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Gregory E. Steinkraus, Ph.D., ABMM

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Title: Gregory E. Steinkraus, Ph.D., ABMM


1
Biology 320 (Immunology)
  • Gregory E. Steinkraus, Ph.D., ABMM
  • Clinical Director and Chief Scientific Officer
  • Department of Pathology and Laboratory Medicine
  • New Hanover Health Network and
  • Wilmington Pathology Associates and
  • NextWave Diagnostic Laboratories
  • Wilmington, North Carolina
  • Clinical Associate Professor
  • South East Area Health Education Center
  • Department of Internal Medicine
  • University of North Carolina Hospitals and School
    of Medicine
  • Chapel Hill, North Carolina
  • Adjunct Faculty
  • Department of Biology and Marine Biology
  • University of North Carolina at Wilmington
  • Wilmington, NC

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INTRODUCTION TO IMMUNOLOGY
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IMMUNOLOGY AND THE IMMUNE SYSTEM
  • Immunology
  • Study of the components and function of the
    immune system
  • Immune System
  • Molecules, cells, tissues and organs which
    provide non-specific and specific protection
    against
  • Microorganisms
  • Microbial toxins
  • Tumor cells
  • Crucial to human survival

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THE IMMUNE RESPONSE AND IMMUNITY
  • Immune response
  • Innate (non-specific)
  • Adaptive (specific)
  • Primary
  • Secondary
  • Immunity
  • State of non-specific and specific protection
  • Acquisition of Immunity
  • Natural
  • Artificial

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NATURALLY ACQUIRED IMMUNITY
  • Active
  • Antigens enter body naturally with response of
  • Innate and adaptive immune systems
  • Provides long term protection
  • Passive
  • Antibodies pass from mother to
  • Fetus across placenta
  • Infant in breast milk
  • Provides immediate short term protection

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ARTIFICIALLY ACQUIRED IMMUNITY
  • Active
  • Antigens enter body through vaccination with
    response of
  • Innate and adaptive immune systems
  • Provides long term protection
  • Passive
  • Antibodies from immune individuals injected into
    body
  • Referred to as
  • Immune serum globulins (ISG)
  • Immune globulins (IG)
  • Gamma globulins
  • Provides immediate short term protection

11
PRINCIPAL FUNCTION OF THE IMMUNE SYSTEM
  • To protect humans from pathogenic microorganisms
  • Pathogenic microorganisms (Pathogens)
  • Microorganisms capable of causing infection
    and/or disease
  • Infection
  • Ability of pathogen to enter host, multiply and
    stimulate an immune response
  • Disease
  • Clinical manifestations associated with infection

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BACTERIA, VIRUSES, FUNGI, PARASITESOH MY!
  • Streptococcus pyogenes (Group A Streptococcus)
  • Klebsiella pneumoniae
  • Mycobacterium tuberculosis
  • Ebola virus
  • Human Immunodeficiency Virus (HIV)
  • Aspergillus fumigatus
  • Candida albicans
  • Cryptococcus neoformans
  • Cryptosporidium parvum
  • Stronglyoides stercoralis
  • Ascaris lumbricoides
  • Plasmodium falciparum

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DEFENSE MECHANISMS OF THE HUMAN HOST
  • Innate Mechanisms (Innate immunity)
  • First line of defense
  • Non-specific
  • Adaptive Mechanisms (Adaptive immunity)
  • Second line of defense
  • Highly specific with memory
  • Cooperation between mechanisms

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ORIGIN OF CELLS OF THE IMMUNE SYSTEM
  • Derived from common progenitor cell in bone
    marrow
  • Pluripotent hematopoietic stem cell
  • Progenitor Stem Cells
  • Erythroid lineage
  • Erythrocytes and Megakaryocytes
  • Myeloid lineage
  • Monocyte/macrophage, dendritic cells, PMNs, mast
    cells
  • Lymphoid lineage
  • Small and large lymphocytes

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CELLS OF INNATE AND ADAPTIVE IMMUNITY
  • Myeloid Lineage
  • Neutrophil
  • Principal phagocytic cell of innate immunity
  • Eosinophil
  • Principal defender against parasites
  • Basophil
  • Functions similar to eosinophils and mast cells
  • Referred to as
  • Polymorphonuclear leukocytes (PMNs)
  • Nuclei are multilobed (2 to 5)
  • Granulocytes
  • Cytoplasmic granules

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CELLS OF INNATE AND ADAPTIVE IMMUNITY
  • Myeloid lineage
  • Monocytes
  • Leukocytes with bean shaped or brain-like
    convoluted nuclei
  • Circulate in blood with half life of 8 hours
  • Precursors of tissue macrophages
  • Macrophages
  • Mononuclear phagocytic cells in tissue
  • Derive from blood monocytes
  • Participate in innate and adaptive immunity

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CELLS OF INNATE AND ADAPTIVE IMMUNITY
  • Myeloid lineage
  • Dendritic cells
  • Cells with dendriform (star shaped) morphology
  • Interdigitating reticular cells (synonym)
  • Capture and present antigens to T lymphocytes
  • Mast cells
  • Located in mucous membrane and connective tissue
    throughout body
  • Major effector cell in allergy
  • Modulation of initial immune response

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CELLS OF INNATE AND ADAPTIVE IMMUNITY
  • Lymphoid Lineage
  • Large lymphocytes (large granular lymphocytes)
  • Natural killer (NK) cells (CD16, CD56)
  • Innate immunity to viruses and other
    intracellular pathogens
  • Participate in antibody-dependent cell-mediated
    cytotoxicity (ADCC)
  • Small lymphocytes
  • B cells (CD19)
  • T cells (CD3, CD4 or CD8)
  • Adaptive immunity
  • Lymphocytes refers to small lymphocytes

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THE CLUSTER OF DIFFERENTIATION (CD)
  • A protocol for identification and investigation
    of cell surface molecules
  • CD number assigned on basis of 1 cell surface
    molecule recognized by 2 specific monoclonal
    antibodies
  • CD nomenclature established in 1982
  • 1st International Workshop and Conference on
    Human Leukocyte Differentiation Antigens (HLDA)

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THE CLUSTER OF DIFFERENTIATION (CD)
  • CD markers on leukocytes
  • Granulocyte CD45,
    CD15
  • Monocyte CD45,
    CD14
  • T lymphocyte CD45, CD3
  • T helper lymphocyte CD45, CD3,
    CD4
  • T cytotoxic lymphocyte CD45, CD3, CD8
  • B lymphocyte CD45,
    CD19
  • Natural killer cell CD45,
    CD16, CD56, CD3-

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COMPLETE BLOOD COUNT WITH DIFFERENTIAL (CBC WITH
DIFF)


  • References Ranges
  • Erythrocytes (RBC)
    4.0 to 5.4 M/uL
  • Thrombocytes (Platelets) 145
    to 400 K/uL
  • Leukocytes (WBC)
    4.8 to 10.8 K/uL
  • Neutrophils
    40 to 74
  • Band neutrophils
    0 to 9
  • Eosinophils
    0 to 6
  • Basophils
    0 to 1
  • Lymphocytes
    15 to 47
  • Monocytes
    0 to 12

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CASE STUDY MARCH OF 2009
  • 47 year old female presents to ER with 24 hour
    history
  • Fever (101.3 F)
  • Rigors (shaking chills)
  • Headache
  • Chest pain
  • Cough
  • Shortness of breath
  • Admitting diagnosis (?)
  • Diagnostic studies
  • Radiology and laboratory

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CASE STUDY MARCH OF 2009
  • Laboratory studies
  • Blood
  • CBC with differential
  • Basic metabolic panel (BMP)
  • C-reactive protein (CRP)
  • Culture
  • Sputum
  • Culture
  • Radiology studies

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CASE STUDY MARCH OF 2009
  • Laboratory studies
  • CBC with diff P
    RR
  • WBC 40.7 K/uL
    4.8 to 10.8
  • Neutrophils 44
    40 to 74
  • Bands 46
    0 to 9
  • Monocytes 1
    0 to 12
  • Lymphocytes 8
    15 to 47
  • BMP mild elevation of BUN and creatinine
  • CRP 24 mg/dL
    gt 1.0 mg/dL

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CASE STUDY MARCH OF 2009
  • Sputum
  • Gram stained smear
  • 50 WBCs per high power field
  • Numerous gram-positive cocci in pairs and short
    chains
  • Culture
  • Heavy Streptococcus pneumoniae
  • Blood cultures
  • Gram stained smear
  • Gram-positive cocci in pairs and short chains
  • Culture
  • Streptococcus pneumoniae

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CASE STUDY JULY OF 2008
  • 22 year old male presents to ED with 36 hour
    history of
  • Fever (100.4 F)
  • Headache
  • Nausea
  • Vomiting
  • AMS (Irritability)
  • Anorexia
  • Nuchal rigidity
  • Photophobia
  • Admitting and differential diagnosis (?)

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CASE STUDY JULY OF 2008
  • Diagnostic studies
  • Radiology
  • Laboratory
  • Blood
  • CBC with diff, BMP and CRP
  • Cerebrospinal fluid (CSF)
  • Glucose, protein and cells (number and type)
  • Culture
  • Polymerase chain reaction (PCR)

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CASE STUDY JULY OF 2008
  • Laboratory studies
  • Cerebrospinal fluid (CSF)
  • Patient
    Reference Range
  • Cell 465 / uL
    0 - 8 / uL
  • Cell type Lymphocytes (97)
  • Protein 117 mg/dL
    15 45 mg/dL
  • Glucose 43 mg/dL
    40 70 mg/dL

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CASE STUDY FEBRUARY OF 2008
  • 50 year old female presents to ER with 15 to18
    hour history
  • Fever (103.1 F)
  • Headache
  • AMS (Irritability and confusion)
  • Nausea
  • Vomiting
  • Nuchal rigidity
  • Sore throat
  • Photophobia
  • Admitting and differential diagnosis (?)

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CASE STUDY FEBRUARY OF 2008
  • Diagnostic studies
  • Radiology
  • Laboratory
  • Blood
  • CBC with diff, BMP and CRP
  • Cerebrospinal fluid
  • Protein, glucose and cells (number and type)
  • Culture
  • Bacterial antigens (latex agglutination)

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CASE STUDY FEBRUARY OF 2008
  • Blood
  • CBC with diff
  • Leukocytes 30.2
    4.8-10.8 K/uL
  • Neutrophils 88
    40-74
  • BMP (normal)
  • CRP 12 mg/dL
    gt1 mg/dL
  • Cerebrospinal fluid
  • Gram stain
  • 50 WBCs per high power field
  • Numerous gram-positive cocci in pairs and chains
  • Culture
  • Heavy Streptococcus pneumoniae

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CASE STUDY FEBRUARY OF 2008
  • Laboratory diagnosis
  • Cerebrospinal
    Fluid (CSF)
  • Patient
    Reference Range
  • Cell 2,100 / uL
    0 8 uL
  • Cell type Neutrophils (98)
  • Protein 325 mg/dL
    15 45 mg/dL
  • Glucose 10 mg/dL
    40 70 mg/dL

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LYMPHOCYTES, LYMPHOID TISSUES AND ORGANS
  • Lymphocytes originate in bone marrow
  • Lymphoid tissues and organs
  • Primary
  • Development and maturation of lymphocytes
  • Bone Marrow (B cells) and thymus gland (T cells)
  • Secondary
  • Mature lymphocytes meet pathogens
  • Spleen, adenoids, tonsils, appendix, lymph nodes,
    Peyers patches, mucosa-associated lymphoid
    tissue (MALT)

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THE LYMPHATIC SYSTEM
  • Lymph
  • Fluid and cells in lymphatic vessels
  • Lymphatic vessels
  • Collect and return interstitial fluid to blood
  • Transport immune cells throughout body
  • Transport lipid from intestine to blood
  • Lymph nodes
  • Kidney shaped organs at intervals along lymphatic
    vessels
  • Other secondary lymphatic tissues and organs

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LYMPHOCYTES AND THE LYMPH NODES
  • Naïve lymphocytes circulate between blood, lymph
    and secondary lymph nodes
  • Pathogens from infected tissue sites are picked
    up by lymphatic vessels and arrive at closest
    lymph node
  • T and B cells congregate at specific regions of
    nodes
  • Architecture and size of nodes change in response
    to activation of lymphocytes

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LYMPHOCYTES AND THE SPLEEN
  • Spleen
  • Lymphoid organ in upper left abdomen
  • Functions
  • Remove damaged or old erythrocytes
  • Activation of lymphocytes from blood borne
    pathogens
  • Architecture of Spleen
  • Red pulp
  • Erythrocytes removed
  • White pulp
  • Lymphocytes stimulated

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SECONDARY LYMPHOID TISSUES ASSOCIATED WITH MUCOUS
MEMBRANES
  • Primary portals of entry for pathogens
  • Respiratory tract
  • Gastrointestinal tract
  • Secondary lymphoid tissues
  • Bronchial-associated lymphoid tissue (BALT)
  • Gut-associated lymphoid tissues (GALT)
  • Tonsils, adenoids, appendix, Peyers patches
  • Pathogens are directly transferred across mucosa
    by M cells

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THE INNATE IMMUNE RESPONSE
  • Mediated (initiated) by phagocytes, NK cells and
    soluble proteins
  • Phagocytes
  • Cells specialized in the process of phagocytosis
  • Macrophages
  • Reside in tissues and recruit neutrophils
  • Neutrophils
  • Enter infected tissues in large numbers
  • Recognize common molecules of bacterial cell
    surface using a few surface receptors
  • Phagocytosis
  • Capture, engulfment and breakdown of bacterial
    pathogen

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THE INNATE IMMUNE RESPONSE
  • Inflammatory response enhances phagocytosis
    through acute phase proteins
  • Mannose-binding lectin (MBL)
  • Binds to bacterial surface with particular
    spatial arrangement of mannose or fucose
  • C-reactive protein (CRP)
  • Binds to phosphorylcholine on bacterial surface
  • Complement
  • Set of proteins which bind to bacterial surface
  • Inflammatory response
  • Accumulation of fluid and cells at infection site
    (swelling, redness, heat and pain)

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THE ADAPTIVE IMMUNE RESPONSE
  • Creates millions of different B and T cells for
    specific antibody-mediated and cell-mediated
    immunity
  • Antibody-Mediated Immunity (AMI)
  • Involves B lymphocytes, plasma cells and
    antibodies
  • Humoral immunity
  • Name derives from antibodies found in body fluids
    (humors - old medical term)
  • Cell-Mediated Immunity (CMI)
  • Involves T lymphocytes, antigen-presenting cells
    and MHC (major histocompatibility complex)
    molecules
  • Cellular immunity

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ANTIBODY-MEDIATED (HUMORAL) IMMUNITY
  • Directed against extracellular microorganisms and
    toxins
  • B-lymphocytes (B cells)
  • Differentiate into plasma cells which produce
    antibodies
  • Function as antigen-presenting cells (APCs)
  • Classification of Antibodies (Immunoglobulins)
  • Immunoglobulin M (IgM)
  • Immunoglobulin G (IgG)
  • Immunoglobulin A (IgA)
  • Immunoglobulin D (IgD)
  • Immunoglobulin E (IgE)

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CELL-MEDIATED IMMUNITY (CMI)
  • Directed against intracellular microorganisms
  • Non-phagocytic cells and phagocytic cells
  • T-lymphocytes (T cells)
  • Differentiate into effector cells following
    antigen presentation by antigen presenting cells
    (APCs)
  • Functional types of T cells
  • Helper (CD4 T cells)
  • TH1 and TH2 cells
  • Cytotoxic (CD8 T cells)
  • Regulatory
  • CD4 and CD8 Tregs

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THE NATURE OF ANTIGENS
  • Historically named as antibody generators
  • Molecule which stimulates production of and binds
    specifically to an antibody
  • Contemporary view distinguishes between
  • Antigen
  • Molecule which can bind to specific antibody but
    cannot elicit adaptive immune response
  • Immunogen
  • Molecule which can stimulate adaptive immune
    response
  • Best immunogens are proteins with MW gt 10,000

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THE NATURE OF ANTIGENS
  • Carbohydrates, nucleic acids and lipids are also
    potential antigens / immunogens
  • Hapten
  • Small (low MW) molecule unable to elicit immune
    response
  • Combines with larger carrier molecule which
    together function as immunogen
  • Antibody may react independently with hapten
    following hapten/carrier adaptive immune response
  • Example
  • Penicillin G (MW of 372)
  • Albumin (MW of 66,000)

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THE NATURE OF ANTIBODIES
  • Antibodies are glycoproteins
  • Exist as monomers, dimers or pentamers of basic
    structure
  • Basic antibody structure has 4 polypeptide chains
  • 2 identical light chains
  • 2 identical heavy chains
  • Regions of heavy and light chains
  • Variable
  • Constant

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THE NATURE OF ANTIBODIES
  • Also referred to as
  • Immune globulins / Immunoglobulins (IG)
  • Immune serum globulins (ISG)
  • Gamma globulins
  • Contemporary immunology
  • Antibody
  • Secreted form of IG made by plasma cells
  • Immunoglobulin
  • Antigen binding molecules of B cells
  • (B cell antigen receptors)

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CLASSIFICATION OF ANTIBODIES (IMMUNOGLOBULINS)
  • Five (5) classes (isotypes)
  • Immunoglobulin A (IgA)
  • Immunoglobulin G (IgG)
  • Immunoglobulin M (IgM)
  • Immunoglobulin D (IgD)
  • Immunoglobulin E (IgE)
  • Based on structural differences in constant
    regions of heavy chains
  • Classes have specialized effector functions

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B LYMPHOCYTES AND HUMORAL IMMUNITY
  • Originate from stem cells in bone marrow
  • Maturation in bone marrow followed by migration
    to secondary lymphoid tissue
  • Antigen exposure in secondary lymphoid tissue
  • Following exposure to antigen, differentiation
    into plasma cells and memory cells
  • Plasma cells produce antibodies of all IG classes

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ACTIVATION OF ANTIBODY PRODUCING CELLS BY CLONAL
SELECTION
  • B lymphocytes recognize intact pathogenic
    microorganisms and toxins
  • B lymphocytes possess specific surface receptors
    for recognition of specific antigen
  • IgM and IgD
  • Binding of specific antigen results in
    proliferation of a clonal population of cells
  • Antigen determines clonal proliferation

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ACTIVATION OF ANTIBODY PROCDUCING CELLS BY CLONAL
SELECTION
  • Proliferation of activated cells is followed by
    differentiation into
  • Plasma cells
  • Life span of
  • 4 to 5 days
  • 1 to 2 months
  • Produce 2,000 antibody molecules / second
  • Memory cells
  • Life span of years to decades
  • Differentiate into plasma cells following
    stimulation by same antigen

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PRIMARY AND SECONDARY ANTIBODY RESPONSE
  • Primary Response
  • Following exposure to an antigen, there is a slow
    rise in IgM followed by a slow rise in IgG
  • Secondary Response
  • Following exposure to previously encountered
    antigen, there is a rapid rise in IgG and slow or
    no rise in IgM
  • Memory or anamnestic response

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T LYMPHOCYTES AND CELL-MEDIATED IMMUNITY
  • Originate from stem cells in bone marrow followed
    by migration to thymus gland
  • Maturation takes place in thymus gland followed
    by migration to secondary lymphoid tissue
  • Respond to antigens on the surface of antigen
    presenting cells (APCs)
  • Antigen presenting cells (APCs)
  • Macrophages
  • Dendritic cells
  • B lymphocytes

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T LYMPHOCYTES AND CELL-MEDIATED IMMUNITY
  • Antigen presenting cells (APCs)
  • Ingest and process antigens then display
    fragments (short peptides) on their surface in
    association with molecules of major
    histocompatibility complex (MHC)
  • Major histocompatibility (MHC) molecules
  • MHC class I molecules
  • Present antigens to CD8 T cells
  • MHC class II molecules
  • Present antigens to CD4 T cells
  • T cells which encounter antigen differentiate
    into effector T cells

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ROLES OF EFFECTOR T CELLS IN IMMUNE RESPONSE
  • CD8 cytotoxic T cells
  • Enter bloodstream and travel to infection site
  • Kill cells infected with viruses and other
    intracellular microorganisms
  • CD4 TH1 helper T cells
  • Enter blood stream and travel to infection site
  • Help activate macrophages
  • CD4 TH2 helper T cells
  • Work within secondary lymphoid tissues
  • Help activate B cells

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DISORDERS OF THE IMMUNE SYSTEM
  • Hypersensitivity Reactions
  • Over-reaction of adaptive immune response to
    harmless antigens
  • Four Types of reactions (I- IV)
  • Autoimmunity
  • Misdirected adaptive immune response
  • Results from a loss of self-tolerance
  • Three Types (II, III, IV) of reactions
  • Immunodeficiencies
  • Components of immune system either absent or
    defective
  • Genetic or acquired etiology

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IMMUNOLOGY FOR DIAGNOSIS OF DISEASES
  • Analytical methods using either antibody or
    antigen with an indicator system for detecting
    specific
  • Antibodies
  • Detected using antigens or antibody
  • Antigens
  • Detected using antibody
  • Indicator systems
  • Latex particles (colored)
  • Microspheres (colored) conjugated with antibody
  • Enzymes conjugated to antibody
  • Fluorochromes conjugated to antibody
  • Nitrocellulose membranes fixed with antigen or
    antibody

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METHODS IN DIAGNOSTIC IMMUNOLOGY
  • Latex agglutination (LA)
  • Latex particles (dyed) coated with antigen,
    antibody or?
  • Read visually for clumping of latex particles
  • Staphyloslide (Becton Dickinson)
  • Identification of Staphylococcus aureus
  • Staphylococcus aureus produces
  • Coagulase (bound and free)
  • Protein A
  • Blue latex particles coated and not coated with
  • Fibrinogen
  • IgG

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METHODS IN DIAGNOSTIC IMMUNOLOGY
  • Immunochromatographic assay (ICA)
  • Antibody or antigen immobilized (Test line)
  • Antibody immobilized (Control line)
  • Membranes
  • Nitrocellulose, cellulose acetate
  • Read visually for colored test and control lines
  • Examples
  • Group A Streptococcus (GAS) antigen
  • Influenza A and B antigens
  • Respiratory syncytial virus (RSV) antigen
  • Rotavirus antigen
  • HIV-1/2 antibody

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PRINCIPLES OF OraQuick RAPID HIV-1/2 ICA ANTIBODY
TEST
  • Antigens and antibody immobilized onto
    nitrocellulose
  • membrane in T and C zones
  • Test (T) Zone
  • Synthetic peptides from HIV envelope region
  • Control (C) Zone
  • Goat anti-human IgG
  • Developer solution
  • Facilitates flow of specimen onto test strip
  • Rehydrates protein-A gold colorimetric reagent

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IMMUNOLOGY FOR PREVENTION OF DISEASE
  • Hepatitis B
  • Pre-exposure prophylaxis
  • Vaccination with hepatitis B surface antigen
    (HBsAg)
  • Post-exposure prophylaxis
  • Administration of
  • Hepatitis B Immune Globulin (HBIG) Human
  • Purified IgG antibody from plasma of donors with
    high titer of antibody to the hepatitis B surface
    antigen (anti-HBs)

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IMMUNOLOGY FOR TREATMENT OF DISEASE
  • Rheumatoid Arthritis
  • Remicade (Infliximab)
  • IgG kappa monoclonal antibody against tumor
    necrosis factor alpha (TNF-alpha)
  • Breast Cancer
  • Herceptin (Trastuzumab)
  • IgG kappa monoclonal antibody against human
    epidermal growth factor receptor 2 (HER2)

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