen Interaction Antigen - any agent capable of eliciting an immune response –Isolated molecules –Molecules on surface of cell or virus A specific antibody molecule will be able to recognize a specific epitope of an antigen –Antibody binds to antigen

14. Clonal Selection The proliferation of lymphocyte cells due to activation by an antigen Useful in primary (first exposure to antigen) and secondary (subsequent exposure to same antigen) immune responses Results in production of many antibodies against the antigen Primary immune response – 10-17 days before maximum response is mounted Secondary immune response – 2-4 days for maximum response

15. Clonal Selection B-lymphocyte binds antigen Stimulates reproduction of B- cells B-cell differentiates into memory cells and plasma cells –Plasma cells produce soluble antibody –Memory cells display antibody on surface

16. Immunoglobulins: Structure and Function

17. Immunoglobulin Structure Heavy & Light Chains Disulfide bonds –Inter-chain –Intra-chain C H1 VLVL CLCL VHVH C H2 C H3 Hinge Region Carbohydrate Disulfide bond

18. Immunoglobulin Structure Variable & Constant Regions –V L & C L –V H & C H Hinge Region C H1 VLVL CLCL VHVH C H2 C H3 Hinge Region Carbohydrate Disulfide bond

19. Immunoglobulin Fragments: Structure/Function Relationships Ag Binding Complement Binding Site Placental Transfer Binding to Fc Receptors

20. Enzyme-Linked Immunosorbent Assays (ELISA)

21. ELISA Used for Ab detection Ag ImmobilizedAb in patient’s sample Ab-enzyme conjugate Immobilize Ag Incubate with patient sample Add antibody-enzyme conjugate Amount of antibody-enzyme conjugate bound is proportional to amount of Ab in the sample Add substrate of enzyme Amount of color is proportional to amount of Ab in patient’s sample X Y

22. ELISA Dilutions of patient sample are placed in adjacent wells of microtiter plate 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Patient # 1 2 + Control  Control More intense color = more Ab present

23. Antibody-Antigen Interaction

24. Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds Reversible Multiple Bonds Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 http://www.med.sc.edu:85/chime2/lyso-abfr.htm