2. Antigen
• Any substance which, when introduced
parentrally into the body stimulates the
production of antibody specifically.
• This is a traditional definition since it had got
some exception
– Polio vaccine – oral administration
– Some antigen may not produce antibodies
• Specificity is the hallmark of all immunological
reactions
3. Function of Antigen
• Immunogenicity : induction of immune response
• Immunological Reactivity: specific reaction with
antibodies or sensitized cells
• Based on this function antigen is classified as
– Complete antigen: induce antibody formation and
specific immune response
– Partial Antigen (Hapten): a specific non-protein
substance incapable of inducing antibody formation
itself but elicit immune response when couple with
carrier protein
• Complex hapten : polyvalent – 2 or more antibody
combining site
• Simple Hapten: univalent
4. Structure of Antigen
• Smallest unit of antigenicity (antigenic
detrminant) – epitope.
• Consist of 4-5 amino residues or monosacharide
residues
• Posses a specific chemical structure, electric
charge and spatial configration
• Capable of sensitizing of an imunocytes
• Paratope: area on antibody molecule on which
epitope binds
– This produces specificity of antigenic reaction
5. Biological Classes of Antigen
• Depending on the ability to produce antibody
formation, antigen are classified as
– T cell dependent (TD): structurally more complex
– T cell independent (TI): simple, limited number of
repeating epitopes
• Although antibody is produced by B-
lymphocytes, but it requires cooperation of T-
lymphocytes
6. Properties of Antigen
• Size
– Antigenicity is related to molecular size
– Larger molecules are highly antigenic where as haptens are low
molecular weight having low antigenicity
• Chemical Nature
– Antigenicity is directly proportional to degree of structural
diversity
– Amino acid> monosachharide> lipid> nucleic acid
• Susceptibility
– Only substance metabolized by tissue enzyme to epitope
fragments – potential to produce antigen
– Polystyrene not antigenic
– Also antigen rapidly broken down doesn’t show Antigenicity
properties
– D-amino acids are not metabolized in the body where as L-
amino acids do - prevention of autoimmunity
7. Properties of Antigen
• Foreignness
– Described by Ehrlich
– Normal body contain numerous antigens or antigen
producing substance which may not produce
antibody.
– But introduction of this into other body produces
immune response
– This response is directly related to degree of
foreignness – antigen from same species shows less
Antigenicity than others
• Specificity
8. Specificity
• Antigen shows various types of specificity.
They are mainly
– Antigenic specificity
– Species specificity
– Iso-specificity
– Auto-specificity
– Organ specificity
– Heterogenic specificity
9. Specificity
• Antigenic specificity
• Chemical nature of antigen makes it different from other
• Its may arise on the difference of group attaches on ortho,
meta or para position
• Also on the basis of spatial arrangement – dextro, levo and
meso
• Species specificity
• All individual contain species-specific antigens
• This has been used in tracing evolutionary relationship
• Ex: a individual sensitive to horse serum will be sensitive
to other equine but not with bovine
10. Specificity
• Iso-specificity
– Antigens found in the some but not all members
of species
– Blood grouping system is based on this
– Genetically determined
• Auto-specificity
• Sometime autologous antigens starts showing antigenicity
• Autoimmunity disorders like Rheumatoid arthritis, Sjogren
Syndrome
12. Introduction
• An antibody is an immunoglobulin (glycoprotein)
molecule formed by the immune system in
response to an antigenic stimulus.
– Found in blood, bodily secretions or on mucous
surface
– Binds to the specific antigen responsible for its
production, thereby inactivating it
• Immunoglobulins provides a structural and
chemical concepts.
• “Antibody” is a biological and functional concept.
• All antibodies are immunoglobulins, but all
immunoglobulins are not antibodies.
13. Antibodies
• Constitute the gamma globulin portion of
blood proteins - 20-25 % of total serum
protein.
• Are soluble proteins secreted by activated B
cells and plasma cells in response to an
antigen
• Are capable of binding specifically with that
antigen
14. Antibody Structure
• Consists of four looping polypeptide chains
linked together with disulfide bonds
– Two identical heavy (H) chains and two identical
light (L) chains
• The four chains bound together form an
antibody monomer
• Each chain has a variable (V) region at one
end and a constant (C) region at the other
• Variable regions of the heavy and light chains
combine to form the antigen-binding site
16. Antibody Structure
• Antibodies responding to different antigens have
different V regions
• C region (either kappa or lambda unit) is the same for all
antibodies in a given class
• C regions form the stem of the Y-shaped antibody and:
– Determine the class of the antibody
– Serve common functions in all antibodies
– Dictate the cells and chemicals that the antibody can
bind to
– Determine how the antibody class will function in
elimination of antigens
17. Classes of Antibodies
• Based on physiochemical and antigenic difference
antibodies are classified as:
– IgG : IgG1, IgG2, IgG3, IgG4
– IgA : Serum IgA or Secretory IgA and IgA1 or IgA2
– IgM
– IgD
– IgE
*** Decreasing order of their concentration.
18. IgG
• Major serum immunoglobulin – 80 % of total
• Molecular weight – 7s
• It exist in polymerised form
• Distributed equally in intravascular and
extravascular compartment
• Half life – 25 days
• Raised in chronic disease like Kala azar, chronic
malaria or myeloma.
• Normal serum concentration: 8-16 mg per ml
19. IgG
• Only maternal immunoglobulin – normally
transported across the placenta and provides
natural passive immunity in the new born
• Not synthesized by fetus
• The function of IgG: general purpose antibody
– Immunological reactions such as complement fixation,
precipitation and neutralization of toxins and viruses
– Protective against infectious agents – active in blood
and tissues
– Binds to MCO and enhances their phaogocytosis
– Surface recognition – Fc fragments
20. IgA
• 2nd most abundant class – 10-13
% of serum immunoglobulin
• Norma serum level: 0.6-4.2
mg/ml
• Half life – 6-8 days
• Found in colostrum, saliva and
tears
• It occurs in two forms
– Serum IgA: principally
monomeric 7s molecule
– Secretory IgA: two monomeric
unit joined together by J-chain
21. IgA : Secretory
• Larger molecule than serum IgA :
11s
• Plasma cells situated near the
mucosal or glandular epithelium
• It contain another glycine-rich
polypeptide – secretory
component or secretory piece.
– Protect IgA from denaturation by
bacterial proteases – sites such as
intestinal mucosa
22. IgA
• Function of IgA
• local immunity – against respiratory and
intestinal pathogens
• inhibits adherens of MCO to surface
mucosa
• Activate the alternative complement
pathway
• Promotes phagocytosis and intracellular
killing of MCO
23. IgM
• 5-8 % of serum immunoglobulins
• Normal level 0.5 – 2 mg/ml
• Hlaf life – 5 days
• Heavy molecule – 19s – millionaire molecule
• Polymer of the four-polypeptide joined by J-
chain.
• Most of IgM – intravascular (80%)
• Phylogenetically oldest immunoglobulin class
– earliest immunoglobulin synthesized by
fetus (20 weeks of age)
24. IgM
• Its not transported across the
placenta
• Presence of IgM in fetus or
newborn: syphilis, rubella,
HIV infection and
toxoplasmosis
• Relatively short lived – hence
its serum demonstration
indicates acute infection
25. IgM
• Its highly potent as compares to IgG
– 1000 times : immune hemolysis
– 500-1000 times : Opsonisation
– 100 times : bacteriocidal
– 20 times : bacterial agglutination
• Less active than IgG : neutralisation of toxins and
viruses
• Largely confined – intravascular space
• Functions
• Protection – blood invasion MCO
• Monomeric IgM – major antibody receptor on the surface of
lymphocytes for antigen recognition
• IgM deficiency - septicemia
26. IgD
• It resembles IgG structurally
• Serum concentration : 3 mg/100 ml mostly
intravascular
• Half life : 3 days
• Recognition receptor for antigen on B
Lymphocytes as that of IgM
• Also it causes stimulation of the B-cell –
activation and cloning to produce antibody or
supression
27. IgE
• Discovered by Ishizaka
• Molecular weight: 8 s
• Half life: 2 days
• Represents IgG structurally
• Heat labile but inactivated at 560 C in 1 hours
• Affinity for the surface of tissue cells – mast cells of
same species
• Mediates Prausnitz-Kustner reaction
• Doesn’t passes placental barrier or fix complement
• Extra-vascular in distribution
28. IgE
• There is very trace (few ng) serum level in normal
condition, but elevated in
– Atopic (type 1 allergic): asthma, hay fever and eczema
– Intestinal parasitic infection
• IgE chiefly produced in the linings of respiratory
and intestinal tracts
• IgE deficiency – undue susceptibility to infection.
• It produces
• Anaphylacttic type of hypersensitivity
• Protection against pathogen by mast cell degranulation and
release of inflammatory mediators
• Defense in helminthic infection
30. Summary
• In general, antibodies protects by
– IgG: body fluid
– IgA: body surface
– IgM: blood stream
– IgD: recognition molecule on the surface of B-
lymphocytes
– IgE: mediates antigenic hypersensitivity
32. Use of Antigen-Antibody Reactions
• In Body
– It forms the basis of antibody
mediates immunity against
infectious disease
– Tissue injury like Hypesensitivity
and autoimmunity
• In the Lab – Serological
Reaction
– Diagnosis of infection
– Identification of infectious
agents and difference between
non infectious antigen
– Quantification of antigen or
antibody
33. Stages of Antigen-Antibody reactions
• It occurs mainly in 3 stages
– Primary Stages
• Initial interaction without any visible effect
• Occurs very rapidly – obeys the laws of chemistry
• Reversible
• Can be represented by estimating free and bound antibodies –
physical and chemical method includind markers like radioistopte,
fluorescent dyes etc
– Secondary Stages: demonstrates the events like
• Precipitation
• Agglutination
• Lysis of Cell
• Neutralisation of toxins and other biologically active antigen
• Complement fixation
• Immobilisation of motile organism
• Enhancement of Phagocytosis
– Tertiary Stages : Complete destruction of injurious antigen and
tissue damage including humoral immunity
34. Features of Antigen-Antibodies
Reaction
• Specific
– antigen combines only with its homologus antibody,
– Sometimes cross-reactivity may occur due to antigenic
similarity
• Completeness: entire molecules reacts and not
fragments
• No denaturation of antigen or anotibodies
• Combination occurs on the surface
• The reaction is firm but reversible
• Varying proportion
• both antigen and antibodies are multivalent
• Antigen have valencies up to 100
• Antibody are generally bivalent – though IgM: 5-10 combining sites
35.
36. Precipitation Reaction
• When a soluble antigen combines with its
antibody in the presence of electrolytes (NaCl)
at suitable temperature and pH forms
insoluble precipitate. It either
– Settles down – Sedimentation
– Suspended as floccules – Flocculation
• It can be carried out either in liquid or gel
media.
• Amount of precipitate is greatly influenced by
antigen/antibody ratio
37. Precipitation Reaction
• Based on the antigen/antibody ratio, the
precipitation reactions can be classified in 3
zones i.e Zone phenomenon
– Prozone : Antibody is in excess. False negative
precipitation may occur.
– Zone of equivalence : equal proportion of antigen
and antibody i.e. optimal proportion, most rapid
and abundant reaction
– Postzone: Antigen is in excess. Precipitation is
again weak or even absent
38.
39. Application of Precipitation Reaction
• Qualitative and quantitative test
• Sensitive in the detection of antigen – as little
as 1μg of protein
• Forensic application
– Identification of blood
– Seminal stains
• Food Adulteration test
• Less sensitive for detection of antibodies
40. Precipitation Reaction
• Various types of precipitation reactions test
– Ring Test
– Slide Test : VDRL test for syphilis
– Tube Test
– Immunodiffucion – precipitation in gel
• Single diffusion in one dimension
• Double diffusion in one dimension
• Single diffusion in two dimension
• Double diffusion in two dimension
• Immunoelectrophoresis
41. Agglutination Reaction
• When a particulate antigen is mixed with its
antibody in the presence of electrolytes at a
suitable temperature and pH, the particles are
clumped or agglutinated
• More sensitive for antibodies detection
• Same Zonal principle applies for agglutination as
that of precipitation
• Incomplete or monovalent antibodies doesn’t
cause agglutination – combine with antigen :
Blocking antibodies
42. Agglutination Reaction
• Positive reaction is flowed by clumping together
of particles
• There are various application for agglutination
reactions
– Slide Agglutination
– Tube agglutionation: Widal test for Typhoid, Paul
Bunnel Test – mononucleosis, Cold agglutination test -
mycoplasma pneumonia
– Antiglobulin (Coombs) test – Rh antibody
– Passive agglutination test – Rose Waaler test for
Rheumatoid arthritis
43. Complement Fixation Test (CFT)
• Complement takes part in many immunological
reactions.
• In the presence of antibodies complement
– Lyses and kills erythrocytes and bacteria
– Immobilises motile organism
– Promotes phagocytosis and immune adherence
– Hypersensitivity
• The ability of antigen-antibody complexes to fix
complement is made use of the Complement
Fixation Test
44. Complement Fixation Test (CFT)
• Very versatile and sensitive test – detecting as
little as 0.04 mg of antibody and 0.1 mg of
antigen.
• Gold standard test for detection of Treponema
pallidum and Vibrio cholerae
• Its carried out in 2 steps and uses five reagents
– Antigen
– Antibody
– Complement
– Sheep Erythrocytes
– Amboceptor – rabbit antibody to sheep erythrocytes
45. Complement Fixation Test (CFT)
• 1ST Step
– Inactivated serum of patient is incubated for 370 C for 1
hr with Wasserman Antigen and fixed amount of
complement (Guinea pig)
– If the serum contains antibody – it will utilizes
complement and there will be no complement left for
further reaction
– If it doesn’t contain any antibody – no reaction will occur
• 2nd Step
• Sensitized cell (Sheep Erythrocytes + Amboceptor) is added to the
mixture and incubated.
• Lysis of erythrocyte will indicate – complement was not fixed in 1st
step therefore no antibody was present : CFT NEGATIVE
• CFT POSITIVE – no lysis will occur
46. Neutralisation test
• Neutralisation of antigen by its antibody can
be demonstrated by various system
• It can be
– Virus neutralisation
– Bacterial Neutralisation
– Toxin Neutralisation – in vivo or in vitro
• Ex: Shick Test – Diptheria, , Antistreptolysin ‘O’
ASO test
47. Oppsonisation
• Given by Wright (1903)
• Heat labile substance present in fresh normal
sera – facilitated phagocytosis
• Opsonic index
– study the progress of resistance during the course
of diseas
– Ratio of the phagocytic activity of the patient’s
blood for a given bacterium to the phagocytic
activity of blood from a normal individual
48. Immunofluorescence
• Fluorescence is the property of absorbing light rays of
one particular wavelength and emitting rays with a
different wavelength
• Coons et al – demonstrated that antibody labelled with
fluorescent dyes can be used to locate and identify
antigen
• Commonly used dyes – Fluorescin isothiocyante (blue
green) and lissamine rhodamine (orange red).
• It can be of two types:
– Direct Immunofluorescence test
– Indirect Immunofluorescence test
49. Direct Immunofluorescence test
• Specific antibody labeled with fluorescent dye –
detection of unknown antigen in a specimen
• If antigen is present it reacts with labeled
antibodies and fluorescence can be observed in UV
light of fluorescent microscope
• Example
– Bacteria, Virus or other antigen in blood, CSF, urine
faeces, tissues and other specimens
– Rabies detection in brain
• Disadvantage: Separate fluorescent antibodies
preparation is required for each antigen detection
51. Indirect Immunofluorescence test
• A known antigen is fixed in a slide with unknown
antibody (Serum),
• If its positive there will be a reaction to form
globullin coating on antigen
• After this the smear is treated with fluorescent
labeled antiserum to humman gammaglobulin
• This will react with antibody globulin bound to
antigen
– Slide is examine under UV-microscope
52. Indirect Immunofluorescence test
• Advantage: a single
antihuman globulin
fluorescent conjugate
can be employed for
detection of antibody
in any antigen
• No separate
conjugation with each
antibody is required
53. Radioimmunoassay (RIA)
• highly sensitive and quantitative procedure
that utilizes radioactively labeled antigen
or antibody
• S. A. Berson and Rosalyn Yalow (Nobel Prize) :
levels of insulin–anti-insulin complexes in
diabetics
• Principle
– Competitive binding of radiolabeled antigen and
unlabeled antigen to a high-affinity antibody.
54. Enzyme Linked Immunoassay (ELISA)
• It is one of immunoassay method used to
detection of
– Antibodies
– Proteins
– Peptides
– Biomolecules
• An absorbing material specific for either one of
the above components
• Ex: Cellulose, agarose or solid phase such as
polysterene, polyvinyl or polycarbonate tubes or
microwells or membranes or discs of
polyacrylamide, paper or plastics
55. ELISA
Advantages
• Reagents are relatively cheap & have a long shelf life
• Highly specific and sensitive
• No radiation hazards occur during labelling or disposal of
waste.
• Easy to perform and quick procedures
• It can be used to a variety of infections.
Disadvantages
• Enzyme activity may be affected by plasma constituents.
• Very specific to a particular antigen. Won’t recognize
any other antigen
• False positives/negatives possible, especially with
mutated/altered antigen
56. Types of ELISA
NON -COMPETETIVE
ELISA
•Direct ELISA
•Indirect ELISA
•S
andwich ELISA
•Competitive ELISA
•OgivesELISA
57. Direct ELISA
• The direct detection method
uses a labeled primary
antibody that reacts directly
with the antigen.
• Direct detection can be
performed with antigen that is
directly immobilized on the
assay plate .
• Direct detection is not widely
used in ELISA
• But is quite common for
immunohistochemical
staining of tissues and cells.
58. • The indirect ELISA
utilizes an unlabeled
primary antibody in
conjunction with a
labeled secondary
antibody.
• The secondary
antibody has
specificity for the
primary antibody
60. Sandwich ELISA
• The sandwich measures the amount of
antigen between two layers of antibodies.
• They are especially useful if the concentration
of antigens is low
• Or they are contained in a mix of high
concentrations of contaminating protein
• Major advantages - the antigen does not need
t
. o be purified prior to use, due to its high
specificity
61. Sandwich ELISA
1. Antibody (capture) is bound to a microtiter plate well.
2. Antigen is then added and bound to the antibody and
unbound products are then removed
3. A second antibody is added (detection) linked to enzyme
4. Lastly substrate to this enzyme is added and converted by
enzyme into colored product
5. The rate of color formation is proportional to amount of
antibody
62. • Antibody is first incubated in solution with a sample
containing antigen.
• The antigen-antibody mixture is then added to an
antigen coated microtiter well.
– The more antigen present in the sample, the less free
antibody will be available to bind to the antigen-coated
well.
• Addition of an enzyme-conjugated secondary antibody
(Ab2) specific for the isotype of the primary antibody
can be used to determine the amount of primary
antibody bound to the well as in an indirect ELISA.
63. COMPETETIVE ELISA
Solid phase coated with antibody
Add unknown amount of unlabeled
antigen and known amount of labeled
antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate
into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the
proportion of labeled antigen in the mixture of labeled and unlabled antigen.
64.
65. • A newer technique
uses an solid phase
made up of an
immuno-sorbent
polystyrene rod with
8-12 protruding o-
gives.
66. ELISA RESULTS
The ELISA assay yields three different types of data output:
Quantitative: ELISA data can be interpreted in
comparison to a standard curve in order to precisely
calculate the concentrations of antigen in various samples.
Qualitative : ELISAs can also be used to achieve a yes
or no answer indicating whether a particular antigen is
present in a sample.
Semi-quantitative: ELISAs can be used to compare
the relative levels of antigen
67. Application
• Screening donated blood for evidence of viral contamination by
– HIV-1 and HIV-2 (presence of anti-HIV antibodies)
– Hepatitis C (presence of antibodies)
– Hepatitis B (testing for both antibodies and a viral antigen)
• Measuring hormone levels
– HCG (as a test for pregnancy)
– LH (determining the time of ovulation)
– TSH, T3 and T4 (for thyroid function)
• Detecting infections
– Sexually-transmitted agents like HIV, syphilis and chlamydia
– Hepatitis B and C
– Toxoplasma gondii
• Detecting illicit drugs.
• Detecting allergens in food and house dust
