Hypersensitivity detail briefly describe each and everything un this gile hshsha jsbshs jejsjs jejsjsj jejsjsb jsjsjs shshhs hshsehs jsjsbshbnnnnnnddnsjs jsjsbsjsns jdjdjsjsjsj djsjsn jsjesjsjsh sjs eue djdbudebhd djd djbduj ujjj jdjdjdj djdndj jdjdjd jdjjdjdh jdjdjdhhd jdjdjdjsh ndjdjdhdhb djdjdjjddbndjdnj djjdjdjdh dndn usususu hsjshshsh jshshsbbshb jshshsh jsjsjsh dnafnsnet bsgnafnsn snsnaan arhabs The clinical manifestations of type I hypersensitivity reactions are related to
the biological effects of the mediators released during mast-cell or basophil
degranulation. These mediators are pharmacologically active agents that act on local
tissues as well as on local populations of secondary effector cells, including eosinophils,
neutrophils, T lymphocytes, monocytes and platelets. The mediators thus serve as an
amplifying terminal effector mechanism, much as the complement system serves as an
amplifier and effector of an antigen-antibody interaction. When generated in response
to parasitic infection, these mediators initiate beneficial defense processes, including
vasodilation and increased vascular permeability, which brings an influx of plasma and
inflammatory cells to attack the pathogen. On the other hand, mediator release induced
by inappropriate antigens, such as allergens, results in unnecessary increases in vascular
permeability and inflammation whose detrimental effects far outweigh any beneficial
effect.
The mediators can be classified as either primary or secondary. The primary
mediators are produced before degranulation and are stored in granules. The most
significant primary mediators are histamine, proteases, eosinophil chemotactic factor,
neutrophil chemotactic factor and heparin. The secondary mediators either are
synthesized after target-cell activation or are released by the breakdown of membrane
phospholipids during the degranulation process. The secondary mediators include
platelet-activating factor, leukotrienes, prostaglandins, bradykinins and various
cytokines. The different manifestations of type I hypersensitivity in different species or
different tissues partly reflect variations in the primary and secondary mediators
present.
Biological effects of several mediators:
Histamine:
Histamine, which is formed by decarboxylation of the amino acid histidine,
is a major component of mast-cell granules, accounting for about 10% of granule
weight. Because it is stored
preformed in the granules, its biological effects are
observed within minutes of mast-cell activation. Once released from mast cells,
histamine initially binds to specific receptors on various target cells. Three types of
histamine receptors―designated H1, H2 and H3―have been identified; these receptors
have different tissue distributions and mediate different effects when they bind
Micromeritics - Fundamental and Derived Properties of Powders
immunology.pdf immunity immune system mechanisms
1. At one time or another in life – suffered and recovered from infectious
diseases- cold, flue, measles, mumps
Recovery- bodies are capable of protecting from harmful effects of
infectious agents
System responsible for such protection – immune system
The state or phenomenon of protection – immunity
Study of immune system, immunity and immune mechanisms –
immunology
Immunity is mainly of two types
Innate/ Non-specific immunity
Acquired/ Specific immunity
Innate/ Non-specific immunity- basic/general resistance/ defense to
any disease that a species possesses by birth, four barriers are
Anatomical/ Physical barriers: Includes skin and mucus membrane
Skin: two distinct layers, thin-epidermis, thick- dermis
Epidermis consists of several layers of epithelial cells, outer layers are
dead, keratinized, epidermis completely renewed in 15-30 days
Dermis- CT, blood vessels, hair follicles, sebaceous & sweat glands
Introduction: Types of Immunity
2. Skin is the first line of defense, prevents the entry of pathogens in to deeper
tissues, low pH inhibit their growth as well
Sebaceous glands- sebum, lactic acids & fatty acids- maintain low pH of skin
between 3 to 5 – prevents the growth of most of the pathogenic bacteria
Any wound, abrasions in intact skin leads to the entry of pathogens
Mucus Membrane: Conjunctiva, GIT, RT, urogenital tract protected by mm,
consist of outer epithelial layer and CT
Saliva, tears, mucus secreted by epithelial cells contains antibacterial &
antiviral substances- protects from pathogens
Cilia in RT traps and propels microorganisms
Microorganisms have evolved their own defense mechanisms
Fimbriae or pilli on Neisseria gonorrhoeae interact with glycoprotein &
glycolipid receptors on epithelial cells of mm
Physiological Barriers: includes Temperature pH and soluble factors
Temperature: Many species are not susceptible to diseases- because of their
body temperature e.g. chicken displays natural immunity to Anthrax – 1070F
pH: Gastric acidity – innate barrier as pathogenic bacteria grow at neutral pH
New born more susceptible- stomach contents are less acidic, gastric flora has
not been fully established
3. Soluble factors: also contribute to non-specific immunity
Lysozyme: a hydrolytic enzyme in mucus secretions – cleave peptidoglycan
of bacterial cell wall
Interferons: group of antiviral proteins produced by virus infected cells
Complement: group of heat labile serum proteins- inactive pro-enzyme form
Upon activation- destroy pathogens, help them clear from the body
Endocytic/ Phagocytic barriers: ingestion of extracellular macromolecules
and particles through endocytosis, phagocytosis
Endocytosis- receptor mediated endocytosis or pinocytosis, Phagocytosis-
ingestion of particulate material including whole microorganisms
Break down into simpler products and eliminated from the cell
Barriers created by inflammatory response: Tissue damage caused by
wounds or invasion by pathogenic microorganisms or by variety of agents/
substances like drugs, pollens, inert physical materials such as wood, pieces of
metals etc. can induce a complex sequence of events- inflammation
In 1st century AD- Roman Physician Celsus- four cardinal signs of
inflammation- rubor (redness), tumor (swelling), calor (heat), dolor (pain)- In
2nd century AD Galen added 5th sign- functio laesa (loss of function)
Inflammation- useful process, results in clearance of pathogen followed by
tissue repair & regeneration
4. Acquired or specific immunity: Reflects the presence of functional immune
system capable of specifically recognizing and selectively eliminating the
pathogens
Four features- specificity, diversity, memory, self/non-self recognition
Developed after birth during life time, may be acquired actively or passively
Active immunity: When an individual is exposed to microorganisms/ foreign
substances- immune system responds- usually long lived
Passive immunity: Through the transfer of antibodies- usually short lived
Both types may be acquired either through natural or artificial means
Naturally acquired immunity: Its is of further two types
Naturally acquired active immunity: Person is exposed to an Ag in daily life/
disease
It may be life long in some disease, for few years, sub-clinical infections-
immunity
Naturally acquired passive immunity: Transfer of maternal antibodies
Trans placental transfer- from placenta to fetus before birth- IgG e.g. if mother
is immune to rubella, polio, diphtheria- new born will also be temporarily
immune, Clostrum after birth- rich in IgA, in poultry through egg yolk- IgG,
amniotic fluid- IgA
In mammals e.g. calf, now trans placental transfer, clostrum is the only source,
FCS is free of maternal Abs- used in research
5. Artificially acquired immunity: Its is of further two types
Artificially acquired active immunity: Through vaccination/ immunization,
same mechanism as pathogens but no disease
Vaccine- live attenuated or inactivated bacterial/ viral- polio vaccine, tetanus
toxoids etc.
Artificially acquired passive immunity: Through introduction of already
prepared purified antibodies
Immediate immunity- short lived, Abs neutralizes Ag- catabolized
Half life is usually up to 3 w, ATS 14-21 days
Abs- serum, antiserum is used, study of serum, Ag-Ab interactions- serology
Electrophoresis- two types of serum proteins, albumin & globulins
Globulins- further three types- Alpha, Beta & gamma
Gamma-globulins- Immunoglobulins (Igs)- Abs
Five classes- IgG, IgM, IgA, IgD and IgE
6. Immune system – structurally & functionally diverse cells, tissues & organs-
throughout the body
Immune/ lymphoid organs – 1. Primary 2. Secondary
Primary- appropriate micro-environment- maturation of lymphocytes
Secondary- trap Ag from tissue & vascular spaces- site for interaction of mature
lymphocytes with Ag
Central cells of immune system- lymphocytes- 25% of WBCs in blood and 99% of
lymph
Approximately 1012 in human = brain + liver
Cells of Immune System
Lymphocytes: Only possesses the four attributes, central cells, all other cells play
accessory role- activation of lymphocytes, phagocytosis, secretion of immune
effector molecules like cytokines
20-40% of WBCs, circulate in blood, lymph, migrate to tissue spaces and lymphoid
organs- lymphocytes re-circulation
Three types- function and cell membrane components- B-cells/ B-lymphocytes, T-
cells/ T-lymphocytes, Null cells
All three types- small (6µm diameter), motile, phagocytic cells- Indistinguishable
morphologically
B/T-cells- not interacted with Ag- naïve, virgin, un-primed cells- in Go phase of
cell cycle, if don’t interact with Ag- die apoptosis (short life span-few days to few
weeks)
Cells of Immune System
7. Interaction of B/T cell with Ag- enter in to cell cycle from Go to G1 (early &
late gene activation stage)- S-stage (lymphoblast stage- 15µm), S-synthesis of
DNA, finally M-division stage- proliferate & differentiate into effector cells &
memory cells
Effector cells- short life span, B-cell lineage- plasma cells (Ab secreting cells)
T-cell lineage- TH cells and CTL-cytotoxic T lymphocytes
Memory cells- long lived cells- Go phase- activated with second encounter of
same Ag
B-lymphocytes: name- site of maturation, bone marrow in mammals & bursa
of Fabricus in birds
Distinguished from the BCR- membrane bounded Immunoglobulins (Abs)-
serve as receptor for Ag, B-220- first marker of B-cell lineage
Express class-II MHC molecule – as antigen presenting cells APC
Interaction Ag and BCR- a naïve B-cell together with T-cell & macrophages-
activated and divides in to plasma & memory cells, plasma cells lack
membrane bounded Igs – secrete one of the five classes of Igs (Abs)-
neutralizes Ag- Humoral immune response
T-lymphocytes: name- site of maturation, thymus, have TCR- not membrane
bound Immunoglobulins, protein receptors which recognize an Ag only when
presented along with MHC molecule- MHC- restriction
Fundamental difference- humoral & cell mediated branches of immunity
8. The earliest marker of T-cell lineage – Thy-1, appear during maturation in
thymus & then remains throughout life span
Two sub-populations of T-cells- T-cells that express CD4 receptors (cluster of
differentiation) – recognize Ag associated with MHC-II called CD4+ or TH
cells- MHC-II restricted
T-cells that express CD8 receptors – recognize Ag associated with MHC-I
called CD8+ or TC cells- MHC-I restricted
TH cells proliferate extensively- recognition of Ag-Class-II MHC complex on
APC- secrete many cytokines- activate B-cells, converted to plasma &
memory cells, plasma cells secrete Abs- humoral immune response
TC cells activated- interaction with Ag-Class-I MHC complex on the surface
of self cells (virus infected cell)- generate CTLs which mediate killing of self/
target cells- CMI
Ration of CD4+ : CD8+ cells is 2:1in normal peripheral blood, may be altered in
immunodeficiency or auto-immune diseases
Null cells: Neither have TCR or BCR- lack four attributes
One functional population – NK cells, large, granulated, 5-10% of peripheral
blood lymphocytes, play important role in defense against tumor cells
Interact with tumor cells in two ways: Direct contact in non-specific Ab
independent process, Specific Ab-dependent cell mediated cytotoxicity
9.
10.
11. Mononuclear Cells: include circulating monocytes- blood & macrophages- tissues
Monocyte- macrophage- number of changes
5-10 fold increase in size, intracellular organelles- increase in number & complexity,
cell acquires more phagocytic ability, produces more hydrolytic enzymes and
secrete more soluble factors
Macrophages: two types 1. Fixed macrophages: Takes residence in a particular
tissues and named accordingly Liver- Kupffer cells, CT- histocytes, Lungs-
alveoler macrophages, Kidney- mesangeal cells, Brain- microglial cells
2. Free/ wandering macrophages- move through amoeboid movement throughout
the tissues
Phagocytosis- ingesting exogenous Ag, insoluble particles, injured & dead cells of
host, cellular debris, activated clotting factors etc.
Granulocytes: three types on the basis of cellular morphology & cytoplasmic
staining characteristics
Neutrophils: produced in bone marrow- hematopoiesis
Granulated cytoplasm- stained with both acidic & basic dyes
Polymorphonulear leukocytes- multilobed nuclei
Released in peripheral blood circulation- 7-10 hours, migrate in to tissues- 3 days
life span
First line of defense against infections, first cells to reach at inflammation site
Neutrophilia – acute infection, Phagocytic activity is more than macrophages
Cells of Immune System
12. Eosinophils: Biolobed nucleus, granulated cytoplasm- stained with acidic dye-
eosin Y
Motile, phagocytic, can move from blood to tissues, Phagocytic role- less than
macrophages
Play major role in defense against parasites- eosinophilic cytoplasmic granules
- secrete certain substances- damage parasite membrane
Basophils: Single lobed nucleus, granulated cytoplasm- stained with basic dye-
methylene blue. Non-phagocytic
Cytoplasmic granules- secrete- pharmacologically active substances, play
major role in allergic responses
Migrate in to tissues- mast cells, secrete histamine – development of allergies
Dendritic cells: named- covered with long membranous processes- resembling
dendrites of nerve cells
Express high level of MHC-II- act as APCs help in activation of TH cells, two
types
Non-lymphoid dendritic cells: found in tissues other than lymphoid, named
accordingly, Langerhans cells- epidermis, interstitial cells- heart, liver, kidney,
GIT etc. capture Ag from tissues spaces- regional lymph nodes
Lymphoid dendritic cells: interdigitating dendritic cells- found in T-cell rich
areas of lymphoid organs- spleen, lymph nodes & thymus- act as APCs
Follicular dendritic cells- found in lymphoid follicles of lymph nodes- B-cells
activation
13.
14.
15. Two types- Primary (central) Secondary (Peripheral)
Immature lymphocytes- hematopoiesis become mature and Ag committed within primary lymphoid
organs
In mammals Bone marrow- B-cell maturation and Thymus- T-cell maturation, in birds Bursa of
Fabricus- B-cell maturation as no bone marrow
Secondary organs trap Ag from tissue & vascular spaces- provide a site where mature
immunocompetent lymphocytes can interact with Ag to generate an immune response
Main secondary lymphoid organs are Lymph Nodes and Spleen
GIT, RT possesses MALT including Peyer’s patches, tonsils, adenoids, appendix- mucosal immunity
Primary Lymphoid Organs:
Thymus: flat, bilobed organ-situated above the heart
Each lobe surrounded by fibrous tissue capsule send projections of CT- trabaculae, divide each lobe
into smaller lobules
Outer portion of each lobe- cortex- densely packed with thymocytes (immature T-cells)
Inner portion medulla- sparsely packed with thymocytes
Both cortex and medulla cris-crossed with three dimensional network of thymic stromal cells
composed of epithelial cells, interdigitating dendritic cells, macrophages- make up the framework of
thymus and help in maturation of T-cells
Cortical epithelial cells (nurse cells) have long membranous processes- hold as many as 50
thymocytes
Organs of Immune System
16. Function: Maturation & selection of T-cells- Epithelial cells secrete many
hormones and cytokines (alpha 1 thymosin, beta 4 thymosin, thymopoeitin,
thymulin)
Maturation, proliferation and differentiation of T-cells
Express Ag binding receptors, differentiate in to sub-populations
Selection process: two types Positive selection and negative selection
T-cells bearing receptors that can recognize self MHC molecule are selected
and other are eliminated through apoptosis
Self reactive T-cells- recognize self MHC with self Ag are eliminated
Both as a result of positive and negative selection only those mature T-cells
whose TCR can recognize self MHC molecule along with foreign Ag are
selected and allowed to move into secondary lymphoid organs
About 95-99% thymocytes die through apoptosis
Bone marrow: Site of maturation of B-cells in mammals, Bursa of Fabricus in
birds
Process of B-cells maturation remains unknown
17.
18.
19. Secondary Lymphoid Organs
Lymph Nodes: encapsulated bean-shaped structures containing reticular network- packed with lymphocytes,
macrophages and dendritic cells
Clustered at the junction of lymphatic vessels, first organized lymphoid structure to encounter Ag that enters
in to tissue spaces
Divided into three regions: Cortex, Para-cortex and Medulla
Cortex: Outermost region contains Lymphocytes (B-cells) and macrophages arranged in Primary follicles, B-
cell rich area/ thymus independent area
Following Antigenic challenge- enlarges in to secondary follicles with germinal center
Intense B-cell maturation and differentiation into plasma and memory cells occurs in germinal center
In childeren with B-cells deficiency cortex lacks primary follicles and germinal centers
Para-cortex: situated beneath cortex, contains T-cells and dendritic cells
Dendritic cells express MHC-II molecule and act as APCs for TH, Para-cortex- T-cell rich area/ thymus
dependent area
Medulla: Inner most portion, sparsely packed with lymphocytes, mostly plasma cells actively secreting Abs
Function: Ag carried to LN by lymph to Para-cortex, trapped, processed and presented along with MHC-II by
dendritic cells- resulting in TH activation which activates B-cells
Ag activated TH cells, B-cells moves to primary follicles of cortex, interaction between follicular dendritic
cells, B-cells, TH cells- development of secondary follicle and germinal center
B-cells- plasma and memory cells, plasma cells move to medulla where secrete Abs
Multiple Afferent Lymphatic vessels pierce capsule of LN and empty lymph in sub-capsular sinuses, single
efferent vessel drains lymph away from LN- contains high conc. of Abs & 50 fold more lymphocytes
Blood supply to LN- lymphatic artery, drainage lymphatic vein, extravasation of lymphocytes at the level of
Post-capillary venuoles
Organs of Immune System
20.
21.
22.
23. Spleen: Large, ovoid organ, left side of abdominal cavity, Unlike LN which trap localized Ag
from regional tissue spaces, spleen- adopted to filter blood and trap blood born Ags and thus
respond to systemic infections
Surrounded by capsule- send trabaculae which divides in to compartments, two compartments
red pulp and white pulp separated by marginal zone
Red Pulp: consists of network of sinusoids filled with RBCs and macrophages
Dead and defective RBCs are destroyed in it- graveyard of RBCs
White Pulp: contains peri-arteoler lymphatic sheath (PALS) around the splenic artery mainly
composed of T-lymphocytes, Around the PALS, marginal zone rich in B-cells organized in
primary follicles
Upon Ag challenge primary follicle develop in to secondary follicle containing germinal
center
Unlike LN, no lymphatic supply, blood born Ags are carried to spleen through splenic artery
which empties in to marginal zone
Ag from marginal zone is trapped by dendritic cells- carries it to PALS, where TH are activated,
which then activates B-cells converted in memory and plasma cells
Abs are carried by splenic vein in to the circulation where it interact with Ag- humoral
immunity
Mucosal-associated lymphoid tissues (MALT):
Mucus membranes lining GIT, RT, urogenital tracts- 400m2 area, protected by MALT
Tonsils: lingual- at the base of tongue, palatine- at the side of the tongue, naso-pharangeal
(adenoids)- nasopharynx
Payer’s patches- 30 to 40 nodules along the outer wall of intestine, BALT, RALT, NALT
24.
25. S U B S T A N C E S C A P A B L E O F I N D U C I N G A S P E C I F I C I M M U N E
R E S P O N S E
F O U R I M M U N O L O G I C A L P R O P E R T I E S
Immunogenicity
Antigenicity
Allerogenicity
Tolerogenicity
F O R P R O T E C T I O N A G A I N S T D I S E A S E S – I M M U N O G E N
P R O P E R T I E S O F A N A N T I G E N
Foreignness
Molecular weight – 100,000 Da good, < 5000-10,000 Da poor
Chemical composition – Protein best followed by
polysaccharides, lipids & NA are not alone
Complexity
Recipient genotype, dose and route of administration
ANTIGENS
26. Adjuvants – Latin word adjuvare – to help
Definition – Not immunogen by themselves, tends to increase the
immunogenicity
Examples: Alum, Freund’s incomplete & complete, Liposome & ISCOMs
Used when, low immunogenicity, costly
Epitopes
Haptens
Mitogens: Induce cell division in B and T lymphocytes irrespective of their
antigenic specificity
Polyclonal activators
B-cell mitogens
T-cell mitogens
Both
Lectins, super-antigens
27. A N T I B O D I E S : A N T I G E N B I N D I N G P R O T E I N S
Membrane bounded – B-cells as receptors
Secreted by plasma cells
Effector molecules of humoral immunity - serum
Structure: Monomer, Y-shape, 2 heavy & 2 light chains
Disulfide bonds, Flexible – T-shape, hinge region
Variable region of heavy & light chain on upper ends of Y- arms
Constant region of heavy & light chain on stem and lower ends of
Y-arms
Fab and crystalizable fragment, Amino and carboxylic terminals
Epitopes: Antigenic determinants on Abs – 3 types
Isotypic, idiotypic and allotypic
Immunoglobulins: Structure & Function
28. Classes of Immunoglobulins: Function
Five classes/ isotypes of Igs: IgG, IgM, IgA, IgD and IgE
IgG, IgD and IgE – Monomer, IgA – Dimer, IgM – Pentamer
IgG: Most abundant, 80% of serum, 4 subclasses
IgG1 (9mg/ml), IgG2 (3mg/ml), IgG3 (1mg/ml), IgG4 (0.5mg/ml)
Monomer, can cross the blood vessels and placenta, protects fetus
Protect against circulating bacteria & viruses, neutralize toxins
Trigger complement system, enhances the effectiveness of phagocytic
cells
IgM: Pentamer, 5-10% of serum
Monomer as receptor on B-cells, Pentamer when secreted by plasma
cells
Ist Ig produced in response to Ag
Ist Ig synthesized by neonate
29. Move less freely, remain in blood
Strong activator of complement than IgG
Enhances phagocytic activity
IgA: 10-15% of serum, major secretary Igs (milk, saliva, tears,
mucus)
Blood – mostly monomer, may be dimer, trimer or tetramer
External secretions- always dimer (J-chain & secretary
component)
Mucosal immunity, clostrum – GIT pathogens
IgD: 0.2%, similar to IgG
No known function in serum, receptor on B-cells
IgE: 0.002%, slightly larger than IgG
Binds by Fc portion to basophiles & mast cells
Pollens react – histamine & other chemical mediators release
Allergic responses and defense against parasites
30. TCR are MHC restricted, CD4 cells are MHC-II, CD8 cells are MHC-I
restricted
Antigen presenting cells (APCs), synthesize & express MHC-II and
present the processed Antigenic peptide to CD4 cells
Self cells: synthesize & express MHC-I and present Antigenic peptide to
CD8 cells
APCs: Monocytes, macrophages, B-cells, dendritic cells, Langerhans
cells, thymic dendritic cells, epithelial cells, venular endothelial cells
Antigen Processing:
Extracellular pathogens – exogenous Ag
Intracellular pathogens – endogenous Ag, posses different challenges
Exogenous Ag – processed through endocytic pathway – Ab production
Endogenous Ag – processed through cytosolic pathway – CMI
Antigen Processing and Presentation
31. Endocytic Processing Pathway
APCs – internalize Ag through phagocytosis, endocytosis or both
Macrophages – phagocytosis, other cells - endocytosis
B-cells – receptor mediated endocytosis using BCR
Ag processed in to peptides of 13-18 residues
Three compartments: early endosome (pH 6-6.5), late endosome/
endolysosome (pH 5-6) and late lysosome (pH 4.5-5)
Acid dependent hydrolytic enzymes in each compartments:
proteases, nucleases, lipases, glycosidases, phospholipases,
phosphatases
Two school of thoughts
1-3 hours – from entry till the appearance of processed peptide in
cleft of MHC-II on the surface of same APC
32. Cytosolic Processing Pathway
Endogenous Ag/ Virus – self cells, processed through cytosolic
pathway
Degraded in to peptide of 8-9 residues
Presented by MHC-I to TC cells – activate CTLs - CMI
Cytosolic proteolytic system: Ag binds with carrier proteins
“ubiquitin” carried to proteosome
Large cylindrical particles containing four subunits with a central
hollow of 10-20Ao
Degradation of Ag inside prevents the proteolysis of other
proteins in side cytoplasm
Low Molecular mass proteins (LMP)- alternate to Cytosolic
proteolytic system
33. Antigen Presentation
Assembly & Stabilization: Processed peptides fit in to the cleft of
MHC-I & MHC-II
Both are synthesized within RER
MHC-II: Two chains - alpha & beta – make cleft together
MHC-I: Two chains – large alpha make cleft & small beta2
microglobulin stabilizes it
Assembly of antigenic peptide processed through cytosolic pathway in
to the cleft of MHC-I takes place within RER – Ag moves to RER
It may misfit in to MHC-II – Occupied by third invariant chain
MHC-II moves from RER to Lysosome – invariant chain degraded by
enzymes and Antigen fit in to the cleft of MHC-II
MHC-II & MHC-I along with processed Ag is displayed on the surface
of APCs & Self cell, respectively – presented to CD4 & CD8 cells for
humoral & CMI, respectively
Clinical Applications: For the design of new vaccines, to understand the
mechanism & treatment of certain autoimmune diseases
34. Used in two ways: either for detection of Ag or Ab if one of them
is known
Highly specific, measurement of Ag or Ab – Serology
Three categories: Primary, Secondary & Tertiary binding tests
Primary: ELISA, RIA, IFA/FAT
Secondary: Precipitation & Agglutination tests, CFT
Tertiary: Neutralization & Protection tests
Reagents required: Serum, -200C, heat inactivation at 560C in
water bath
Complement: group of heat labile serum proteins, source guinaea
pig serum
Antiglobulins: Anti-Ab, Anti-species Ab, Anti-isotypic Ab
Monoclonal Ab: highly specific & pure
Antigen-Antibody Interactions/
Immunodiagnostics
35. Primary Binding Tests
Direct: for detection of Ag with known Ab
Indirect: for detection of Ab with known Ag
Named on the basis of Indicator system used
Radio-immunoassays (RIA): Radioisotopes are used as indicator like 3H, C14,
I125
RIA for Ab/ Indirect: Known Ag is impregnated to nitrocellulose filter paper
Unknown serum sample added and incubated
Radiolabeled Anti-Ab are added and radioactivity is observed under X-ray film
RIA for Ag/ Direct or Competitive RIA: Unlabeled Ag will displace
radiolabeled Ag from immune-complexes
Known Ab and Radiolabeled Ag are added in test tube, incubated, radioactivity
of supernatant is observed
Unknown Ag/ sample is added, it will replace the radiolabeled Ag which will
come in supernatant showing radioactivity
Amount of radioactivity is directly proportional to Ag present in sample
Highly sensitive, qualitative as well as quantitative but costly and not safe
36. Immunofluorescence assays (IFA): Fluorochromes/ fluorescent dyes
are used as indicator like FITC, RITC, Oramine etc.
FITC yellow color compound – UV invisible – fluoresce with bright
green color
Direct FAT for Ag: Unknown Ag/ sample is attached on to
nitrocellulose paper
Known Ab conjugated with FITC added, incubated and fluorescence is
observed under UV invisible or fluorescent microscope
Can detect low level of Ag directly from infected body tissues like
Negri bodies in rabbied brain
Indirect FAT for Ab: Known Ag is attached on to nitrocellulose paper
Unknown serum sample is added, incubated
Known Anti-Ab conjugated with FITC added, incubated and
fluorescence is observed under UV invisible or fluorescent microscope
Highly sensitive, qualitative as well as quantitative but costly
37. Enzyme immunoassays (EIA): Enzymes along with respective
substrates are used as indicator like HRP, Alkaline phosphatase etc.
On addition of substrate, color development/ OD value is measured
Direct ELISA for Ag: Known Ab is attached on to the surface of ELISA
plate wells, washed
Unknown Ag/ sample is added, incubated, washed
Known Ab conjugated with enzyme is added, incubated
Substrate added, OD value is observed through ELISA reader
Sandwich ELISA as Ag is sandwiched between two Ab molecules
Indirect ELISA for Ab: Known Ag is attached on to the surface of
ELISA plate wells, washed
Unknown serum sample is added, incubated
Known Anti-Ab conjugated with enzyme added, incubated and OD
value is observed through ELISA reader
Highly sensitive, qualitative as well as quantitative, comparatively
economical, safe and most commonly used
38. Ag or Ab is detected on the basis of the results of primary
reaction
Precipitation Tests: Soluble Ag + Ab, mixture cloudy after few
minutes, precipitate in 1 hour if +ve
Slide and tube precipitation tests
Optimum level of both is required for precipitation to occur
AGID/ AGPT, Immuno-electrophoresis, CCIE, Western blotting
Simple, economical, specific but less sensitive, difficult to read
Agglutination Tests: Particulate Ag + Ab – Agglutination/
clumping
Simple, economical, more sensitive, easy to read
Slide & tube agglutination tests/ macro-agglutination tests
Micro-agglutination tests – Haemagglutination tests: HA, HI,
IHA etc.
Antigen-Antibody Interactions/ Secondary
Binding Tests
39. Complement Fixation Test
Two systems: Test system & Indicator system
Major/ Test system: Ag + test serum + complement
Minor/ Indicator system Sheep RBCs + Amboceptors
If no hemolysis - +ve
If hemolysis - -ve
Tertiary Binding Tests:
Neutralization Test:
To test the neutralization ability of Ab against an Ag in-vitro
Protection Test:
To test the protective ability of Ab against an Ag in-vivo
40. Development of effective immune response – lymphoid cells,
inflammatory & hematopoietic cells
Complex interaction between these cells is mediated by a group
of low molecular weight (30KDa) regulatory proteins - cytokines
Secreted by WBCs, particularly lymphocytes in response to
inducing stimuli
Binds to receptors on target cells, signal transduction, biological
effect
Hormones – messengers of endocrine system, Cytokines –
messengers of immune system
Cytokines may act in three ways
Autocrine: same cell act as cytokine producing & target cell
Paracrine: target cell is near by to producing cell
Endocrine: binding to target cells in distant part of the body
CYTOKINES
41. Cytokine may exhibit four properties
Pleotropy: Cytokine has biological effect on different target cells –
pleotropic e.g. IL-4
Redundancy: Two or more cytokines – same biological effect on single
target cell – redundant e.g. IL-2, IL-4, IL-5
Synergy: When combined effect of two or more cytokines is greater
than their individual effect – synergism e.g. IL4 + IL-5
Antagonism: Effect of one cytokines is inhibited by the other –
antagonistic e.g. IL-4 by IFN-γ
Nomenclature: Previously on source of release e.g. lymphocytes –
lymphokines, monocytes – monokines
Technically incorrect- as monokines and lymphokines also secreted
from other cells
Now named on the basis of function
Interleukins: Cellular communication among leukocytes, IL1 to IL-17...
Interferons: Glycoproteins – in response to virus infection – antiviral
IFN-α, IFN-β, IFN-γ
42. Tumor Necrosis factor: anti-tumor TNF-α, TNF-β
Growth factors: Stimulate the growth of many cells, CSF, G-CSF, M-
CSF, GM-CSF
Transforming Growth factors: TGF-β
Chemokines: Inflammation, IL-8
Structure: Proteins, four structural groups
I: Four α helix – IL2,3,4,5,6,7,10,11,13, G-CSF, GM-CSF, Interferons
II: Long β sheets – TNF-α, β, IL-1, TGF-β
III: Both α helix & β sheets: Chemokines, IL-8
IV: Mosaic, mixed structure- IL-12
Discovery & Purification: up to 1960s chromatography
Disadvantages: low yield- sub-nanomolar level, less purity
Recombinant DNA technology: in 1960 1st compound – cDNA cloning
techniques
To date genes IL-1 to 13, IFNs, TNF, TGF, LIF, Oncostatin M cloned
43. Functions of Cytokines: Main source TH , macrophages, B-cells
Development of humoral and cellular immune responses
Induction of inflammatory response
Regulation of hematopoiesis
Control of cellular proliferation & differentiation
Induction of wound healing
In-vitro diagnostic and in-vivo therapeutic uses
Cytokines antagonists: Inhibit the action of cytokines
Binds directly to cytokines
Binds to their receptors on target cells
44. Antibodies produced – purified Ag - Polyclonal
Advantages in localization, phagocytosis & complement
mediated lysis of Ag
Due to undesirable, non-specific, cross-reacting Ab - In-vitro
diagnostic and in-vivo therapeutic uses
Purified through chromatographic techniques – very difficult
Alternate – purified clones of mono-specific plasma cells in-vitro
– but short life span
Solution by George Kohler & Cesar Milstein in 1975 by fusing
B-cells, myeloma cells – hybridoma, Nobel Prize - 1984
Formation & selection of hybrid cells: 1970s two somatic cells
fused – heterokaryon, fusion by Sendai virus, PEG
Initially 2-5 nuclei, loss of chromosomes until stabilizes
Hybridoma and Monoclonal Antibodies
45. Selection of hybridoma: HAT medium
Denovo pathway: Phosphoribosyl pyrophosphate + uradylate –
nucleotide and DNA - blocked by aminopterin
Salvage pathway: Hypoxanthine + Thymidine – catalyzed by HGPRT +
TK enzymes, only hybrid cell will survive
Production of Monoclonal Antibodies: Three steps
Generating B-cells hybridoma
Screening for Monoclonal antibody specificity
Propagating hybridoma secreting specific monoclonal antibodies
Ag – mice – Primed B-cells from spleen (HGPRT+, Ig+) + Myeloma
cells ((HGPRT-, Ig-, immortal) – PEG – Heterokaryon along with un-
fused B-cell and un-fused myeloma cells
HAT selection – only B-cells – myeloma cell hybridoma will grow
Assay for desired Ab in culture supernatant – ELISA, RIA, FAT
Reclone Ab+ hybridoma: Expand in Tissue culture (10-100µg/ml),
peritoneal cavity of histocompatable mice (1-25mg/ml), Alginate gel
(100 fold more than tissue culture, Fermenter (1000L – 100g in 2w)
46. Purification of Proteins: Can purify any protein out of complex
mixture, present at extremely low level with 100% purity
Previously IFN – chromatographic techniques
Disadvantages – 1% purity, 99% impurities
DS Secher & DC Burke prepared anti-IFN monoclonal antibodies
Fixed on sephadex in an Immuno-adsorbant column
Identification and isolation of lymphocytes sub-populations:
Monoclonal antibodies can be produced to different membrane
bound receptors or proteins – surface of lymphocytes sub-
populations
CD4+ on TH and CD8+ on TC, anti-CD8 Ab conjugated with
FITC and anti-CD4 Ab with PE
Identified and isolated in FAC
Uses of Monoclonal Antibodies
47. Tumor detection and imaging:
Tumor specific membrane proteins/ receptors – only on tumor cells
Monoclonal Ab are prepared against them – detect tumor
Ab are radiolabeled and injected in the body
Used to image metastatic tumors
Tumor Killing: in two ways
Complement mediated lysis: a large no of tumors are resistant
Immunotoxins: ricin, shigella & diphtheria toxins – potent toxins
Two components: Binding polypeptide & inhibitor chain
Binding component is removed & replaced with specific Ab
Monoclonal Ab will guide toxic component to tumor cell
Safe: will not damage the normal cells
Diagnostic Reagents: More than 100 diagnostic agents available
Home pregnancy diagnosis using anti-HCG monoclonal Ab coated
strips
Cancerous T-cells – Thymoma cells
48. Immunity – Active and Passive
Passive – Natural passive – Maternal Ab (tetanus, mumps, polio,
diphtheria), Artificial passive – ATS, anti-snake venom
Active – Natural active – disease, Artificial active –
immunization/ vaccination
Vaccine – Live modified or killed whole or a part of
microorganism which when administered in to the body do not
cause disease rather elicit a high humoral or CMI
As prophylaxis – infectious diseases
Whole organism vaccines – bacterial or viral
Live attenuated vaccines – viral or bacterial
Attenuation – losses pathogenicity, retains immunogenicity
Bacillus Calmattee Guerin (BCG)–M. bovis, increased conc. of
bile – 13 years – live attenuated vaccine against TB, Polio
vaccine – Sabin and Salk strains
VACCINES
49. Advantages: increased, prolonged immunity, no boosters required
Both humoral and CMI
Disadvantages: Reversion back to virulence, stability less
Contaminants may cause problems, post-vaccination reactions
Irreversible attenuation through gene deletion technology, losses
immunogenicity along with pathogenicity
Inactivated bacterial / viral vaccines:
Inactivation - mechanical, chemical
Heat – degradation of epitopes, chemical – formaldehyde, BEI, β-
propiolactone
Advantages: More stable, safe, no reversion back to virulent form
Disadvantages: Require booster, short immunity, less CMI more
humoral immunity
Post-vaccination complications
50. Purified Macro-molecules as vaccines:
Keeping in view the disadvantages of both
Specific surface macromolecules used as vaccine
e.g. Capsular LPS as vaccine against meningitis
Advantages: more specific, less un toward reactions
Disadvantages: activates B-cells directly, no activation of TH cells
Poor immunogenic - LPS + Protein carriers
e.g. Capsular LPS + Tetanus toxoids – more immunogenic,
vaccine against two or more diseases in single shot
But difficulty in obtaining purified macromolecules in large
quantity
51. Recombinant Antigen Vaccine – Most immunogenic Ag is
cloned in to an appropriate cloning vector, expressed in an
expression host and used as vaccine after purification
First ever – FMDV, VPI cDNA – plasmid – E. coli
First in human – HBV – gene for Major surface protein – YAC –
Yeast cells, used after harvesting and purification
Advantages: Specific immune response, no un to ward reaction,
minimum post-vaccination complications, safe , stable
Disadvantages: Exogenous Ag – only humoral immune response
Recombinant Vector Vaccine – Biological vectors are used –
attenuated poliovirus, adenovirus, Salmonella, BCG etc.
Recombinant vaccinia vector vaccine of small pox
Advantages: specific, safe, stable, CMI
Disadvantages: Less immunogenic
RECOMBINANT VACCINES
52. Synthetic peptide vaccine: Most immunogenic peptide is identified
Amino acid sequence of highly conserved region is identified which
must have
Receptors on cell surface
Capable of producing neutralizing Ab
Influenza – HA sialic acid residues
HBV, malaria, diphtheria, HIV in process
Multivalent Subunit Vaccine:
SMAA - Immunogenic peptide of multiple Ag activating B-cells and T-
cells are attached to their Monoclonal Abs fixed on sephadex
Liposomes – Ag is incorporated in to lipid vesicles using detergents
ISCOMs – Ag is incorporated in to protein miscles
Advantages: Better immunogenicity, both humoral and CMI,
53. Anti-idiotypic vaccine:
Unique a.a. sequence of VH and VL chain of Ab not only act as Ag
binding site but also antigenic determinant
Ab against VH and VL chain of a specific Ab can be used as vaccine
Adjuvants:
Freund’s incomplete & Freund’s complete adjuvants
Alum adsorbed vaccines, Liposome and ISCOMs
Oil adjuvants: surfactants to prepare stable emulsion, Tween 80, Span
80, HLB = 7
Methodology:
Isolation, identification, characterization, purification
Seed cultivation, attenuation, inactivation
Quantification (CFU/ml, PFU/ml)
Infectivity testing (LD50/ EID50/ TCID50)
Adjuvant, HLB
Tests (Safety, stability, sterility, potency/ efficacy)
Lyophilization, packing, labeling
Marketing