Immunity

Syllabus

• Immunity, vaccines
• Immunization schedule
• Definition of Antigen, antibody, list of antigen
antibody reactions.
 Specific Learning Obective

At the end of session, students should be able to

Define Immunity
Classify the types of immunity
Outline types of immunity with examples.
Explain mechanism of innate
 Immunity

• Definition:
Immunity is the capability of multicellular
organisms to resist harmful microorganisms
from entering it.
Humans have Two types of Immunity
Innate immunity is classified on Level
of Species , race or Individual
1. Species Immunity: Species immunity (species resistance) is that in
which a disease affecting one species does not affect the other species.
Example : Humans do not contract cattle plague, chicken cholera, hog
cholera, infectious horse anaemia, etc., while animals are not affected
by many human diseases such as enteric fever, scarlet fever, syphilis,
gonorrhoea, measles, etc.
2. Racial Immunity: Immunity shown by specific race within a species..
Example Black Africans affected by sickle cell anaemia, a genetic disease,
are resistant to malaria while malaria affects other human races.
3. Individual Immunity: Some Individuals of the race experience fewer or
less severe infections than other individuals of the same race.
Example: children are more susceptible to diseases such as measles and
chicken pox, while aged individuals are susceptible to other diseases like
pneumonia.
Factors Influencing the level of innate immunity
in individual
1. Age
2. Hormone
3. Nutrition
 Age

1. The foetus-in-uterus is usually protected from maternal infections by the

placental barrier. However, human immunodeficiency virus (HIV), rubella
virus, cytomegalovirus, and Toxoplasma gondii cross the placental
barrier and cause congenital infections.

2. Very old people are susceptible to suffer more than young people from a
disease (e.g., pneumonia) and have high mortality. This is due to warning
of immune system

3. Measles, mumps, poliomyelitis, and chicken pox are few examples of the
diseases that cause more severe clinical illness in adults than in young
children.
 Hormonal Influences

1. Individuals with certain hormonal disorders become increasingly

susceptible to infection.

2. For example, individuals suffering from diabetes mellitus,

hypothyroidism, and adrenal dysfunction are increasingly
susceptible to staphylococcal infection, streptococcal infection,
candidiasis, aspergillosis, zygomycosis and many other microbial
infections.

3. Similarly, pregnant women are more susceptible to many

infections due to higher level of steroid during pregnancy.
 Nutrition

• Nutritional status of the host plays an important role in innate immunity.

• Both humoral and cell-mediated immunities are lowered in malnutrition.

Examples are:

1. Neutrophil activity is reduced, interferon response is decreased, and C3

and factor B of the complement are decreased in protein–calorie
malnutrition.

2. Deficiency of vitamin A, vitamin C, and folic acid makes an individual

highly susceptible to infection by many microbial pathogens.
 Mechanism of Innate Immunity

1. Mechanical Barrier / Epithelial Surfaces

2. Antibacterial Substances in Blood and tissues
3. Microbial Antagonisms
4. Cellular Factors in Innate immunity
5. Inflammation
6. Fever
7. Acute phase proteins
• The principal components of innate immunity are:

(1) Mechanical Barrier / Surface Secretion:

Physical and chemical barriers, such as epithelia and antimicrobial chemicals
produced at epithelial surfaces.
For Example : Skin , Acidic pH in stomach , Cilia

(2)Humoral Mechanism
Blood proteins ,Lysozyme, including members of the complement system ,
Interferon and other mediators of inflammation.

(3)Cellular Defence Mechanism

Phagocytic cells (neutrophils, macrophages), dendritic cells, and natural killer
(NK) cells , Mast Cells , Eosinophil, Basophil and other innate lymphoid cells
1) Mechanical Barrier / Epithelial Surfaces

1. Skin : It is primary barrier to the entrance of microorganisms into the

body. It is covered with a layer of dead, keratinized epithelium that is too
dry for bacteria to grow and enter inside body.

Sweat and other skin secretions may lower pH, contain toxic lipids, and
physically wash microbes away.

2. Mucus: Mucus layer of the gastrointestinal tract, respiratory tract,

reproductive tract, eyes, ears, and nose traps both microbes and debris, and
facilitates their removal.
1) Gastrointestinal tract:
Saliva in the mouth is rich in lysozyme (an enzyme) that destroys bacteria by
digesting their cell walls.
Stomach contains gastric juices that are fatal to many pathogens.

2) Respiratory tract:
Hairs : Filter outs microbes and dust from nose
Cilia : Cilia along with mucous ,traps microbes and dust and removes from
upper respiratory tract

3) Reproductive and Urinary tract:

Urine : Washes microbes from Urethra
Acidic pH of Vagina keeps it free of pathogens
In Male Semen contain antimicrobial susbtances
2) Humoral Mechanism / Antibacterial
Substances in Blood and tissues
Antibacterial Substances in Blood and tissues are
1. Compliment System : Compliment system has bactericidal activity that
kills foreign bacteria in blood and tissues.
2. Interferon: Interferons are named for their ability to "interfere" with
viral replication by protecting cells from virus infections. They are
produced by WBC in blood.
3. Lysozymes
4. Beta Lysin: It is thermostable substance which is active against anthrax
and other related bacilli .
5. Basic Polypeptide: such as leukin from Leukocytes and Plakin from
platelets
6. Acidic Substance : Lactic acid found in muscles and inflammatory zone
 3) Microbial Antagonisms

• The inhibition of one bacterial organism by another is called microbial

Antagonism.
• Through microbial antagonism, the normal bacterial flora of the body
provides some defence against disease causing organisms.

Example : The Skin and mucous surface have resident bacterial flora which
prevent colonisation by pathogens.
4) Cellular Factors in Innate immunity

• When the infective agent has crossed the barriers of epithelial surfaces,
the tissue factors come into play for defence. These substances are

• Phagocytic Cells: They engulf microbes and destroy them

• Fibrin : It is protein substance that is arranged in long fibrous chains found

in blood plasma. When tissue damage results in bleeding, fibrinogen is
converted at the wound into fibrin by the action of thrombin, a clotting
enzyme.

• Natural Killer Cells : NK cells play important role in non-specific defence

against viral infections and tumours.
 Phagocytes
• Macrophages, neutrophils, and dendritic cells are the major phagocytes
of the immune system.

Monocytes
(Present in Blood)
Mononuclear
Phagocytes
Macrophages
Phagocytic cells (Present in Tissues)

Polymorphonuclear
Microphages
Leukocytes(PMN)
 Phagocytosis

• Mechanism can be divided in to four parts

1. Chemotaxis : Phagocytes reach the site of infection
attracted by chemotactic substances
2. Attachment: The infective agent get attached to phagocytic
membrane
3. Ingestion: Phagocyte engulfs the infective materials into
vacuole(Phagosome). The membrane of phagosome fuses
with lysosome to form phagolysosome
4. Intracellular Killing : Most bacteria are destroyed by
phagolysosome. However some bacteria
including Mycobacteria tuberculosis, the cause of
tuberculosis, may be resistant to these enzymes and are
therefore much more difficult to clear from the body.
 5) Inflammation
• Inflammation is a defence
mechanism in the body.

• It is response of vascular
connective tissue towards injury.

• The immune system recognizes

damaged cells, irritants and
pathogens, and begins healing
process .
When tissue is injured, the small blood vessels in
the damaged area constrict this process called
vasoconstriction.

Blood vessels dilate (vasodilation). Blood flow

increases into the area

Walls of the blood vessels, which normally allow

only water and salts to pass through easily,
become more permeable.

clotting factors, which help prevent the spread of

infectious agents throughout the body. Other
proteins include antibodies that help destroy
invading microorganisms.
 6) Fever

• Fever is rise in body temperature which destroys the infecting micro-

organism.

• Fever also stimulates the production of interferon. These interferon helps

in fighting against viral infection.
 7) Acute phase proteins

• Acute-phase proteins (APPs) are a class of proteins whose

plasma concentrations increase (positive acute-phase proteins) or
decrease (negative acute-phase proteins) in response to inflammation.
Example : 1) C reactive protein
2) Mannose Binding Protein

• Uses:
They Prevent tissue injury and helps in repair of inflammatory lesion
Mechanism Summary
 Questions

1. Discuss the mechanism of innate immunity ?

2. Write Short note on
• Innate immunity
• Inflammation
• Phagocytosis
• Microbial Antagonism
 Acquired Immunity
1. The second kind of protection
2. Develops throughout our lives.
3. Involves the lymphocytes
4. Develops as people are exposed to diseases or immunized
against diseases through vaccination.

It is of two types
1) Active Immunity
2) Passive Immunity
 Active Immunity

Definition:
Immunity develops after exposure to a disease-causing infectious
microorganism or other foreign substance, antigen, infection or vaccination.

Types
1. Natural Active Immunity – Induced Clinical or subclinical infection
Example: Persons recovering from smallpox infection
2. Artificial Active immunity – Induced by vaccination

Mechanism :
1. Humoral immunity
2. Cell Mediated immunity
 Humoral immunity

• Humoral immunity means Immunity to infection due to

antibodies, which circulate in the blood and lymph, and
which are produced by B cells
Flow Chart-CMI response
Summary- Humoral & CMI Response
 Passive Immunity

• Passive immunity is "borrowed" from another source and it

lasts for a short time.

• For example, antibodies in a mother's breast milk provide a

baby with temporary immunity to diseases the mother has
been exposed to. This can help protect the baby against
infection during the early years of childhood
 Similarities Between Active and Passive
Immunity
• Both active and passive immunity are two types of adaptive
immunity.
• Both active and passive immunity deal with antibodies.
• Both active and passive immunity can be either naturally-
acquired or artificially-acquired.
 Difference Between Active and Passive
Active Passive
Definition Active immunity results Passive immunity refers to a short-
from the production of term immunity which results from
antibodies by the the introduction of antibodies from
person’s own immune the outside
system in response to a
direct contact of an
antigen.
Antibodies Active immunity is Passive immunity is mediated by the
mediated by the antibodies produced outside the
antibodies produced by body.
the person’s own cells.
Pathogen The pathogen has direct The pathogen has no direct contact
contact with the body. with the body.
Response Time Active immunity does Passive immunity generates a rapid
not generate a rapid response.
response.
Lasts for Active immunity may last Passive immunity may not last for a
 Difference Between Active and Passive
Immunity Continue..
Active Passive
Immunological Memory Active immunity Passive immunity does
generates an not generate an
immunological memory. immunological memory.
Side Effects Side effects of the The body may react to
adaptive immunity are antisera.
very low.
In Immunodeficient Hosts Active immunity does Passive immunity works
not work in in immunodeficient hosts.
immunodeficient hosts
 Miscellaneous

• Combined Immunisation
• Adoptive Immunity
• Local Immunity
• Herd Immunity
 Combined Immunisation

• A Combination of active and passive immunisation is

employed simultaneously is called as combined
immunisation.

• Passive immunity provides the protection necessary till the

active immunity becomes effective.
 Adoptive Immunity

• Immunity produced by transferring lymphoid cells (e.g. T

cells) from an immune donor to a genetically identical
recipient.

• In cancer patients, for example, adoptive immunization is

performed to boost immunity following intensive
radiotherapy, using the patient's own T cells harvested prior
to the treatment.
 Local Immunity

• Natural or acquired immunity to an infectious agent that is

limited to a particular organ or tissue.
 Herd Immunity
• Herd immunity is a form
of indirect protection
from infectious disease
that occurs when a large
percentage of a
population has become
immune to an infection,
thereby providing a
measure of protection
for individuals who are
not immune.
 Fundamental Difference between Innate and
Acquired Immunity

Feature Innate Immunity Acquired Immunity

Response Time Rapid (hours) Slow(Days)
Specificity Limited and fixed Diversifies and improves
during course of immune
response
Response to re-exposure Identical to Primary Much more faster than
of Pathogen response primary response
 Questions

1. What is immunity? Explain various factors

influence the level of innate immunity in an
individual ?
 Antigen
• Definition: Antigens are molecules capable of stimulating an
immune response, especially Antibodies.

• Antigens include toxins, chemicals, bacteria, viruses, or other

substances that come from outside the body.

• Sometimes antigens are part of the host itself in an

autoimmune disease.
 Type of Antigen

Antigen

Complete Antigen Incomplete Antigen/

Haptens

Complete Antigen: A complete antigen is one that is sufficient

to induce a full immune response.
• It has both immunogenicity and immunoreactivity.
 Hapten / Incomplete Antigen
• An incomplete antigen is one that is not sufficient to induce a
full immune response.
• It has does not have immunogenicity and but it has
immunoreactivity.

• Hapten when binds to carrier molecule then it can bind to

antibody and produce immune response.
COMPLETE ANTIGEN INCOMPLETE ANTIGEN / HAPTENS
These are antigen which have both These are substance which does not have
immunogenicity and immunoreactivity immunogenicity but have
immunoreactivity
This does not require any carrier Hapten + Carrier Protein = Complete
Protein Antigen (Immunogen)

Note: Carrier proteins helps to make

hapten immunoreactive
Antibody can directly bind to them Antibody can bind to hapten (incomplete
Antigen) once they bind to carrier protein
and become complete antigen.
 Factors of Antigenicity or
Property of antigens
1. Foreignness
2. Size
3. Chemical Nature
4. Susceptibility to tissue Enzymes
5. Antigenic Specificity
6. Species Specificity
7. Isospecificity
8. Autospecificity
9. Organ Specificity
10. Heterophile Specificity
 Immunogenicity is determined by
1. Foreignness
• An antigen must be a foreign substances to the animal to
elicit an immune response.

2. Molecular Size
• The most active immunogens tend to have a molecular mass
of 14,000 to 6,00,000 Da.
• Examples: tetanus toxoid, egg albumin, thyroglobulin are
highly antigenic.
• Insulin (5700 ) are either non-antigenic or weakly antigenic.
3. Chemical Nature and Composition
• In general, the more complex the substance is chemically the
more immunogenic it will be.
• Antigens are mainly proteins and some are polysaccharides.
• It is presumed that presence of an aromatic radical is
essential for rigidity and antigenicity of a substance.

4. Physical Form
• In general particulate antigens are more immunogenic than
soluble ones.
• Denatured antigens are more immunogenic than the native
form.
5. Antigen Specificity
• Antigen Specificity depends on the specific actives sites on
the antigenic molecules (Antigenic determinants).
• Antigenic determinants or epitopes are the regions of
antigen which specifically binds with the antibody molecule

6. Species Specificity
• Tissues of all individuals in a particular species possess,
species specific antigen.
• Human Blood proteins can be differentiated from animal
protein by specific antigen-antibody reaction.
7. Organ Specificity
• Organ specific antigens are confined to particular organ or tissue.
• Certain proteins of brain, kidney, thyroglobulin and lens protein of one
species share specificity with that of another species.

8. Auto-specificity
• The autologous or self antigens are ordinarily not immunogenic, but
under certain circumstances lens protein, thyroglobulin and others may
act as autoantigens.

9. Isospecificity
• Isoantigens are Antigen found in some but not all members of a species
• Best example: human RBC antigens based on which individuals can be
classified into different blood groups.
 10. Heterophile Specificity
Heterophile antigens are closely related antigen present in
different tissues in different biological species, classes or
kingdoms.
Antibodies to these antigens produces by one species cross
react with other species

Some heterophile antigens are responsible for some diagnostic

serological tests such as
• Weil felix reaction for typhus fever
• Paul bunnell test for infectious mononucleosis
 Forssman Antigen

• Forssman Antigen are lipoprotein-polysaccharide complexes

• They are cross reacting microbial antigen so antibodies to

these antigens produced by one species cross react with
antigens of other species.

• It is widely present in some plants bacteria animal and birds.

However it is not present in rabbit.

• Therefore antibodies are produced in rabbit serum by

injecting the antigen (antiforssman antibodies).
 SuperAntigen
• Super Antigens are the molecules that can interact with Antigen-
presenting cells(APC) and T-Lymphocytes in a non-specific manner.

• Examples: Staphylococcal enterotoxins (food poisoning), Staphylococcal

toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins
(scalded skin syndrome) and Streptococcal pyrogenic exotoxins (shock).
 Immunization schedule
• The Immunization schedule is designed to protect infants
and children early in life when they are most vulnerable and
before they are exposed to potentially life-threatening
diseases.
 Age Vaccines Content Tag
Birth Bacillus Calmette–Guérin (BCG) BCG
Oral polio vaccine (OPV 0) OPV
Hepatitis B (Hep – B1) Hep -B
6 weeks Diptheria, Tetanus and Pertussis vaccine (DTwP 1) DTP
Inactivated polio vaccine (IPV 1) IPV
Hepatitis B (Hep – B2) Hep -B
Haemophilus influenzae type B (Hib 1) Hib
Rotavirus 1 Rotavirus
Pneumococcal conjugate vaccine (PCV 1) PCV
10 weeks Diptheria, Tetanus and Pertussis vaccine (DTwP 2) DTP
Inactivated polio vaccine (IPV 2) IPV
Haemophilus influenzae type B (Hib 2) Hib
Rotavirus 2 Rotavirus
Pneumococcal conjugate vaccine (PCV 2) PCV
14 weeks Diptheria, Tetanus and Pertussis vaccine (DTwP 3) DTP
Inactivated polio vaccine (IPV 3) IPV
Haemophilus influenzae type B (Hib 3) Hib
Rotavirus 3 Rotavirus
Pneumococcal conjugate vaccine (PCV 3) PCV
 Age Vaccines Content Tag
Oral polio vaccine (OPV 1) OPV
6 months
Hepatitis B (Hep – B3) Hep -B
Oral polio vaccine (OPV 2) OPV
9 months
Measles, Mumps, and Rubella (MMR – 1) MMR

9 – 12 months Typhoid Conjugate Vaccine Typhoid Conjugate Vaccine

12 months Hepatitis A (Hep – A1) Hep -A

Measles, Mumps, and Rubella (MMR 2) MMR

15 months
Varicella 1 Varicella
PCV booster PCV

Diphtheria, Perussis, and Tetanus (DTwP B1/DTaP B1) DTP

16 to 18 months Inactivated polio vaccine (IPV B1) IPV

Haemophilus influenzae type B (Hib B1) Hib

18 months Hepatitis A (Hep – A2) Hep -A

2 years Booster of Typhoid Typhoid Conjugate Vaccine

Conjugate Vaccine

Diphtheria, Perussis, and Tetanus (DTwP B2/DTaP B2) DTP

Oral polio vaccine (OPV 3) OPV

4 to 6 years
Varicella 2 Varicella

Measles, Mumps, and Rubella (MMR 3) MMR

Tdap/Td Tdap
10 to 12 years
Human Papilloma Virus (HPV) HPV
 Questions
Question 1) What is Antigen ? Discuss briefly about various
factors of antigenicity.

Question 2) Define Following:

1) Forssman Antigen
2) Super Antigen
3) Haptens
4) Hetrophile Antigens
 ANTIBODIES
• Antibodies, also known as immunoglobulins,
• Y-shaped proteins
• produced by the immune system to help stop foreign particle
(Antigen) from harming the body.
• When an Antigen enters the body, the immune system
springs into action.
Structure of Immunoglobulin
 Structure of Immunoglobulin
They are Heavy globular proteins found in the plasma.
• Made of four polypeptide chains –
Two heavy chains
Two light chains.

• Together the heavy and light chains are held together by

disulphide bonds, giving the structure of the antibody molecule,
a Y shape.
• The portion of the heavy and light chains that come in contact
with the antigen is called the variable region. It consists of
100-110 amino acids that differ in each antibody molecule
depending on the antigen encountered.
Structure of Immunoglobulin
Heavy Chain:
• Each heavy chain is made up of large polypeptides of around
50,000 Da.
• There are five types of heavy chains – Alpha, Delta, Epsilon,
Gamma, and Mu.

Light Chain:
Each light chain is made up of polypeptides of around 20,000Da.
• There are two types of light chains – Lambda and Kappa.
 Immunoglobulin Antigen Determinants
• The epitopes (or antigenic determinants) of the antibody
molecule are described in three categories:

1. Isotypic determinant
2. Allotypic determinants
3. Idiotypic determinant
 Immunoglobulin Classes
• There are five classes of antibodies or immunoglobulins
categorized by differences in their constant region.
• These are IgG, IgA, IgM, IgE, and IgD. TRICK TO REMEMBER–
GAMED
• They differ in prevalence, function and their constant regions.
Immunoglobulin Classes
Properties of Antibodies
 Structure of IgG
• Gamma heavy chains

• Two antigen binding

sites.

• Monomer

• Molecular wt.
150,000 Da. Approx
 Properties Of IgG
• IgG is the most predominant antibody found in the body and
constitutes for 80% of the total antibody content in the serum.

• It is the only antibody with the ability to cross the placental

membrane and provide immunity to the fetus.

• There are four sub-classes of the IgG molecule: IgG1, IgG2, IgG3,
and IgG4.

• Among these, IgG 3 and IgG 4 possess the ability to cross the
placenta. IgG1 is the most common antibody subclass among the
four.
 Functions of IgG
1. It provides immunity to the developing fetus.
2. Activates the complement pathway of immune response.

3. Mediates a process known as opsonization which refers to

antibodies coating a pathogenic cell to lure the phagocyte
towards the antigenic surface of the pathogen.
4. Facilitates the process of phagocytosis.

5. Neutralization of toxins and pathogens.

6. Since it is widely found circulating in the blood, it offers
protection against pathogens in the blood and tissues.
 Structure of IgA
• The heavy chains- Alpha

• Four antigen binding sites.

• The molecular weight is 385,000 Da.

• Dimeric forms.
 Properties of IgA
• IgA is found in the mucous membranes of the gastrointestinal
and respiratory tracts. It is located in mucus secretions, saliva,
tears, and the colostrum.

• It constitutes 13% of total antibody content found in the

serum.

• There are two subclasses of the IgA antibody – IgA1 and IgA2.

• The IgA1 antibody is the most prevalent and is also called

secretory immunoglobulin or sIgA, and is most commonly
found in secretions in high quantities.
 Functions of IgA
• IgA is found in the secretions and provides the first line of
defense against the uptake of microbes and antigens into the
body.

• It limits inflammation.

• Participates in the immune response through the activation

of the complement pathway.

• Provides immunity to the fetus and the newborn infant.

 Structure of IgM
• The heavy chains -Mu
subclass.

• 10 antigen binding
sites.

• The molecular weight

900,000 Da.

• Pentameric form.

• Largest antibody
 Properties of IgM
• largest antibody

• First to be produced after an antigen enters the body.

• It is found in the blood and the lymph fluid.

• It constitutes 6% of the total antibody content of the serum.

 Functions of IgM
• Found on the surface of the B-cell and helps in antigenic
recognition.

• Activation of the complement pathway.

• It is involved in opsonization and agglutination.

• Facilitates efficient activation of the immune system due to

the more significant number of antigenic sites on its surface.

• The ABO system of blood grouping consists of IgM antibodies

that are specific to the ABO antigens expressed on the
surface of the RBCs.
 Structure of IgE
• The heavy chains - Epsilon
• Two antigenic binding sites.
• Monomer
• Molecular weight - 200,000 Da.
 Properties of IgE

• It is found in the linings of the respiratory and intestinal tracts.

• least abundant

• Involved in hypersensitivity reactions.

 Functions of IgE
• Immune protection against pathogens is invading through the
gastrointestinal or respiratory barriers.

• It has a vital role in Type 1 hypersensitivity reactions or

allergic response
 Structure of IgD
• The heavy chains –Delta

• Two antigen binding sites.

• Monomer

• Molecular wt. 180,000 Da.

Function:
It is present on the surface of the B cell and acts as a receptor
and participate in B cell activation and differentiation.
 Properties of IgD

• IgD antibody makes up less than 1% of the total antibody

content of serum.

• It is usually co-expressed on the surface of B cells with IgM.

• Its specific function is still unknown. However, it is thought to

be involved in the process of B cell activation.
 Abnormal Immunoglobulins
• Multiple Myeloma
• Heavy Chain Disease
• Cryoglobulinaemia
 Multiple Myeloma
• Multiple myeloma is a cancer that forms in a type of white blood
cell called a plasma cell.

• Multiple myeloma causes cancer cells to accumulate in the bone

marrow, where they crowd out healthy blood cells.
 Antigen-Antibody reactions
• The important antigen-antibody reactions are:

1. Precipitation Reactions
2. Immunodiffusion Test
3. Counter Current Immunoelectroptioresis Test
4. Agglutination Reactions
5. Complement Fixation Reactions
6. Neutralization Reactions
7. Radioimmunoassay
8. Enzyme-Linked Immunosorbent Assay
9. Fluorescent Antibody Technique.
 1. Precipitation Reactions
• The reaction of soluble antigens with IgG or IgM antibodies to form a
large interlocking aggregates (lattices) is called precipitation reaction.

• The precipitates formed by antibodies are known as precipitins .

In precipitation test, a precipitation ring

appears which display the creation of optimal
ratio.

This zone is known as the zone of equivalence

 The precipitation reactions occur in two stages
(i) Rapid interactions within a second between antigen and antibodies and
formation of complex.

(ii) Slow rate of reaction completing even within a few minutes or hours and
forming lattices from antigen-antibody complexes.

Note : When the antibodies and antigens are in proper ratio, precipitation
reactions normally occur. When there is excess amount of either of two, no
visible precipitate is formed
 2. Immunodiffusion Test (IDT)
1. Immunodiffusion tests are performed in a gelled agar medium.
2. One of the IDTs is Ouchterlony test.
3. In Ouchterlony test wells are cut, into which a purified antiserum (a
serum containing antibodies) is added, and to each surrounding well,
soluble form of test antigens are added.
4. Line of visible precipitate is formed between the wells where after
diffusion optimal ratio of antigen-antibody is formed.
Uses of Immunodiffusion tests :

• The presence of antibodies in the serum against more than one antigen at
a time can be demonstrated.

• Identical, partially identical and different types of antigens can also be

found out.
 3.Counter Current Immunoelectroptioresis Test
Principle:
• It is based on the movement of antigens and antibodies to opposite poles
after applying electric current in buffers of correct electric strength and
pH, because some of the antigens and antibodies have the opposite
charges.

• If a reaction occurs, a precipitation line appears within an hour.

 Uses
• Rapid movement of antigen and antibody.

• Protein can be separated within an hour.

• CIE is useful for the diagnosis of bacterial meningitis and the

other diseases.
 4.Agglutination Reactions
• Agglutination is the process of linking together of antigens by
antibodies and formation of visible aggregates.

• It involve particulate antigens i.e. soluble antigens adhering

to particles.

• It is of two types

1. Direct agglutination tests

2. Indirect agglutination tests
 Direct agglutination test
• This test diagnose antibodies against a large number of
cellular antigens such as RBCs, bacteria and fungi.
• Test is carried out in plastic microtiter plates that have several
small shallow wells.
• Each well acts as small test tube
 Indirect (Passive) Agglutination Tests
• This type of diagnostic tests are very rapid particularly for the detection of
streptococci.

• If the antigens are adsorbed onto particles (e.g. RBCs, latex beads,
bentomile clay), soluble antigens can respond to agglutination test.

• Antibody reacts with the soluble antigen adhering to the particles.

• Therefore, the particles agglutinate with each other as these do in the

direct agglutination tests
 Haemagglutination
• It is process of clumping of RBCs.
• When the RBCs are agglutinated by certain viruses such as
those causing mumps, measles, influenza, etc. it is called viral
haemagglutination.

Uses:

• This test is used for the diagnosis of a number of viruses

affecting human body.
 5. Complement Fixation Reactions
• A group of 20 or more serum protein is collectively known as
complement.
• During reaction, the complement binds to antigen-antibody
complex and is used up or fixed.
Uses:
• Used to measure even very small amount of antibody that
does not produce a visible reaction such as precipitation or
agglutination.
• Used in diagnosis of diseases such as leptospirosis,
mycoplasmal pneumonia, Q fever, polio, rubella,
histoplasmosis, coccidiodomycosis and streptococcal
infections.
The test is accomplished in the two stages
Stage 1:
• For example, if a patient is suffering from a disease caused by
streptococci the test antigen would be the streptococcal antigen.

• If the patient’s serum contains antibodies against streptococci, the

test antigen will form complement sequence.

• This mixture is again incubated for about 30 minutes.

• At this point, no antigen-antibody reaction occurs.

 Stage 2:
Procedure for
complement fixation
test
 Stage-2
• In stage 2, the complement fixed by antigen-antibody reaction is
detected by an indication system.

• This system consists of sheep RBCs containing specific antibodies

attached to their surfaces.

• When these are added to complement, haemolysis of RBCs occurs

that impart changes in colour of the mixture.

• This shows that the complements have not been fixed during the
first stage; therefore, these become available to cause haemolysis.
• This indicates that the patient has no streptococcal pneumonia .
 6. Neutralization Reactions
• It involves elimination of harmful effects of bacterial exotoxins or a virus
by specific antibodies.

• These neutralizing substances i.e. antibodies are known as antitoxins.

• This specific antibody is produced by a host cell in response to a bacterial

exotoxin or corresponding toxoid (inactivated toxin).

• The antitoxin reacts with exotoxin and neutralizes it.

Example:
• These antitoxins can be artificially induced in animals such as horses. Thus,
the antitoxin of animal sources in turn can be injected into human which
provides a passive immunity against a toxin present in human body
produced by the pathogens causing diphtheria, tetanus, etc.
 Uses of Neutralisation Reaction
1) Diagnosis of Viral Infections
• After introduction of a virus, antibodies are produced in
response and bind to receptor sites present on the viral
surfaces.
• After binding of antibodies, viral particles fail to reach to the
cells. Thereafter, the virus is destroyed.
 Opsonisation
It is the process where opsonin protein make an invading microorganism more
susceptible to phagocytosis
 Immunofluorescence
Principle: Immunofluorescence is an assay which is used primarily on
biological samples and use to detect antigens in cellular contexts using
antibodies.
The specificity of antibodies to their antigen is the base for
immunofluorescence.
 Types of Immunofluorescence
Two types of Immunofluorescence
1) Direct (Primary) Immunofluorescence
2) Indirect (Secondary) Immunofluorescence
 Radioimmunoassay
Principle : Radioimmunoassay (RIA) is a highly sensitive way to measure the
concentration of antigen in a sample.

Steps:
• In this assay, a quantity of the antigen of interest is tagged with a
radioactive isotope (typically of iodine-125 or iodine-131) and mixed with
a known amount of its cognate antibody.
• Sample is then added and any antigen in the sample matching the
radiolabeled antigen will compete for binding to the added
antibody—effectively drawing antibody away from the labeled antigen.
• Bound and unbound antigen are separated, and the amount of
radioactivity in the unbound fraction measured.
• The level of radioactivity in this fraction is proportional to the amount of
antigen in the sample
Radioimmunoassay

A standard binding curve, shown in red,

can be drawn.
Enzyme-linked immunosorbent assay
(ELISA)
• It is a method of target antigen (or antibody) capture in samples using a
specific antibody (or antigen), and of target molecule detection/
quantitation using an enzyme reaction with its substrate

Principle:
• Specific antibodies bind the target antigen, and a detection system
indicate the presence and quantity of antigen binding.
• In order to maximize the sensitivity and precision of the assay, the plate
must be carefully coated with high-affinity antibodies – a process that
Boster Bio has mastered.
 Most Common ELISA Types
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
 Direct ELISA
• A target protein (or a target antibody) is immobilized on the surface of
microplate wells and incubated with an enzyme-labelled antibody to the
target protein (or a specific antigen to the target antibody).

• After washing, the activity of the microplate well-bound enzyme is

measured
 Indirect ELISA
• A target protein is immobilized on the surface of microplate wells and
incubated with an antibody to the target protein (the primary antibody),
followed by a secondary antibody against the primary antibody.
• After washing, the activity of the microplate well-bound enzyme is
measured.
 Sandwich ELISA
• The immobilized antibody (orange) and
the enzyme-labelled antibody (green)
must recognize different epitopes of
the target protein.

• After washing, the activity of the

microplate well-bound enzyme is
measured.
 Competitive ELISA
• An antibody specific for a target protein is immobilized on the surface of
microplate wells and incubated with samples containing the target
protein and a known amount of enzyme-labelled target protein.
After the reaction, the activity of the microplate well-bound enzyme is
measured.
 Summary of Key Steps in Different ELISA
Indirect Direct Sandwich Competitive
Capture Ab X X √ X
Coating
Antigen Coating √ √ X √
Blocking √ √ √ √
Sample X X √ √
(Antigen)
Incubation
Primary Ab √ √ √ √
Incubation
Secondary Ab √ X √ √
Incubation
Substrate Prep √ √ √ √
Signal Detection √ √ √ √
Data Analysis √ √ √ √
 Chemiluminescence Immunoassay (CLIA)
Principle:
• Luminescence means that light is emitted by a substance when it returns
from an excited state to a ground state
• The enzymes used in chemiluminescent immunoassay convert a substrate
to a reaction product, which emits a photon of light instead of developing
a particular colour.
 Immunoblotting
• Principle. Western blotting (protein blotting or immunoblotting) is a rapid
and sensitive assay for detection and characterization of proteins.
• It is based on the principle of immunochromatography where proteins are
separated into polyacrylamide gel according to their molecular weight.