The document provides an extensive overview of the immune system, detailing the types of immunity (innate and acquired), the various cells and organs involved, and how the body defends against pathogens. It explains the roles of lymphocytes, macrophages, and other immune cells, as well as the processes of maturation and activation within primary and secondary lymphoid organs. Additionally, it describes the mechanisms of both humoral and cell-mediated immune responses, illustrating how the immune system is structured to recognize and eliminate pathogens effectively.

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

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

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

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

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

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