Immunlogy (As a Speciality only)
Immunology is a branch of biology and medicine that covers the study of immune systems (Defence system) in all organisms. Immunology is molecular & cellular in nature. It contextualizes the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders (such as auto-immune diseases, hypersensitivities, immune deficiency, and transplant rejection; and the physical, chemical, and physiological characteristics of the components of the immune system in vitro, in situ, and in vivo.
Immunology has applications in numerous disciplines of medicine, particularly in the fields of organ transplantation, oncology, rheumatology, virology, bacteriology, parasitology, psychiatry, and dermatology.
The immune system consists of functional thymus, bone marrow, and chief lymphatic tissues such as spleen, tonsils, lymph vessels, lymph nodes, adenoids, and liver. These components are cellular in nature, and embedded or circulating in various tissues located throughout the body.
Topics /Lesson Coverage – Immunology
Chapter 1
Overview of the Immune System
A Historical Perspective of Immunology
Early vaccination studies led the way to immunology
Vaccination is an ongoing, worldwide enterprise
Immunology is about more than just vaccines and
Infectious disease
Immunity involves both humoral and cellular Components
How are foreign substances recognized by the Immune system?
Important Concepts for Understanding the Mammalian
Immune Response
Pathogens come in many forms and must firstbreach natural
barriers
The Immune response quickly becomes tailored to suit
the assault
Pathogen recognition molecules can be conceded in the
germline or randomly generated
Tolerance ensures that the immune system avoids destroying
the host
The Immune response is composed of two interconnected
arms: innate immunity and adaptive immunity
Adaptive immune responses typically generate memory
The Good, Bad, and Ugly of the immune system
Inappropriate or dysfunctional immune responses can result in a
range of disorders
The immune response renders tissue transplantation challenging
Cancer presents a unique challenge to the immune response
Chapter 2
Cells, Organs and Micro-environments of the Immune
System
Cells of the Immune System
Hematopoietic stem cells have the ability to differentiate into
many types of blood cells
Hematopoiesis is the process by which hematopoietic stem cell
Develop into mature blood cells
Cells of the myeloid lineage are the first responder to infection
Cells of the lymphoid lineage regulate the adaptive immune response
Primary Lymphoid Organs-
Where Immune Cells Develop
The bone marrow provides niches for hematopoietic stem cell to
self-renew and differentiate into myeloid cells and B lymphocytes
The thymus is a primary lymphoid organ where T cell mature
Secondary Lymphoid Organs-
Where the Immune Response is initiated
Secondary lymphoid organs are distributed through-out the body
and share some anatomical features
Lymphoid organs are connected to each other and to infected tissue
by two different circulatory systems blood and lymphatic
The lymph node is a highly specialized secondary lymphoid organ
The Spleen organizes the immune response against blood-bone
pathogens
MALT organizes the response to antigen that enters mucosal tissues
The skin is an innate immune barrier and also includes lymphoid
tissues
Tertiary lymphoid tissues also organize and maintain an immune
response
Chapter 3
Receptors and Signaling: B and T-Cell Receptors
Receptor- Ligand Interactions
Receptor-ligand binding occurs via multiple noncovalent bonds
How do we quantitate the strength of receptor-ligand interactions?
Interactions between receptors and ligand can be multivalent
Receptor and ligand expression can vary during the course of an
immune response
Local concentrations of cytokines and other ligands may be
extremely high
Common strategies Used in many Signaling Pathways
Ligand binding can induce conformational changes in and/or
clustering of the receptor
Some receptors require receptor-associated molecules to
signal cell activation
Ligand-induced receptor clustering can alter receptor location
Tyrosine phosphorylation is an early step in many signaling pathways
Adapter proteins gather members of signaling pathways
Phosphorylation on serine and threonine residues is also a common
step is signaling pathways
Phosphorylation of membrane phospholipids recruits PH domain-
containing proteins to the cell membrane
Signal-induced PIP2 breakdown by PLC causes an increase in
cytoplasmic calcium ion concentration
Ubiquitination may inhibit or enhance signal transduction
Frequently Encountered Signaling Pathways
The PLC pathway induces calcium release and PKC activation
The Ras/Map kinase cascade activates transcription through AP-1
PKC activates the NF-kB transcription factor
The Structure of Antibodies
Antibodies are made up to multiple immunoglobulin domains
Antibodies share a common structure of two light chains and
two heavy chains
There are two major classes of antibody light chains
There are five major classes of antibody heave chains
Antibodies and antibody fragments can serve as antigens
Each of the domains of the antibody heavy and light chains
mediate specific functions
X-ray crystallography has been used to define the structural basis
of antigen-antibody binding
Signal Transduction in B Cells
Antigen binding results in docking of adapter molecules and enzymes
into the BCR-lga/lgB membrane complex
B Cells use many of the downstream signaling pathways described
above
B Cells also receive signals through co-receptor
T-Cells Receptor and Signaling
The T-cell receptor is a heterodimer with variable and constant regions
The T-cell signal transduction complex includes CD3
The T-cell co-receptors CD4 and CD8 also bind the MHC
Lck is the first tyrosine kinase activated in T cell signaling
T cells use downstream signaling strategies similar to those of B cells
Chapter 4
Receptors and Signaling: Cytokines and Chemokines
General properties of Cytokines and Chemokines
Cytokines mediate the activation, proliferation and differentiation
of target cells
Cytokines have numerous biological functions
Cytokines can elicit and support the activation of specific
T-cell subpopulations
Cell activation may alter the expression of receptors and adhesion
molecules
Cytokines are concentrated between secreting and target cells
Signaling through multiple receptors can fine tune a cellular response
Six Families of Cytokines and Associated Receptor Molecules
Cytokines of the IL-1 family promote proinflammatory signals
Hematopoietic (Class I) family cytokines share three-dimensional
structural motifs, but induce a diversity of functions in target cells
The Interferon (Class II) cytokine family was the first to be discovered
Members of the TNF cytokine family can signal development, activation,
or death
The IL-17 family is a recently discovered, proinflammatory cytokine
cluster
Chemokines direct the migration of leukocytes through the body
Cytokine Antagonists
The IL-1 receptor antagonist blocks the IL-1 cytokine receptor
Cytokine antagonists can be derived from cleavage of the cytokine
receptor
Some viruses have developed strategies to exploit cytokine activity
Cytokine-Related Disease
Septic shock is relatively common and potentially lethal
Bacterial toxic shock is caused by superantigen induction of
T-cell cytokine secretion
Cytokine activity is implicated in lymphoid and myeloid cancers
Cytokine storms may have caused many deaths in the 1918
Spanish influenza
Cytokine-Based Therapies
Chapter 5
Innate Immunity
Anatomical Barriers to Infection
Epithelial Barriers prevent pathogen entry into the body’s interior
Antimicrobial proteins and peptides kills would be invaders
Phagocytosis
Microbes are recognized by receptors on phagocytic cells
Phagocytized microbes are killed by multiple mechanisms
Phagocytosis contributes to cell turnover and the clearance of
dead cells
Induced Cellular Innate Responses
Cellular pattern recognition receptors activate responses to
microbes and cell damage
Toll-like receptors recognize many types of Pathogen molecules
C-Type lectin receptors bind carbohydrates on the surfaces of
extracellular pathogens
Retinoic acid inducible gene-I-like receptors bind viral RNA in the
cytosol of Infected cells
Nod-like receptors are activated by a variety of PAMPs, DAMPS,
and other harmful substances
Expression of innate immunity proteins is induced by PRR signaling
Inflammatory Responses
Inflammation results from innate responses triggered by infection,
tissue damage or harmful substances
Proteins of the acute phase response contribute to innate immunity
and inflammation
Natural Killer Cells
Regulation and Evasion of Innate and Inflammatory Responses
Innate and inflammatory responses can be harmful
Innate and inflammatory responses are regulated both positively
and negatively
Pathogens have evolved mechanisms to evade innate and
inflammatory responses
Interactions Between the Innate and Adaptive Immune Systems
The innate immune system activates and regulates adaptive
immune responses
Adjuvants activate innate immune responses to increase the
effectiveness of immunizations
Some pathogen clearance mechanisms are common to both innate
and adoptive immune responses
Ubiquity of Innate Immunity
Plants rely and vertebrate immune responses to combat infections
Invertebrate and vertebrate innate immune responses show both
similarities and differences
Chapter 6
The Complement System Major Pathways of Complement Activation
The classical pathway is initiated by antibody binding
The lectin pathway is initiated when soluble proteins recognize
microbial antigens
The alternative pathway is initiated in three district ways
The three complement pathways converge at the formation of the
C5 convertase
C5 initiates the generation of the MAC
The Diverse Functions of Complement
Complement receptors connect compliment-tagged pathogens
to effector cells
Complement enhances host defense against infection
Complement mediates the interface between innate and
adaptive immunities
Complement aids in the contraction phase of the immune
response
Complement mediates CNS synapse elimination
The Regulation of Complement Activity
Complement activity is passively regulated by protein stability
and cell surface composition
The C1 inhibitor, C1NH, promotes dissociation of C1 components
Decay Accelerating Factors promote decay of C3 Convertases
Factor I degrade C3b and C4b
Protectin inhibits the MAC attack
Carboxypeptidases can inactivate the anaphylatoxins C3a & C5a
Complement Deficiencies
Microbial Complement Evasion Strategies
Some pathogens interfere with the first step of immunoglobulin
mediated complement activation
Microbial proteins bind and inactivate complement proteins
Microbial proteases destroy complement proteins
Some microbes mimic or bind complement regulatory proteins
The Evolutionary Origins of the Complement System
Chapter 7
The Organization and Expression of Lymphocyte
Receptor Genes The Puzzle of immunoglobulin Gene Structure Investigators proposed two early theoretical models of antibody
genetics
Breakthrough experiments revealed that multiple gene segments
encode the light chain
Multigene Organization of Ig Genes
Kappa light-chain genes include V,J, and C segments
Lambda light-chain genes pair each J segment with a particular
C segment
Heavy-chain gene organization includes V, D, J and C, segments
The Mechanism of V(D) J Recombination
Recombination is directed by signal sequences
Gene segments are joined by the RAG ½ recombinase combination
V(D)J recombination results in a functional lg variable regiongene
V(D)J recombination can occur between segments transcribed in either the same or opposite directions
Five mechanisms generate antibody diversity in naïve B cells.
T-Cell Receptor Genes and Expression
Understanding the protein structure of the TCR was critical to the process of discovering the genes
The B-chain in gene was discovered simultaneously in two different laboratories
A search for the a-chain gene led to the y-chain gene instead
TCR genes undergo a process of rearrangement very similar to that of lg genes
TCR expression is controlled by allelic exclusion
TCR gene expression is tightly regulated
Chapter 8
The Major Histocompatibility Complex and Antigen Presentation
The Structure and Function of MHC Molecules
Class l molecules have a glycoprotein heavy chain and a small protein light chain
Class ll molecules have two non-identical glycoprotein chains
Class l and ll molecules exhibit polymorphism in the region that binds to peptides
General Organization and Inheritance of the MHC
The MHC locus encodes three major classes of molecules
The exon/Intron arrangement of class l and ll genes reflects their domain structure
Allelic forms of MHC genes are inherited in linked groups called haplotypes
MHC molecules are codominantly expressed
Class l and class ll molecules exhibit diversity at both the individual and species levels
MHC polymorphism has functional relevance
The Role of the MHC and Expression Patterns
MHC molecules present both intracellular and extracellular antigens
MHC class l expression is found throughout the body
Expression of MHC class ll molecules is primarily restricted to antigen-presenting cells
MHC expression can change with changing conditions
T cells are restricted to recognizing peptides presented in the context of self-MHC alleles
Evidence suggests different antigen processing and presentation pathways.
The Endogenous Pathway of Antigen Processing and Presentation
Peptides are generated by protease complexes called proteasomes
Peptides are transported from the cytosol to the RER
Chaperones aid peptide assembly with MHC class l molecules.
The exogenous Pathway of Antigen Processing and Presentation
Peptides are generated from internalized molecules in endocytic vesicles
The invariant chain guides transport of class ll MHC molecules to endocytic vesicles
Peptides assemble with class ll MHC molecules by displacing CLIP
Cross-Presentation of Exogenous Antigens
Dendritic cells appear to be the primary cross-presentation cell type
Mechanisms and Functions of Cross-Presentation
Presentation of Nonpeptide Antigens
Chapter 9
T-Cell Development
Early Thymocyte Development
Thymocytes progress through four double-negative stages
Thymocytes can express either TCRαβ or TCRyδ receptors
DN thymocytes undergo β-selection, which results in proliferation and differentiation
Positive and Negative Selection
Thymocytes “learn” MHC restriction in the thymus
T Cells undergo positive and negative selection
Positive selection ensures MHC restriction
Negative selection (central tolerance) ensures self-tolerance
The selection paradox: Why don’t we delete all cells we positively select?
An alternative model can explain the thymic selection paradox.
Do positive and negative selection occur at the same stage of development, or in sequence?
Lineage Commitment
Several models have been proposed to explain lineage commitment
Double-positive thymocytes may commit to other types of lymphocytes
Exit from the Thymus and Final Maturation
Other Mechanisms That Maintain Self-Tolerance
TREG cells negatively regulate Immune responses
Peripheral mechanisms of tolerance also protect against auto reactive thymocytes
Apoptosis
Apoptosis allows cells to die without triggering an inflammatory response
Different stimuli initiate apoptosis, but all activate caspases
Apoptosis of peripheral T cells is mediated by the extrinsic (Fas) pathway
TCR-mediated negative selection in the thymus induces the intrinsic (mitochondria-mediated) apoptotic pathway
Bcl-2 family members can inhibit or induce apoptosis
Chapter 10
B-Cell Development
The Site of Hematopoiesis
The site of B-cell generation changes during gestation
Hematopoiesis in the fetal liver differs from that in the adult bone marrow
B-Cell Development in the Bone Marrow
The stages of hematopoiesis are defined by cell-surface markers, transcription-factor expression, and immunoglobulin gene rearrangements
The earliest steps in lymphocyte differentiation culminate in the generation of a common lymphoid progenitor
The later steps of B-cell development result in commitment to the B-cell phenotype
Immature B cells in the bone marrow are exquisitely sensitive to tolerance induction
Many, but not all, self-reactive B cells are deleted within the bone marrow
B cells exported from the bone marrow are still functionally immature
Mature, primary B-2B cells migrate to the lymphoid follicles
The Development of B-1 and Marginal-Zone B Cells
B-1 Cells are derived from a separate developmental lineage
Marginal-zone cells share phenotypic and functional characteristics with B-1B cells and arise at the T2 stage
Comparison of B-and T-Cell Development
Chapter 11
T-Cell Activation, Differentiation, and Memory
T-Cell Activation and the Two-Signal Hypothesis
Costimulatory signals are required for optional T-cell activation and proliferation
Clonal anergy results if a costimulatory signal is absent
Cytokines provide Signal 3
Antigen-presenting cells have characteristic costimulatory properties
Superantigens are special class of T-cell activators
T-Cell Differentiation
Helper T cells can be divided into district subsets
The differentiation of T helper cell subsets is regulated by polarizing cytokines
Effector T helper cell subsets are distinguished by three properties
Helper T cells may not be irrevocably committed to a lineage
Helper T-Cell subsets play critical roles in immune health and disease
T-Cell Memory
Naïve, effector, and memory T cells display broad differences in surface protein expression
TCM and TEM are distinguished by their locale and commitment to effector function
How and when do memory cells arise?
What signals induce memory cell commitment?
Do memory cells reflect the heterogeneity of effector cells generated during a primary response?
Are there differences between CD4* and CD8* memory T cells?
How are memory cells maintained over many years?
Chapter 12
B-Cell Activation, Differentiation, and Memory Generation
T-Dependent B-Cell Responses
T-dependent antigens require T-cell help to generate an antibody response
Antigen recognition by mature B cells provides a survival signal
B cells encounter antigen in the lymph nodes and spleen
B-cell recognition of cell-bound antigen results in membrane spreading
What causes the clustering of the B-cell receptors upon antigen binding?
Antigen receptor clustering induces internalization and antigen presentation by the B cell
Activated B cells migrate to find antigen-specific T cells
Activated B cells move either into the extra-follicular space or into the follicles to form germinal centers
Plasma cells form with in the primary focus
Other activated B cells move into the follicles and initiate a germinal center response
Somatic hypermutation and affinity selection occur within the germinal center
Class switch recombination occurs within the germinal center after antigen contact
Most newly generated B cells are lost at the end of the primary immune response
Some germinal center cells complete their maturation as plasma cells
B-cell memory provides a rapid and strong response to secondary infection
T-Independent B-Cell Responses
T-Independent antigen stimulate antibody production without the need for T-cells help
Two novel subclasses of B cells mediate the response to T-Independent antigens
Negative Regulation of B Cells
Negative signaling through CD22 shuts down unnecessary BCR signaling
Negative signaling through the FcyRllb receptor inhibits B-cell activation
B-10B cells act as negative regulator by secreting lL-10
Chapter 13
Effector Responses: Cell-and Antibody-Mediated Immunity
Antibody-Mediated Effector Functions
Antibodies mediate the clearance and destruction of pathogen in a variety of ways
Antibody isotypes mediate different effector functions
Fc receptors mediate many effector functions of antibodies
Cell-Mediated Effector Responses
Cytotoxic T lymphocytes recognize and kill infected or tumor cells via T-cell receptor activation
Natural killer cells recognize and kill infected cells and tumor cells by their absence of MHC class l
NKT cells bridge the innate and adaptive immune systems
Experimental Assessment of Cell-Medicated Cytotoxicity
Co-culturing T cells with foreign cells stimulates the mixed-lymphocyte reaction
CTL activity can be demonstrated by cell-mediated lympholysis
The graft-versus-host reaction is an in vivo indication of cell-mediated cytotoxicity
Chapter 14
The Immune Response in Space and Time
Immune Cell Behavior before Antigen Is Introduced
Naïve lymphocytes Circulate between secondary and tertiary lymphoid tissues
Naïve lymphocytes sample stromal cells in the lymph nodes
Naïve lymphocytes browse for antigen along reticula networks in the lymph node
Immune Cell Behavior during the Innate Immune Response
Antigen-presenting cells travel to lymph nodes and present processed antigen to T cells
Unprocessed antigen also gains access to lymph-node B cells
Immune Cell Behavior during Adaptive Immune Response
Naïve C4+T Cells arrest their movements after engaging antigens
B cells seek help from CD4+ T cells at the border between the follicle and paracortex of the Lymph Node
Dynamic imaging approaches have been used to address a controversy about B-cells behavior in germinal centers
CDB+ T cells are activated in the lymph node via a multicellular interaction
Activated lymphocytes exit the lymph node and recirculate
A summary of our current understanding
The immune response contracts with 10 to 14 days
Immune Cell Behavior in Peripheral Tissues
Chemokine receptors and Integrins regulate homing of effector lymphocytes to peripheral tissues
Effector lymphocytes respond to antigen in multiple tissues
Chapter 15
Allergy, Hypersensitivities, and Chronic Inflammation
Allergy: A Type l Hypersensitivity Reaction
lgE antibody are responsible for type l hypersensitivity
Many allergens can elicit a type l response
IgE antibodies act by cross-lining FCE receptors on the surfaces of innate immune cells
IgE receptor signaling is tightly regulated
Innate immune cells produce molecules responsible for type I hypersensitivity symptoms
Type l hypersensitivities are characterized by both early and late responses
There are several categories of type l hypersensitivity reactions
There is genetic basis for type l hypersensitivity
Diagnostic tests and treatments are available for type I hypersensitivity reactions
The hygiene hypothesis has been advanced to explain increases in allergy incidence
Antibody-Mediated (Type ll) Hypersensitivity Reactions
Transfusion reactions are an example of type ll hypersensitivity
Hemolytic disease of the newborn is caused by type ll reactions
Hemolytic anemia can be drug induced
Immune complex-Mediated (Type lll) Hypersensitivity
Immune complexs can damage various tissues
Immune complex-mediated hypersensitivity can resolve spontaneously
Autoantigens can be involved in immune complex-mediated reactions
Arthus reactions are localized type lll hypersensitivity reactions
Delayed-Type (Type lV) Hypersensitivity (DTH)
The initiation of a type lV DTH response involves sensitization by antigen
The effector phase of a classical DTH response is induced by second exposure to a sensitizing antigen
The DTH reaction can be detected by a skin test
Contact dermatitis is a type lV hypersensitivity response
Chronic Inflammation
Infections can cause chronic inflammation
There are noninfectious causes of chronic inflammation
Obesity is associated with chronic inflammation
Chronic inflammation can cause systemic disease
Chapter 16
Tolerance, Autoimmunity, and Transplantation
Establishment and Maintenance of Tolerance
Antigen sequestration is one to protect self-antigens from attack
Central tolerance limits development of autoreactive T cells and B cells
Peripheral tolerance regulates autoreactive cells in the circulation
Autoimmunity
Some autoimmune diseases target specific organs
Some autoimmune diseases are systemic
Both intrinsic and extrinsic factors can favour susceptibility to autoimmune disease
Several possible mechanisms have been proposed for the induction of autoimmunity
Autoimmune diseases can be treated by general or pathway-specific immunosuppression
Transplantation Immunology
Graft rejected occurs based on immunologic principals
Graft rejection follows a predictable clinical course
Immunosuppressive therapy can be either general or target-specific
Immune tolerance to allografts is favored in certain instances
Some organs are more amenable to clinical transplantation than others
Chapter 17
Infectious Diseases and Vaccines
The importance of Barriers to Infection and the Innate Response
Many viruses are neutralized by antibodies
Cell-mediated immunity is important for viral control and clearance
Viruses employ several different strategies to evade host defense mechanism
Influenza has been responsible for some of the worst pandemics in history
Bacterial Infections
Immune responses to extracellular and intracellular bacteria can differ
Bacteria can evade host defense mechanism at several different stages
Tuberculosis is primarily controlled by CD4* T cells
Diphtheria can be controlled by immunization with inactivated toxoid
Parasitic Infections
Protozoan parasites account for huge worldwide disease burdens
A variety of diseases are caused by parasitic worms (helminthes)
Fungal Infections
Innate immunity controls most fungal infections
Immunity against fungal pathogens can be acquired
Emerging and Re-emerging Infectious Diseases
Some noteworthy new infectious diseases have appeared recently
Diseases may re-emerge for various reasons
Vaccines
Protective immunity can be achieved by active or passive immunization
There are several vaccine strategies, each with unique advantages and challenges
Conjugate or multivalent vaccines can improve immunogenicity and outcome
Adjuvants are included to enhance the immune response to a vaccine
Chapter 18
Immunodeficiency Disorders
Primary Immunodeficiencies
Combined immunodeficiencies disrupt adaptive immunity
B-cell immunodeficiencies exhibit depressed production of one or more antibody isotypes
Disruptions to innate components may also impact adaptive responses
Complement deficiencies are relatively common
Immunodeficiencies that disrupts Immune regulation can manifest as autoimmunity
Immunodeficiency disorders are treated by replacement therapy
Animal models of immunodeficiency have been used to study basic immune function
Secondary Immunodeficiencies
HIV/AIDS has claimed millions of lives worldwide
The retrovirus HIV-1 is the causative agent of AIDS
HIV-1 is spread by intimate contact with infected body fluids
In vitro studies have revealed the structure and life cycle of HIV-1
Infection with HIV-1 leads to gradual impairment of immune function
Active research investigates the mechanism of progression to AIDS
Therapeutic agents inhibit retrovirus replication
A vaccine may be the only way to stop the HIV/AIDS epidemic
Chapter 19
Cancer and the Immune System
Terminology and Common Types of Cancer Malignant Transformation of cells
DNA alterations can induce malignant transformation
The discovery of oncogenes paved the way for our understanding of cancer induction
Genes associated with cancer control cell proliferation and survival
Malignant transformation involves multiple steps
Tumor Antigens
Tumor-specific antigens are unique to tumor cells
Tumor-associated antigens are normal cellular proteins with unique expression patterns
The Immune Response to Cancer
Immunoediting both protects against and promotes tumor growth
Key immunologic pathways mediating tumor eradication have been identified
Some inflammatory responses can promote cancer
Some tumor cells evade immune recognition and activation
Cancer Immunotherapy
Monoclonal antibodies can be targeted to tumor cells
Cytokines can be used to augment the immune response to tumors
Tumor-specific T cells can be expanded and reintroduced into patients
New therapeutic vaccines may enhance the anti-tumor immune response
Manipulation of costimulatory signals can improve cancer immunity
Combination cancer therapies are yielding surprising results
Chapter 20
Experimental Systems and Methods
Antibody Generation
Polyclonal antibodies are secreted by multiple clones of antigen-specific B cells
A monoclonal antibody is the product of a single stimulated B cell
Monoclonal antibodies can be modified for use in the laboratory or the clinic
Immunoprecipitation- Based Techniques
Immunoprecipitation can be performed in solution
Immunoprecipitation of soluble antigens can be performed in gel matrices
Immunoprecipitation allows characterization of cell-bound molecules
Agglutination Reactions
Hemagglutination reactions can used to detect antigen conjugated to the surface of red blood cells
Hemagglutination inhibition reactions are used to detect the presence of viruses and of antiviral antibodies
Bacterial agglutination can be used to detect antibodies to bacteria
Antibody Assays Based on Antigen Binding to Solid-Phase Supports
Radioimmunoassay are used to measure the concentrations of biologically relevant proteins and hormones in bodily fluids
ELISA assays used antibodies or antigens covalently bound to enzymes
The design of an ELISA assay must consider various methodological options
ELISPOT assays measure molecules secreted by individual cells
Western blotting can identify a specific protein in a complex protein mixture
Methods to Determine the Affinity of Antigen-Antibody Interactions
Equilibrium dialysis can be used to measure antibody affinity for antigen
Surface plasmon resonance is commonly used for measurements of antibody affinity
Microscopic Visualization of Cells and Subcellular Structures
Immunocytochemistry and immunohistochemistry used enzyme-conjugated antibodies to create images of fixed tissues
Immunoelectron microscopy used gold beads to visualize antibody-bound antigens
Immunofluorescence-Based imaging Techniques
Fluorescence can be used to visualize cells and molecules
Immunofluorescence microscopy used antibodies conjugated with fluorescent dyes
Confocal fluorescence microscopy provides three dimensional images of extraordinary clarity
Multiphoton fluorescence microscopy is a variation of confocal microscopy
Intravital imaging allows observation of immune responses in vivo
Flow Cytometry
Magnetic Activated Cell Sorting
Cell Cycle Analysis
Tritiated (H) thymidine uptake was one of the first methods used to assess cells division
Colorimetric assays for cell division are rapid and eliminate the use of radioactive isotopes
Bromodeoxyuridine-based assays for cells division used antibodies to detect newly synthesized DNA
Propidium iodide enables analysis of the cells cycle status of cell populations
Carboxy-fluorescein succinimidyl ester can be used to follow cell division
Assays of Cell Death
The 51Cr Release assay was the first assay used to measure cell death
Fluorescently labeled annexin V measures phosphatidyl serine in the outer lipid envelope of apoptotic cells
The TUNEL assay measure apoptotically generated DNA fragmentation
Caspase assays measure the activity of enzymes involved in apoptosis
Biochemical Approaches Used to Elucidate Signal Transduction Pathways
Biochemical inhibitors are often used to identify intermediates in signaling pathways
Many methods are used to identify proteins that interact with molecules of interest
Whole Animal Experimental Systems
Animal research is subject to federal guidelines that protect nonhuman research subjects
Inbred stains can reduce experimental variation
Congenic resistant strains are used to study the effects of particular gene loci on immune responses
Adoptive transfer experiments allow in vivo examination of isolated cells populations
Transgenic animals carry genes that have been artificially introduced
Knock-in and knockout technologies replace an endogenous with a nonfunctional or engineered gene copy
The cre/lox system enables inducible gene deletion in selected tissues
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