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In Search of the Body’s Antibodies:

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.

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In Search of the Body’s Antibodies:

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  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.som.tulane.edu/classware /pathology/Krause/Blood/BL11a.html http://www.cat.cc.md.us/courses/ bio141/lecguide/unit3/nknomhc.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

  13. 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

  14. 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

  15. Immunoglobulins:Structure and Function

  16. Disulfide bond Carbohydrate CL VL CH2 CH3 CH1 Hinge Region VH Immunoglobulin Structure • Heavy & Light Chains • Disulfide bonds • Inter-chain • Intra-chain

  17. Disulfide bond Carbohydrate CL VL CH2 CH3 CH1 Hinge Region VH Immunoglobulin Structure • Variable & Constant Regions • VL & CL • VH & CH • Hinge Region

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

  19. Enzyme-Linked Immunosorbent Assays (ELISA)

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

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

  22. Antibody-Antigen Interaction

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

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