Gregory E. Steinkraus, Ph.D., ABMM

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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

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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)

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  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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