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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 The human body has developed intricate means of defense against infections, tissue damage, and abnormal body cells. Outline •Non-specific immunity •Specific immunity •Immunoglobulin Structure and Function •Enzyme Linked Immunosorbent Assay Non-specific Immunity 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 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. 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 Non-phagocytic Cells Killing is by means other than phagocytosis • • Eosinophils – effective against larger parasites; attach to parasite and discharge destructive enzymes http://www.som.tulane.edu/classware /pathology/Krause/Blood/BL11a.html 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 Complement System • • • Made up of about 20 serum proteins Form pores in microbial cells that cause them to lyse Also functions in Specific Immunity Interferons • • Proteins secreted by virus-infected cells Inhibit virus reproduction in neighboring cells Specific Immunity 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 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 Clonal Selection • B-lymphocyte binds antigen • Stimulates reproduction of Bcells • B-cell differentiates into memory cells and plasma cells –Plasma cells produce soluble antibody –Memory cells display antibody on surface Immunoglobulins: Structure and Function Immunoglobulin Structure • Heavy & Light Chains • Disulfide bonds – Inter-chain – Intra-chain Disulfide bond Carbohydrate CL VL CH2 CH1 VH Hinge Region CH3 Immunoglobulin Structure Disulfide bond • Variable & Constant Regions – VL & CL – VH & CH Carbohydrate CL VL • Hinge Region CH2 CH1 VH Hinge Region CH3 Immunoglobulin Fragments: Structure/Function Relationships Ag Binding Complement Binding Site Binding to Fc Receptors Placental Transfer Enzyme-Linked Immunosorbent Assays (ELISA) ELISA Used for Ab detection • 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 Ab-enzyme conjugate X Ab in patient’s sample Immobilized Y Ag ELISA 1/512 1/256 1/128 1/64 1/32 1/16 1/8 1/4 Patient # 1 2 + Control Control 1/2 Dilutions of patient sample are placed in adjacent wells of microtiter plate More intense color = more Ab present Antibody-Antigen Interaction Nature of Ag/Ab Reactions • Lock and Key Concept http://www.med.sc.edu:85/chime2/lyso-abfr.htm • Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds • Multiple Bonds • Reversible Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000