Edward Jenner(1749–1822) Often termed the founder of immunology
Louis Pasteur(1822–1895) French. Father of immunology
Immunological reactions [ ANTIGEN-ANTIBODY Reactions]
• Any substance which introduced parenterally into the body, stimulates the production of an antibody with which it reacts specifically and in an observable manner
ANTIGEN
1. Induction of an immune response
2. Specific reaction with antibodies
Antigen
1. Induction of an immune response
2. Specific reaction with antibodies
Antigen
Complete antigen Hapten
Antigen
Epitope or Antigenic determinant:
The smallest unit of antigenicity is known as epitope
Smallest area 4-5 amino acids or monosaccharide residuesSpecific chemical structure, charge and configurationAntibodies are specific for epitopes
Antibodies or Immunoglobulins
Immunoglobulin Provides Structural and Chemical concept
Antibody Is a biological and functional concept
All antibodies are Immunoglobulins, but all Ig may not antibodies
Antigen-Antibody Reaction
• Ag& Ab by definition combines with each other specifically& in an observable manner
The reaction serve several purposes
In vivo In vitro
Immunity , Autoimmune diseases Serological reactions
Strength of Antigen-AntibodyInteractions
The reactions b/w Ag-Ab occur in 3 stages
Primary Secondary Tertiary
General features of Ag-Ab reaction
The reaction is specific
Entire molecule react and not fragment
There is no denaturation of Ag or Ab
Combination occurs at the surface
Combination is firm but reversible
Both Ag and Ab participate in the reaction
Ag-Ab can combine in varying proportions
Measurement of Ag and Ab
• The Ab titer of a serum is the highest dilution
of the serum that shows an observable
reaction with the Ag in the particular test
In terms of MASS(mg nitrogen ) or as units or titer
Sensitivity
• Highly sensitive – False negative reactions are absent or minimal
Specificity
• Highly specific – False positive reactions are absent or minimal
Measurement of Ag and Ab
Precipitation reaction
Agglutination
Complement fixation test
Neutralisation test
Opsonisation
Immunoflurorescence
Radioimmunoassay
Enzyme Immunoassays
Chemiluminescence immunoassay
Immunoelectroblot Techniques
Immunochromatographic tests
Immunoelectronmicroscopic tests
Serological Reactions
Precipitation occurs
Electrolyte
Antibody Soluble antigen
Precipitation reaction
• Flocculation • Medium • The amount of precipitation influenced by
relative proportion of Ag and Ab• Mechanism of precipitation - Prozone - Zone of equivalence - Postzone
Precipitation reaction
Prozone—Ab Excess
Prozone: Too much Antibody relative to Antigen
LATTICE HYPOTHESIS
Precipitation reactions in fluids
Precipitation reaction
Application:
Both qualitative and quantitative tests
Very sensitive in detecting antigens
Forensic application
Precipitation reaction
TYPES
Ring test
Slide test
Tube test
Immunodiffusion
Electroimmunodiffusion
Immunodiffusion
1. Single diffusion in one direction
2. Double diffusion in one direction
3. Single diffusion in two dimension
4. Double diffusion in two dimension
5. Immunoelectrophoresis
Radial Immunodiffusion (Mancini)
• Interpretation– Diameter of ring is
proportional to the concentration
• Quantitative– Ig levels– Screaning sera for Abs
to influenza virus among others
• Method– Ab in gel– Ag in a well
Ag Concentration
Dia
met
er2
AgAgAgAg
Ab in gel
Double diffusion in 2 dimensions
IMMUNOELECTROPHORESIS
• First do electrophoretic separation of a mixture of soluble antigens in a gel medium.
• Individual antigens are detected using an antiserum containing antibodies for all antigens in the mixture diffused at a right angle to the direction of electrophoresis.
Antigen applied to wells;Separation by charge
Antibody in trough, Protein diffusion in gel
Precipitin complex indicating identity and recognition
Immunoelectrophoresis Result1 2
3
Example: Human sera probed with rabbit anti-human antibodies
AgAg
Electroimmunodiffusion
• Development of precipitin lines can be speeded up by electrically
Various methods
A. One dimensional double electroimmunodiffusion
• Counter immunoelectrophoresis
B. One dimensional single electroimmunodiffusion
• Rocket electrophoresis
A. One dimensional double electroimmunodiffusion [Counter immunoelectrophoresis]
Clinical application: detecting various Ags; α-fetoprotein in serum& specific Ags of Cryptococcus & Meningococcus in the CSF
ROCKET ELECTROPHORESIS
AGGLUTINATION REACTIONS
Interaction between antibody and a particulate antigen resulting in visible clumping
Slide agglutination Tube agglutination Antiglobulin test Passive agglutination Haemagglutination
Applications
+
Patient’s RBCs Coombs Reagent(Antiglobulin)
Patient’s Serum
TargetRBCs
+ Step 1
+ Coombs Reagent
(Antiglobulin)
Step 2
Complement fixation test
• The ability of Ag-Ab complex to fix complement is made use of in CFT
• Looking for evidence of infection.
• The test requires five reagents and is carried out in two steps.
• The Wassermann test is one form of complement fixation test: serodiagnosis of syphilis.
NEUTRALISATION TESTS
• Based on the concept that antibodies can neutralize biological activity of many pathogens and their toxins
Viral neutralisation test Toxin neutralisation test
Opsonisation
Heat labile substance
Heat stable substance
Complement Bacteriotropin
Opsonin
Opsonisation
Immunofluorescence
Coons -1942Albert Hewett Coons (1912–1978)
Direct Immunofluorescence test
Indirect Immunofluorescence test
Radioimmunoassay [RIA]
• 1957 – Berson and Yalow
• Application – Hormone - Drugs - Tumour markers - IgE - Viral antigens
Nobel Prize 1977
Radioimmunoassay [RIA]
Enzyme immunoassay
• Enzyme labeled conjugate
• Advantage – Sensitivity - Simplicity - Economy - Absence of radiation - Availability of test kit - Facility for automation
Enzyme immunoassay [EIA]
• Includes all assays based on measurement of enzyme labeled antigen, hepten or antibody
2 types
Homogeneous EIA Heterogeneous EIA
EMIT ELISA
One step Multistep
ELISA
• Technique involves the use of an IMMUNOSORBENT
• Usually done using 96-well microtitre plates suitable for automation
• Application: – Rota virus - Anti -HIV antibody test
ELISA
Enzyme-linked Immunosorbent Assay
Chemiluminescence Immune Assay
• Refers to a chemical reaction emitting energy in the form of light
• Chemiluminescent compounds - LUMINOL - ACRIDINIUM ESTERS
• The method has been fully automated
Immunoelectroblot technique
• Combine the sensitivity of EIA with much greater specificity
• Combination of 3 separate procedures
Separation Blotting EIA or RIA
Immunochromatographic test
• One step• Qualitative • Wide application - Simplicity - Economy - Reliability • HBSAg detection
Immune response
THE IMMUNE SYSTEM
We are constantly exposed to an incredible diversity of bacteria, viruses, and parasites
How does the immune system protect us?
The key isour ability to produce more than 108 distinct antibodies and more than 1012 T-cell receptors
The immune system has 2 arms
Innate immunity
Adaptive immunity
The Immune System is the Third Line of Defense Against Infection
The immune system of the body consists of two major components
B lymphocytes T lymphocytes
Mainly derived from bone marrow
T lymphocytes are of thymic origin
Responsible for the synthesis of circulating, humoral antibodies, also known as immunoglobulins.
Cellmediated immunologic processes
Immune response
• The specific reactivity induced in a host by an antigenic stimulus
2 TYPES:
1. Humoral (AMI)
2. Cellular (CMI)
• The production of antibodies consists of 3 steps
Humoral Immune response(AMI)
Entry Processing Secretion
Antibody production follows a characteristic pattern
Lag phase Log phase Plateau or steady state
Phase of decline
ANTIBODY RESPONSE AFTER EXPOSURE TO ANTIGEN
Fate of antigen in tissues
• Antigens are removed from circulation in 2 phases
Non-immune phase Phase of immune elimination
PRODUCTION OF ANTIBODIES
• Immune response to an antigen is brought about by 3 type of cells
Lymphocytes APC Effector cells
© Ranjith’s
Central Role of Helper T Cells
© Ranjith’s
Cytotoxic T Cells Lyse Infected Cells
Humoral Response to Antigens
Factors influencing antibody production
Genetic Age Nutrition
Route of administration
Size & number of doses
Multiple antigens
Adjuvants Immunosuppressive drugs
Effect of antibody
CELLULAR IMMUNE RESPONSE (CMI)
Specific immune response that do not involve antibodies.
Delayed hypersensitivity
Immunity in infections
Transplantation immunity
Immunological surveillance
Autoimmune disease
Scope of CMI
Induction of CMI
• Antigenic stimulus• Only T-cell dependant antigens lead to CMI• T-cell bears specific receptor on its surface• T cells recognize antigens only when
presented with MHC molecules• Helper T-cells• Cytotoxic T-cells
T Cells Only Recognize Antigen Associated with MHC Molecules on Cell Surfaces
Central Role of Helper T Cells
CYTOTOXIC T CELLS LYSE INFECTED CELLS
Cytokines • Peptide mediators or intracellular messengers
which regulate immunological, inflammatory and reparative host responses
• They are potent hormone like substances
• Produced by lymphocytes, macrophages, platelets & fibroblasts
CYTOKINES - CLASSIFICATION
Interleukins
Colony stimulating factors
Tumour necrosis factors
Interferons
Others
• Source: Macrophages & other cells• IL-1α and IL-1β• Functions: Proliferation & differentiation of T, B and other
cells Pyrogenic Induce acute phase proteins Bone marrow cell proliferation
Interleukin-1
• Source: T-cells• Powerful modulator of the immune response• Functions: Promote growth and differentiation of T and B
cells Cytotoxicity of T and NK cells Secretions of other lymphokines
Interleukin-2[TCGF]
• Stimulates the growth & differentiation of pleuripotent stem cells in the bone marrow
• Responsible for adjusting the rate of production of blood cells
• Treating hematopoietic dysfunctions in infections & malignancies
Colony stimulating factors
• Induce hemorrhagic necrosis in certain tumours
• 2 types: TNFα and TNFβ• Cachectin is TNFα
Tumour necrosis factors
• Originally identified as antiviral agentsα-Produced by leucocytesβ-Produced by fibroblastsγ-Produced by T-cells• Functions: Macrophage activation Antitumour activity
Interferons
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