Immunology & Immunological

Preparations

By: Bijaya Kumar Uprety

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Immunology
Branch of biological science
concerned with the study of immunity,

Or concerned with the structure and

function of immune system.

Immunity
Latin term immunis meaning
exempt.
Immunity means the state of

protection from infectious disease.

Year

Name

Event

430 B.C Earliest written

reference to the
phenomenon of immunity.

Thucydides (great
historian of the
Peloponnesian war).

In describing plague in Athens, he wrote during the war, only who had recovered from the
plague could nurse the sick because they would not get the disease for the second time.

Chinese and Turks

Dried crust derived from small pox pustules were either inhaled into the nostrils or
inserted into small cuts in the skin (technique known as variolation).

Lady Mary Wortley

(wife of british
ambassador to
Constantinople)

Performed variolation on her own children after realizing the technique was effective
among her native people.

Edward Jenner
(physician)

Propounded an idea that introducing fluid from a cowpox pustule into people might
protect them from smallpox and he tested his idea on eight year old kid which was
successful.

15th century- First record

to induce immunity
deliberately.

1718

1798

 Louis Pasteur grew the fowl cholera causing

bacterium in culture and when this was injected it into

chicken they developed cholera but later on when he
once again injected them with the same old culture
they got ill but recovered later on.
He grew fresh culture and tried it on same chickens.
They completely recovered.
Hence, Hypothesized and proved that aging had
weakened the virulence of the pathogen and
concluded that the attenuated strain might be
administered to protect against the disease. He called
this attenuated strain a vaccine.

Continue..
Later extended his findings to other diseases

and
demonstrated it is possible to attenuate, or weaken a
pathogen and administer them to use it as a vaccine.

In 1881, Pasteur vaccinated one group of sheep with

heat-attenuated anthrax bacillus (bacillus anthracis)

and left another group of unvaccinated sheep . All
unvaccinated sheep died while other lived.
This was the beginnings of

the discipline of
immunology. In 1885, he first administered his first
vaccine to a human (a young boy) against rabies.
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 Pasteur proved vaccination worked but didnt know

how it worked.
In 1890, Emil von Behring and Shibasaburo Kitasato

gave first insight into the mechanism of immunity

Got nobel prize in 1901.
They demonstrated that serum from animals

previously immunized to diptheria could transfer the

immune state to unimmunized animals.

 During next decade, it was demonstrated by various

researchers that an active component from immune

serum could neutralize toxins, precipitate toxins and
agglutinate bacteria and active agent was named for its
activity it exhibited: antitoxin, precipitin and
agglutinin resp.
Initially different serum component was thought to be

responsible for each activity but during 1930, Elvin

Kabat (mainly him) found that gamma-globulin (now
immunoglobulin, also a fraction of serum) was
responsible for all these.
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 The active molecules in the immunoglobulin fraction

are called antibodies.

Because the immunity was mediated by antibodies
contained in the fluids (known at that time as
humors), it was called humoral immunity.
In 1883, Elie Metchnikoff demonstrated that cells also
contribute to the immune state of an animal. He
hypothesized that cells rather than serum components
were major effector of immunity.(term phagocytes was
coined and an idea of cell-mediated immunity dvpt).
Controversy developed between two concepts.
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 But latter proved that both were correct.

Immunity requires both humoral and cellular responses.
In 1950, lymphocyte was identified as the cell responsible

for both cellular and humoral immunity and experiments

on chicken pioneered by Bruce Click at Mississippi State
University indicated that there are two types of
lymphocytes.
1. T- lymphocytes derived from thymus mediated cellular
immunity.
2. B-lymphocytes from bursa of Fabricius were involved in
humoral immunity.
Both these systems work hand in hand to protect our
body against various foreign attack.
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Introduction to Immune system

Remarkably versatile defense system that protect

animals against various invading micro-organisms and

cancer.
Able to generate enormous variety of cells and

molecules capable of specifically recognizing and

eliminating large variety of foreign invaders.
Invaders human body immune system
respond eliminate or destroys the invaders
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 Body endowed with different defense system.

At first, external defense system comes into play which

includes, skin, secretion of mucus, ciliary action,

lavaging action of bactericidal fluids (e.g. tears),
gastric acid and microbial antagonism.
If penetration occurs, bacteria are destroyed by

soluble factors such as lysozyme and by phagocytosis

with intracellular digestion.
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 Functionally, immune response can be divided into

two related activities1. Recognition Remarkable for its specificity.

2. Response.

Immune system is able to recognize subtle chemical

differences that recognize one foreign pathogen
from another.

Able to discriminate between foreign molecules

and the bodys own cells and proteins.
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 Once a foreign organism has been recognized, it recruits a

variety of cells and molecules to mount an appropriate

response, called effector response, to eliminate or
neutralize the organism.
The immune response enables the elimination or

neutralization of the cells/molecules (pathogens) from the

body.
Convert initial recognition event variety of effector
responses eliminate or neutralize particular
pathogen.
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 Later exposure to the same foreign organism induces a

memory response, characterized by a more rapid and

heightened immune reaction that serves to eliminate
the pathogen and prevent disease.

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Types of immunity
Two types of immunity:

1. Innate or nonspecific immunity.

2. Acquired or specific immunity.
Innate immunity:
It is the basic resistance to diseases that an individual
has from the time of its birth.
Not specific to any one pathogen but rather constitutes
a first line of defense.
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 It consists of following four types of defensive barriers:

Anatomic barriers
2. Physiologic barriers
3. Endocytosis /phagocytosis barriers
4. Inflammatory barriers
1.

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 Endocytosis-

Process of cellular
ingestion of
macromolecules by
invagination of plasma membrane to
produce an intracellular vesicle which
encloses the ingested material.
3 types Phagocytosis (for particulates),
Pinocytosis (liquid),
Receptor mediated
endocytosis

(LDL) .
Most phagocytosis (most common) is

done by blood monocytes, neutrophils,

and tissue macrophages.

Fig. Steps in phagocytosis of a

bacterium.
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Fig 2 showing the major events in the inflammatory response.[ vasoactive and
chemotactic factors i.e kinin and histamine. Additionally , bradykinins which is
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a type of kinin stimulate pain receptors and fibrin-clot]

Acquired Immunity
Also known as adaptive immunity.
Capable of recognizing and selectively eliminating

specific foreign microorganisms and molecules (i.e.

foreign antigens).
Displays four characteristic attributes:
1. Antigenic specificity
2. Diversity
3. Immunologic memory
4. Self/nonself recognition.
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Components of Acquired Immunity

Involves the following two major groups of cells
1. Lymphocytes which includes B and T lymphocytes.

2. Antigen presenting cells (APCs) Group of B-cells, dendritic cells and macrophages.
They express class II MHC molecules on their
membranes &
They are able to deliver a co-stimulatory signal that is
necessary for TH cell activation.

APC have Class II MHC (major histocompatibility

complex) molecules on their surface MHC molecules
bind to antigen derived peptides present them to
lymphocytes immune system activated.
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B lymphocytes
B lymphocytes mature within the bone marrow; when they leave it, each expresses a

unique antigen-binding receptor on its membrane . This antigen-binding or B-cell

receptor is a membrane-bound antibody molecule.
Antibodies are glycoproteins that consist of two identical heavy polypeptide chains
and two identical light polypeptide chains. Each heavy chain is joined with a light
chain by disulfide bonds, and additional disulfide bonds hold the two pairs
together.
The amino-terminal ends of the pairs of heavy and light chains form a cleft within
which antigen binds.
When a naive B cell (one that has not previously encountered antigen) first
encounters the antigen that matches its membrane bound antibody, the binding of
the antigen to the antibody causes the cell to divide rapidly; its progeny differentiate
into memory B cells and effector B cells called plasma cells.
Memory B cells have a longer life span than naive cells, and they express the same
membrane-bound antibody as their parent B cell.
Although plasma cells live for only a few days, they secrete enormous amounts of
antibody during this time. It has been estimated that a single plasma cell can secrete
more than 2000 molecules of antibody per second. Secreted antibodies are the
major effector molecules of humoral immunity.
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T lymphocytes
T lymphocytes also arise in the bone marrow. Unlike B cells, which mature
within the bone marrow, T cells migrate to the thymus gland to mature.
During its maturation within the thymus, the T cell comes to express a unique

antigen-binding molecule, called the T-cell receptor, on its membrane.

Unlike membrane-bound antibodies on B cells, which can recognize antigen

alone, T-cell receptors can recognize only antigen that is bound to cellmembrane proteins called major histocompatibility complex (MHC)
molecules.
MHC molecules that function in this recognition event, which is termed
antigen presentation, are polymorphic (genetically diverse) glycoproteins
found on cell membranes.

There are two major types of MHC molecules:

Class I MHC molecules, which are expressed by nearly all nucleated cells of
vertebrate species, consist of a heavy chain linked to a small invariant protein
called 2-microglobulin.
Class II MHC molecules, which consist of an alpha and a beta glycoprotein
chain, are expressed only by antigen-presenting cells.
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 When a naive T cell encounters antigen combined with a MHC molecule on a cell,

the T cell proliferates and differentiates into memory T cells and various effector T
cells.
There are two well-defined subpopulations of T cells: T helper (TH) and T

cytotoxic (TC) cells. Although a third type of T cell, called a T suppressor (TS) cell,
has been postulated, recent evidence suggests that it may not be distinct from TH
and TC subpopulations.

T helper and T cytotoxic cells can be distinguished from one another by the

presence of either CD4 or CD8 membrane glycoproteins on their surfaces . T

cells displaying CD4 generally function as TH cells, whereas those displaying CD8
generally function as TC cells. TH cells generally recognize antigen combined with
class II molecules, whereas TC cells generally recognize antigen combined with class
I molecules.
After a TH cell recognizes and interacts with an antigen MHC class II molecule

complex, the cell is activatedit becomes an effector cell that secretes various
growth factors known collectively as cytokines. The secreted cytokines play an
important role in activating B cells, TC cells, macrophages, and various other cells
that participate in the immune response.

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 Differences in the pattern of cytokines produced by activated

TH cells result in different types of immune response.

Under the influence of TH-derived cytokines, a TC cell that
recognizes an antigenMHC class I molecule complex
proliferates and differentiates into an effector cell called a
cytotoxic T lymphocyte (CTL).
In contrast to the TH cell, the CTL generally does not
secrete many cytokines and instead exhibits cell-killing or
cytotoxic activity.
The CTL has a vital function in monitoring the cells of the
body and eliminating any that display antigen, such as virusinfected cells, tumor cells, and cells of a foreign tissue graft.
Cells that display foreign antigen complexed with a class I
MHC molecule are called altered self-cells; these are targets of
CTLs.
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ANTIGEN-PRESENTING CELLS
Activation of both the humoral and cell-mediated branches of the

immune system requires cytokines produced by TH cells.

It is essential that activation of TH cells themselves be carefully
regulated, because an inappropriate T-cell response to selfcomponents can have fatal autoimmune consequences.
To ensure carefully regulated activation of TH cells, they can recognize
only antigen that is displayed together with class MHC II molecules on
the surface of antigen-presenting cells (APCs).
These specialized cells, which include macrophages, B lymphocytes,
and dendritic cells, are distinguished by two properties: (1) they express
class II MHC molecules on their membranes, and (2) they are able to
deliver a co-stimulatory signal that is necessary for TH-cell activation.
Antigen-presenting cells first internalize antigen, either by
phagocytosis or by endocytosis, and then display a part of that antigen
on their membrane bound to a class II MHC molecule. The TH cell
recognizes and interacts with the antigenclass II MHC molecule
complex on the membrane of the antigen-presenting cell .An
additional costimulatory signal is then produced by the antigenpresenting cell, leading to activation of the TH cell.
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Humoral Immune Responses

It is based on antibodies.
It can be conferred on nonimmune individuals by

administration of serum antibodies from an immune

individual.
Antibodies act as an effector of humoral response.
They bind to the antigens and facilitate their
elimination.
Elimination could be in various ways.

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Fig showing the structure of Antibody.

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1. By forming clusters through cross-linking of antigen

molecules, which are readily ingested by phagocytic
cells.

2. By binding of antibodies to a microorganism can

activate the complement system, which lyses the
mos.
3. Antibodies bind to toxins and viral particles, and
prevent their subsequent binding to host cells.

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Cell-mediated Immune Responses

Based on T cells, which are a type of lymphocyte.
T cells are of the following two types:

T helper (TH)
2. T cytotoxic (TC) cells.
1.

TH cell interacts with an antigen MHC II molecule

complex present on an APC cytokines secreted
cytokines activate B cells, Tc Cells, and various
phagocytic cells.

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 Activated phagocytic cells able to kill mos (bacteria

and protozoa).
When Tc cell interacts with an antigen-MHC I

complex, the Tc cell proliferates under the influence of

cytokines produced by activated TH cells.
These

Tc cells differentiate into cytotoxic T

lymphocytes (CTLs). The CTLs kill all such cells that
display foreign antigens complexed with MHC I
molecules. Such cells are called altered self-cells, they
are usually virus-infected cells, tumor cells and foreign
tissue cells.
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 Thus TH cells and CTLs are effectors of the cell-

mediated immune response.

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Fig 2. Overview of humoral and cell mediated immune responses.

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Passive Immunization
It is the administration of preformed antibodies (usually

IgG) either intravenously or intramuscularly.

Used to provide rapid protection in certain infections such

as diptheria or tetanus or in the event of accidental

exposure to certain pathogens such as hepatitis B.
Also used to provide protection in immune compromised

individuals who are unable to produce appropriate

antibody response or in some instances incapable of
making any antibody at all (i.e. severe combined
immunodeficiency).
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 Antibodies given to immune deficient patients are

usually IgG- derived from pooled normal plasma and

are administered on a continuous basis (ideally every
three weeks) as they are continuously catabolized and
are effective only for short duration.
Preformed antibodies from animals, notably horse are

also administered for some diseases but it presents a

danger of immune complex formation and serum
sickness (if repetitively injected).

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Active immunization
Administration of vaccines containing microbial

products with or without adjuvants in order to obtain

long term immunological protection against the
offending microbe.
2 types:

Systemic Immunization.
2. Mucosal Immunization.
1.

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Systemic Immunization
This is the method of choice at present for most

vaccinations.
Carried out by injecting vaccine subcutaneously or

intramuscularly into the deltoid muscle.

Ideally all vaccines given soon after birth but some

deliberately delayed.
Common eg includes vaccines for measles, mumps, and

rubella usually given at the age of 1. If given earlier

maternal antibody would decrease their effectiveness.
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 But

carbohydrate vaccines for Pneumococcus,

Meningococcus, and Haemophilus infections are given
at about 2 years as before this age they respond poorly
to polysaccharides unless they are associated with
protein components that can act to recruit T cell help
for development of anti- polysaccharide antibody.

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Mucosal Immunization
Most of the infectious agents gain entry to the systemic

system through mucosal route and the largest source of

lymphoid tissue is also present at the mucosal surfaces.
Thus recent vaccination approaches have focussed on the

mucosal route as the site of choice for immunization either

orally or through the nasal associated immune tissue
(NALT).
Moreover, it eliminate the need for painful injection and

allow for self-administration of certain vaccines such as

those for immunization against influenza.
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 Adjuvant vaccines and live vector vaccines have been

used to target mucosal immune system with some

success.
Attenuated strains of salmonella can act as a powerful
immune stimulus as well as acting as carriers of
foreign antigens.
This approach has been used to immunize against
mucosal surfaces against herpes simplex virus and
human papilloma virus.
Bacterial toxins, eg those derived from cholera, E. coli
etc which posses immunomodulatory properties are
also being exploited in the dvpt of mucosally active
adjuvants.
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Table 1. Passive immunization

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Vaccine
Vaccine is a preparation containing

a pathogen
(disease producing organism) either in attenuated or
inactivated state.
This preparation is introduced into an individual to
induce adequate antibody production against the
pathogen in question so that the individual becomes
protected against infection, at a later date, by that
pathogen.
The introduction of a vaccine in an individual is called
vaccination or immunization as it leads to the
development of immunity in the vaccinated
individuals to the concerned pathogen.
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 The immunity is induced by the antigens of pathogen

origin present in the vaccine.

Conventionally, various vaccines can be broadly
classified into two groups:
1. Vaccines containing killed or inactivated pathogens,
i.e. most bacteria vaccines and some virus vaccines
(e.g. influenza virus inactivated by formalin, rabies
virus inactivated by phenol and - priolactone).
2. Those containing live but attenuated pathogens, e.g.,
most virus vaccines.

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 Attenuation means a drastic reduction in the virulence

of a pathogen which is achieved as follows:

Several consecutive passages through an animal,
which is not the usual host of the pathogen, e.g., small
pox virus in calf.
Several passages through cultured cells of the host,
e.g., rabies virus in human diploid cell culture, or of a
different species, e.g., rabies virus, yellow fever virus in
chick embryo cell culture.
Selection of less virulent strains of pathogens, e.g., a
mutant strain of polio virus.
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 Treatment of the pathogen with some chemicals, e.g.,

B.C.G. (Bacillus of Calmette Guerien) vaccine

produced by culturing the bacteria on a medium
containing bile.
Culturing pathogens under unfavourable conditions
like high temp, e.g., anthrax vaccine obtained by
cultivation of the bacterium (Bacillus anthracis) at 4050 0C.
In general, inactivation of virus is always coupled with
attenuation to minimize the accidental presence of
active virulent particles which could cause disease in
the vaccinated individuals.
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 The different vaccines differ in their composition,

efficacy and the duration of effective protection to the

vaccinated individuals. These are one of the earliest
examples of biotechnological intervention in human
and animal health care.

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Types of various vaccines

There are various types of vaccines,
1.

Whole-Organism Vaccines (Conventional vaccines)

2.

Purified Macromolecules as Vaccines (Conventional )

3.

Subunit Vaccine

4. Recombinant- Vector Vaccines

5.

DNA Vaccines
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Whole organism vaccines

Many vaccines now available for humans, and animal

use are made using whole organisms (bacteria or

virus), either in the inactivated (killed) form or
attenuated (live but avirulent) form.
Examples: Salmonella typhi (killed bacteria)against

Typhoid, Salmonella paratyphi (killed bacteria) against

paratyphoid, Vibrio cholerae (killed cells or cell
extract) against cholera, Attenuated virus against
yellow fever, measles, mumps, rubella and polio.

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Preparation and storage of Typhoid-Paratyphoid A and B Vaccine [TABVaccine]

Typhoid fever (enteric fever) is an acute generalized infection caused by
Salmonella typhi ; whereas, paratyphoid fever is caused by Salmonella paratyphi
A and Salmonella paratyphi B.
Preparation

(1) The vaccine is prepared by the general process and contains the following in
each millilitre : Typhoid bacilli (Salmonella typhi) : 1000 million Paratyphi A
bacilli (S. paratyphi A) and Paratyphi B bacilli (S. paratyphi B) : 500 or 750
million.
(2) The smooth strains of the three organisms known to produce the full
complement of O somatic antigens should be used. This specific strain of S.
typhi must contain the virusassociated antigens (Vi-antigen).
(3) It has been duly established that when the organisms were killed with 75%
ethanol and the resulting vaccine preserved with 22.5% ethanol, the potency of
the alcohol treated vaccine was found to be almost double to that of the
heat-treated vaccine, there by minimizing the possibility of both local and
constitutional reaction with the relatively smaller dose. Besides, alcohol treated
vaccines did possess definitely and predominantly longer life under the
optimal storage conditions [viz., storage between 2-4 C without allowing the
vaccine to freeze].
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Purified macromolecules as vaccine

The purified antigenic portions from the bacterial cell

wall, or viral coat protein are used as vaccines, and

they can elicit immune reaction. Examples of
macromolecule vaccines are:
1. Capsular polysaccharides
2. Surface antigens
3. And inactivated exotoxins called toxoids.

The macromolecule vaccines are generally safe since

they dont contain live organism. Example is given in
next slide.
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Preparation of Meningococcal Polysaccharide Vaccine

The Meningococcal Polysaccharide Vaccine consists

of one or more purified polysaccharides obtained

from appropriate strains of Neisseria meingitidis group A,
group C, group Y and group W135 that have been
adequately proved to be capable of producing
polysaccharides that are absolutely safe and also capable
of inducing the production of satisfactory levels of
specific antibody in humans.
The vaccine is prepared immediately before use by
reconstitution from the stabilized dried vaccine with
an appropriate prescribed sterile liquid. It may either
contain a single type of polysaccharide or any mixture of
the types.
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The various Preparation steps adopted are as stated under :

(1) The preparation of the vacccine is based on a seed-lot system. Each
seed-lot is subjected to microbiological examination by culture in an
appropriate media and microscopic examination of Gram-stained
smears.
(2) The polysaccharide shown to be free from contaminating bacteria is
precipitated by the addition of cetrimonium bromide and then
purified.
(3) Each polysaccharide is dissolved under aseptic conditions in a sterile
solution containing lactose or another suitable stabilizing medium for
freeze drying.
(4) The solution is blended, if appropriate, with solution of the
polysaccharides of any or all of the other groups and passed through a
bacteria-retentive filter.
(5) Finally, the filtrate is freeze dried to a moisture content shown to be
favourable to the stability of the vaccine

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Limitation of Conventional Vaccines

Not all infectious agents can be grown in culture and no vaccines

have been developed for a number of diseases, where the

infectious agent is nonculturable.
Production of animal and human viruses requires animal cell
culture, which is expensive.
Yield and rate of production of animal viruses is low.
Extensive laboratory precautions are needed while dealing with
highly infectious agents.
In spite of the best precautions, some batches of vaccines may
not be completely killed or attenuated.
Attenuated strains may revert to pathogenic state, occasionally,
and may cause actual disease against which protection was
sought.
Not all infectious disease are preventable by traditional vaccines
(e.g. AIDS).
Have limited Shelf-life thus requires refrigeration.

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Vaccines made through recombinant DNA technology

Recombinant DNA technology can be best used in the

following ways in vaccine development.

1. Virulence genes can be deleted from the infectious agent
retaining the immunogenic properties.
2. An organism (non-pathogenic) carrying antigenic
determinants can be created by insertion of the genes
coding for the antigenic proteins.
3. For non-culturable agents, genes for the protein (critical
antigenic determinants) can be cloned and expressed in
an expression vector (e.g. E. Coli or a mammalian cell
line).
4. A targeted cell-specific killing system that kills only the
infected cells can be designed. In this technique, gene for
a fusion protein is constructed. First, one part of the
fusion protein binds to the infected cell. Then the other
part kills the infected cell.
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Subunit Vaccines
For viruses, it has been shown that specific protein from

the coat or envelope is enough to elicit the immune

response.
Vaccines with components of a pathogenic organism rather
than the whole organism are called subunit vaccines. rDNA
technology is best suited to develop subunit vaccines.
Purified proteins are more stable and are chemically
precise and safe from side effects. However, purification of
protein can be expensive and sometimes purification can
alter the configuration of protein and alter its
antigenicity!!!
These factors have to be assessed before making a protein
preparation.
One of the example of subunit vaccines developed through
rDNA tech will be discussed in the upcoming slides.
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Subunit vaccine for foot and mouth disease virus (FMDV)

Formalin-killed FMDV was used as vaccine earlier. The

genome of FMDV is single stranded RNA (ssRNA).

The cDNA complementary to this ssRNA, 8000

nucleotide long is prepared . It is digested with

restriction enzymes, and the fragments are cloned in
E. coli.

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Recombinant vector vaccines

Genes that encode major antigens of especially virulent

pathogens can be introduced into attenuated viruses or

bacteria.
The attenuated organism serves as a vector, replicating within

the host and expressing the gene product of the pathogen.

A number of organisms have been used for vector vaccines,

including vaccinia virus (it is most strong candidate as it

is efficient in delivery and expression of cloned genes),
the canarypox virus, attenuated poliovirus, adenovirus,
attenuated strains of Salmonella, the BCG strain of
Mycobacterium bovis and certain strains of streptococcus that
normally exist in the oral cavity.
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 Vaccinia virus has been widely employed as a vector

vaccine. This large, complex virus, with a genome of

about 200 genes, can be engineered to carry several
dozen foreign genes without impairing its capacity to
infect host cells and replicate.
The process of producing a vaccinia vector that carries
a foreign gene from a pathogen is outlined in figure
below.
The genetically engineered vaccinia expresses high
level of the inserted gene product, which can then
serve as a potent immunogen in an inoculated host.
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63

 Like the smallpox vaccine, genetically engineered vaccinia

vector vaccines can be administered simply by scratching

the skin, causing localized infection in the host cells.
Antigen genes introduced into animal cells through
vaccinia virus genome inclues Rabies virus G protein,
Hepatitis B surface antigen, Influenza virus NP and HA
proteins, etc.
If the foreign gene product expressed by the vaccinia is a
viral envelope protein, it is inserted into the membrane of
the infected host cell, inducing development of cellmediated immunity as well as antibody mediated
immunity.
Similar to vaccinia vector vaccines other vector vaccines
which have been recently tried include canarypox virus.
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DNA vaccines/ Gene vaccine (Genetic Immunization)

Plasmid DNA encoding antigenic proteins is injected

directly into the muscle of the recipient. Muscle cells take

up the DNA and the encoded protein antigen is expressed,
leading to both a humoral and cell-mediated response.
The DNA either integrate into the chromosomal DNA or to

be maintained for long periods in an episomal form.

It offers advantage over many of the existing vaccines few of

which are listed below:

1 . The encoded protein is expressed in the host in its natural
form- there is no denaturation or modification . Due to this
the immune response is therefore directed to the antigen
exactly as it is expressed by the pathogen.
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2. It induces both humoral and cell mediated immunity.

3. DNA vaccines cause prolonged expression of the
antigen, which generates significant immunological
memory.
4. Refrigeration is not required for handling and storage
of the plasmid DNA (thus lowers cost and complexity
of delivery).
5. The same plasmid vector could be custom tailored to
make variety of proteins, so the same manufacturing
techniques can be used for different DNA vaccines,
each encoding an antigen from a different pathogen.
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 An improved method of administering these vaccines

involves coating microscopic gold beads with the

plasmid DNA and then delivering the coated particles
through the skin into the underlying muscle with an
air gun (called gene gun). This will allow rapid delivery
of a vaccine to large populations without the
requirement for huge supplies of needles and syringes.
Test of DNA vaccines in animal models have shown

these vaccines to be effective against various viral

diseases including influenza virus.
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Antigen-Antibody Interaction
The antigen-antibody interaction is a biomolecular

association similar to an enzyme-substrate interaction.

However, it doesnt lead to an irreversible chemical

alteration in either the antibody or the antigen.

The association between an antigen and antibody

involves various noncovalent interactions between the

antigenic determinant (epitope) of the antigen and the
variable-region (VH/VL ) domain of the antibody
molecule, particularly the hypervariable regions, or
complementarity- determining regions (CDRs).
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 The noncovalent interactions that form the basis of

antigen-antibody binding include hydrogen bonds,

ionic bonds, hydrophobic interactions, and vander
Waals interactions.
Since these interactions are individually weak
(compared with a covalent bond), a large number of
such interactions are required to form a strong Ag-Ab
interaction.
Furthermore, each of these noncovalent interactions
operates over a very short distance (1 angstrom or 1 x
10-7 mm). Hence a strong Ag-Ab interaction depends
on a very close fit between the antigen and antibody.
Such fit require a high degree of complementarity
between antigen and antibody.
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Cross-reactivity
Although Ag-Ab reactions are highly specific, in some cases

antibody elicited by one antigen can cross- react with an

unrelated antigen. Such cross-reactivity occurs if two
different antigens share an identical or very similar epitope.
Cross- reactivity is often observed among polysaccharide
antigens that contains similar oligosaccharide residues.
The ABO blood-group antigens, for example, are
glycoproteins expressed on RBCs. Subtle differences in the
terminal residues of the sugars attached to these surface
proteins distinguish the A and B blood group antigens.
RBC glycoprotein sugars attached to the terminal end
of it subtle difference in the terminal residues of these
sugars distinguish A and B blood group antigens.
71

 An individual lacking one or both of these antigens will

have serum antibodies to the missing antigen(s).

The antibodies are induced not by exposure to red blood
cell antigens but by exposure to cross-reacting microbial
antigens present on common intestinal bacteria.
These microbial antigens induce formation of antibodies
in individuals lacking the similar blood-group antigens on
their RBCs.
The blood-group antibodies, although elicited by microbial
antigens, will cross-react with similar oligosaccharides on
foreign RBCs. This provides the basis for blood typing tests
and accounts for the necessity of compatible blood types
during blood transfusions. Type A individual has anti- B
antibodies, type B has anti- A and type O has anti- A & B .
72

 Numerous viruses and bacteria have epitopes identical

or similar to normal host-cell components. In some

cases, these microbial antigens have shown to elicit
antibody that cross-reacts with the host-cell
components, resulting in a tissue- damaging
autoimmune reaction.
For e.g. bacterium Streptococcus pyrogenes, express
cell wall proteins called M antigens . Abs produced
against these antigens have shown to cross react with
several myocardial and skeletal muscle proteins
causing kidney and heart damage following
streptococcal infections.
Some vaccines also exhibit cross-reactivity.
73

74

1. Precipitation Reactions
Antibody and soluble antigen interacting in aqueous solution form a

lattice that eventually develops into a variable precipitate.

Antibodies that aggregate soluble antigens are called precipitins.

Although formation of the soluble Ag-Ab complex occurs within

minutes, formation of the visible precipitate occurs more slowly and

often takes a day or two to reach completion.

Formation of Ag-Ab lattice depends upon the valency of both:

1.
Ab should be bivalent; a precipitate will not form with monovalent

Fab fragments.

2.

Ag must be either bivalent or polyvalent i.e. it must have at least two

copies of the same epitope or have different epitopes that react with
different antibodies present in polyclonal antisera.

75

A. Precipitation reaction in fluids

Precipitation reaction in fluids yields a precipitin

Curve.
A quantitative precipitation reaction can be performed
by placing a constant amount of antibody in a series of
tubes and adding increasing amount of antigen to the
tubes. At one time this method was used to measure
the amount of antigen or antibody present in a sample
of interest.
Once precipitate is formed each tube centrifuged to
pellet the precipitate supernatant poured off
amount of precipitate is measured.
76

 Plotting

the
amount
of
precipitate against increasing
antigen concentrations yields a
precipitin curve.
The figure below shows that the
excess of either
antigen or
antibodies
interferes
with
maximal precipitation, which
occurs at equivalence point.
Maximal precipitation occurs
at equivalence point.
As a large macromolecular
lattice is formed at equivalence,
complex increases in size and
precipitate out.
Follow figure and draw it!!!!it is
important to draw figure.
77

78

B. Precipitation Reaction in Gels

Precipitation rxn in gels yields visible precipitin Lines.
Immune precipitates can form not only in solution but also

in agar matrix.
When antigen and antibody diffuses towards one another
in agar, or when Ab is incorporated into the agar and
antigen diffuses into the antibody containing matrix, a
visible line of precipitation will form.
As in precipitation reaction in fluid, visible precipitation
occurs in the region of antibody or antigen excess.
Two types of immunodiffusion reactions can be used to
determine relative concentration of antibodies or antigen ,
to compare antigens, or to determine the relative purity of
an antigen preparation.
79

 Two types of immunodiffusion reactions both of which

are carried out semisolid medium such as agar.

1 Radial immunodiffusion(Mancini method): In this
method, an Ag sample is placed in a well and allowed
to diffuse into agar containing a suitable dilution of
antiserum. As antigen diffuses into the agar, the region
of equivalence is established and a ring of
precipitation, a precipitin ring, forms around the well.
The area of precipitin ring is proportional to the
concentration of antigen. By comparing the area of the
precipitin ring with a standard curve (obtained by
measuring
the
precipitin
areas
of
known
concentrations of the antigen), the concentration of the
antigen sample can be determined.
80

2. Double immunodiffusion (the Ouchterlony method):

In this method both the antigen and antibody diffuse,
radially from the wells towards each other , thereby
establishing a concentration gradient. As equivalence
is reached, a visible line of precipitation, a precipitin
line is formed.

Please refer figure in next slide!!!!!!!!!!!!!!

81

82

2. Agglutination Reaction
The interaction between antibody and antigen results

in visible clumping called agglutination.

Antibodies that produce such reactions are called
agglutinins.
Agglutination reaction are similar in principle to
precipitation reactions; they depend on the
crosslinking of polyvalent antigens.
Just as an excess of antibody inhibits precipitation
reactions, such excess can also inhibit agglutination
reactions; this inhibition is called prozone effect.

83

Its application

a. Hemagglutination is used in blood typing:

Agglutination reactions are routinely performed to type
RBCs.
In typing for the ABO antigens, RBCs are mixed on a slide
with antisera to the A or B blood-group antigens.
If the antigen is present on the cells, they agglutinate,
forming a visible clump on the slide.
Determination of which antigens are present on donor and
recipient RBCs is the basis for matching blood types for
transfusions.
b. Bacterial agglutination is used to diagnose infection.
c. Passive agglutination is useful with soluble antigens.
84

Immuno-assay Techniques
Radioimmunoassay

ELISA
Western Blotting
Immunofluorescence

Immunoelectron Microscopy.

85

Immunoassays
The

exquisite specificity of antigen-antibody

interactions has led to the development of a variety of
immunologic assays, which can be used to detect the
presence of either antibody or antigen.

Immunoassays have played vital roles in diagnosing

diseases, monitoring the level of the humoral immune

response, and identifying molecules of biological or
medical interest. These assays differ in their speed and
sensitivity, some are strictly qualitative, other are
quantitative.

86

Radioimmunoassay
One of the most sensitive techniques for detecting antigen or

antibody is radioimmunoassay (RIA).

Principle: The principle of RIA involves competitive binding of

radiolabeled antigen(usually labeled with gamma emitting

isotope such as 125I but beta emitting isotopes such as tritium 3H
are also routinely used as labels) to a high- affinity antibody. The
labeled antigen is mixed with antibody at a concentration that
saturates the antigen-binding sites of the antibody.
Then

test samples of unlabeled antigen of unknown

concentration are added in progressively larger amounts. The
antibody doesnt distinguish labeled from unlabeled antigen, so
the two kinds of antigen compete for available binding sites on
the antibody.
87

 As the concentration of unlabeled antigen increases, more

labeled antigen will be displaced from the binding sites.

The decrease in the amount of radiolabeled antigen bound to

specific antibody in the presence of the test sample is measured

in order to determine the amount of antigen present in the test
sample.[Note: To determine the amount of labeled antigen
bound, the Ag-Ab complex is precipitated to separate it from
free antigen, and the radioactivity in the precipitate is measured.
A standard curve can be generated using unlabeled antigen
samples of known concentration (in place of test sample), and
from this plot the amount of antigen in the test mixture may be
precisely determined.]
A microtiter RIA has been widely used to screen for the presence

of the hepatitis B virus.

88

89

Enzyme-Linked Immunosorbent Assay

Commonly known as ELISA (or EIA).
Its principle is similar to RIA but depends on an enzyme

rather than a radioactive label.

An enzyme conjugated with an antibody reacts with a

colorless substrate to generate a colored reaction product.

Such a substrate is called a chromogenic substrate.
A number of enzymes have been employed for ELISA,
including alkaline phosphatase, horseradish peroxidase,
and - galactosidase. These assays approach the sensitivity
of RIAs and have the advantage of being safe and less
costly.
90

 Types: 3 types.
Indirect ELISA: used in determination of serum Abs

against HIV.
Sandwich ELISA
Competitive ELISA
In

one of the version of ELISA using

chemiluminescence, a luxogenic (light-generating)
substrate is used in place of chromogenic substrate
(used in conventional ELISA). Its advantage is
increased sensitivity.
91

92

Indirect ELISA
Antibody can be detected or quantitatively determined
with an indirect ELISA (Figure 6-10a). Serum or some other
sample containing primary antibody (Ab1) is added to an
antigen-coated microtiter well and allowed to react with
the antigen attached to the well.
After any free Ab1 is washed away, the presence of antibody

bound to the antigen is detected by adding an enzymeconjugated secondary anti-isotype antibody (Ab2), which
binds to the primary antibody.

Any free Ab2 then is washed away, and a substrate for the

enzyme is added. The amount of colored reaction product

that forms is measured by specialized spectrophotometric
plate readers, which can measure the absorbance of all of
the wells of a 96-well plate in seconds.
93

 Indirect ELISA is the method of choice to detect the presence of serum

antibodies against human immunodeficiency

virus (HIV), the
causative agent of AIDS. In this assay, recombinant envelope and core
proteins of HIV are adsorbed as solid-phase antigens to microtiter
wells.
Individuals infected with HIV will produce serum antibodies to

epitopes on these viral proteins. Generally, serum antibodies to HIV

can be detected by indirect ELISA within 6 weeks of infection.

94

SANDWICH ELISA
Antigen can be detected or measured by a sandwich ELISA

(Figure 6-10b). In this technique, the antibody (rather than

the antigen) is immobilized on a microtiter well.
A sample containing antigen is added and allowed to react

with the immobilized antibody.

After the well is washed, a second enzyme- linked antibody

specific for a different epitope on the antigen is added and

allowed to react with the bound antigen. After any free
second antibody is removed by washing, substrate is added,
and the colored reaction product is measured.

95

COMPETITIVE ELISA
Another variation for measuring amounts of antigen is competitive
ELISA (Figure 6-10c). In this technique, 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.
In the competitive assay, however, the higher the concentration of

antigen in the original sample, the lower the absorbance.

96

Western blotting
Indentification of a specific protein in a complex mixture of proteins can be

accomplished by a technique known as Western blotting.

In western blotting, a protein mixture is electrophoretically separated on an SDSpolyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate
(SDS), a dissociating agent.
The protein bands are transferred to a nylon membrane by electrophoresis and the
individual protein bands are identified by flooding the nitrocellulose membrane
with radiolabeled or enzyme linked polyclonal or monoclonal antibody specific for
the protein of interest.
The Ag-Ab complexes that is formed on the band ,containing the protein
recognized by the antibody, can be visualized in a variety of ways. If the protein of
interest was bound by a radioactive antibody, its position on the blot can be
determined by exposing the membrane to a sheet of x-ray film, a procedure called
autoradiography.
However, the most generally used detection procedures employ enzyme-linked
antibodies against the protein. After binding of the enzyme antibody conjugate,
addition of a chromogenic substrate that produces a highly colored and insoluble
product causes the appearance of a colored band at the site of the target antigen.
The site of the protein of interest can be determined with much higher sensitivity if
a chemiluminescent compound along with suitable enhancing agents is used to
produce light at the antigen site.

97

 Western blotting can also identify a specific antibody in a mixture. In

this case, known antigens of well-defined molecular weight are

separated by SDS-PAGE and blotted onto nitrocellulose.
The separated bands of known antigens are then probed with the
sample suspected of containing antibody specific for one or more of
these antigens.
Reaction of an antibody with a band is detected by using either
radiolabeled or enzyme-linked secondary antibody that is specific for
the species of the antibodies in the test sample.
The most widely used application of this procedure is in confirmatory
testing for HIV, where Western blotting is used to determine whether
the patient has antibodies that react with one or more viral proteins.

98

99

Immunoprecipitation

The immunoprecipitation technique has the advantage of

allowing the isolation of the antigen of interest for further

analysis.
It also provides a sensitive assay for the presence of a particular
antigen in a given cell or tissue type.
An extract produced by disruption of cells or tissues is mixed
with an antibody against the antigen of interest in order to form
an antigen-antibody complex that will precipitate.
However, if the antigen concentration is low (often the case in
cell and tissue extracts), the assembly of antigen-antibody
complexes into precipitates can take hours, even days, and it is
difficult to isolate the small amount of immunoprecipitate that
forms.
When used in conjugation with biosynthetic radioisotope
labelling, immunoprecipitation can also be used to determine
whether a particular antigen is actually synthesized by a cell or
tissue.
100

101

Immunofluorescence
In 1944, Albert Coons showed that antibodies could be

labeled with molecules that have the property to

fluorescence.
If antibody molecules are tagged with fluorescent dye,
or fluorochrome, immune complexes containing these
fluorescently labeled antibodies can be detected by
colored light emission when excited by light of
appropriate wavelength.
Antibody molecules bound to antigens in cells or
tissue sections can similarly be visualized.
The emitted light could be viewed with fluorescence
microscope, which is equipped with UV light source.
102

 In this technique (immunofluorescence), various

fluorescent compounds in use include Fluorescein,

Rhodamine, Phycoerythrin.
Fluorescent-antibody staining of cell membrane
molecules or tissue sections can be direct or indirect.
In direct staining, the specific antibody (the primary
Ab) is directly conjugated with fluorescein.
In indirect staining, the primary Ab is unlabeled and is
detected with an additional flurochrome-labeled
reagent.

103

Immunoelectron microscopy
Specificity of antibody has made them powerful tools

for visualizing specific intracellular tissue components

by immunoelectron microscopy.
In this technique,

Electron-dense label conjugated to Fc portion of a

specific antibody for direct staining or conjugated to
an anti-immunoglobulin reagent for indirect staining
Electron dense label(commonly used are ferritin
and colloidal gold) absorbs electrons it can be
visualised with electron microscope as small black
dots.
104

Monoclonal Antibodies
Monoclonal Antibodies

are usually produced from

hybridoma clones.
Each hybridoma clone is derived by the fusion of a
myeloma cell and an antibody producing lymphocyte,
and the hybridoma clone producing the desired
antibody is identified and isolated.
Hybridoma cells are mass-cultured for the production
of monoclonal antibodies either (1) in vivo in the
peritoneal cavity of mice or (2) in vitro in large scale
culture vessels.

105

Application
When Mabs are used to detect the presence of a

specific antigen or of antibodies specific to an antigen

in a sample or samples, this constitutes a diagnosic
application.
Antibodies specific to a cell type, say, tumor cells, can
be linked with a toxin polypeptide to yield a conjugate
molecule called immunotoxin. This immunotoxin will
bind to tumor cell and kills it.
Immunopurification.

106

ANTIGENS AND HAPTENS

The two terminologies viz., antigens and haptens are intimately
associated with immunology ; and, hence one may understand and have a
clear concept about them as far as possible.

Antigens
An antigen is either a cell or molecule which will bind with preexiting
antibody but will not definitely cause induction of antibody production.
Antigen may also be defined as a macromolecular entity that
essentially elicits an immune response via the formation of specific
antibodies in the body of the host.
In a broader perspective the antigen (or immunogen) is invariably regarded
as the afferent branch of the prevailing immune system, and is any cell or
molecule which would provoke an immune response very much in an
immunologically viable and competent individual. Generally, immunogens
(antigens) must fulfill the following two characteristic features, namely:
(a) should be larger than 2000 in molecular weight, e.g., protein, glycoprotein and
carbohydrates, and
(b) must be absolutely foreign to the individual into whom they have been
introduced appropriately.

107

 Example : The best example of an antigen is ones own

erythrocytes. Because, they will not induce antibody

formation in oneself but will definitely react with an
antibody essentially contained in an improperly matched
blood transfusion.
Quite often an antigen is a protein, but it could also be a
polysaccharide or nucleic acid or any other substance.
Importantly, it may also be possible that a foreign
substance (e.g., protein)-not necessarily belonging to a
pathogenic microorganism, may act as an antigen so
that on being injected into a host, it may induce antibody
formation.
Besides, they may turn out to be antigenic and thereby
cause stimulation of antibody production, incase they are
intimately and lightly get bound to certain
macromolecules, for instance : proteins, carbohydrates and
nucleic acids.
108

Haptens
In usual practice, the relatively smaller, less rigid or rather less

complex molecules usually are not immunogenetic in their purest

form, but may be made so by simply linking them strategically to
either larger or more complex structures. Consequently, the smaller
molecules are invariably termed as haptens ; whereas, the larger
molecules or cells are known as carriers.
Hapten may also be definedas a substance that normally does
not act as an antigen or stimulate an immune response but that can
be combined with an antigen and, at a later time, initiate a specific
antibody response on its own.
Furthermore, small molecules (micromolecular), such as : drug
substances, that may serve as haptens and can normally be made
antigenic by coupling them chemically to a macromolecular
substance e.g., protein, polysaccharide, carbohydrate etc. The hapten
is obtained from a non-antigenic compound (micromolecule) e.g.,
morphine, carteolol etc., which is ultimately conjugated,
covalently to a carrier macromolecule to render it antigenic.

109

 One of the good example is of gastrin (hapten) which is duly

coupled to albumin (i.e., protein carrier) by treatment with

carbodiimides (CCD), which couple functional carboxyl,
amino, alcohol, phosphate or thiol moieties.
Importantly, the hapten-conjugate thus obtained is normally
subjected to emulsification in a highly refined mineral oil
preparation containing-killed Mycobacterium (Complete
Freunds Adjuvant), and subsequently injected intradermally
either in healthy rabbits or guinea pigs on several occasions
at intervals.
Evidently, the serum antibody should have not only high
degree of specificity but also a reasonably strong affinity for
the prevailing antigens.

110

Hypersensitivity
Immune system mobilizes variety of effector molecules that act to

remove antigen by various mechanisms.

Generally, these effector molecules induce a localized inflammatory

response that eliminates antigen without extensively damaging the

hosts tissue. Under certain circumstances, however, this inflammatory
response can have deleterious effects, resulting in significant tissue
damage or even death. This inappropriate immune response is termed
hypersensitivity or allergy.
Although the word hypersensitivity implies an increased response, the

response is not always heightened but may, instead, be an inappropriate

immune response to an antigen. Hypersensitive reactions may develop
in the course of either humoral or cell-mediated responses.
Hypersensitivity may be defined as an abnormal sensitivity to a
stimulus of any kind.
111

 There are four types of hypersensitivity reaction:

Type I hypersensitivity (IgE Mediated

Hypersensitivity)
2. Type II (IgG Mediated Hypersensitivity)
3. Type III (Immune complex mediated
hypersensitivity)
4. Type IV (Cell Mediated Hypersensitivity)
1.

112

1. IgE mediated Hypersensitivity

A type I hypersensitive reaction is induced by certain types of antigens (such as foreign
serum, vaccine, penicillin, rye grass, ant venom, bee venom, etc) referred to as allergens,
and has all the characteristics of a normal humoral response.

That is, an allergen induces a humoral antibody response by the same mechanisms as for
other soluble antigens, resulting in the generation of antibody-secreting plasma cells and
memory cells.

What distinguishes a type I hypersensitive response from a normal humoral response is

that the plasma cells secrete IgE. This class of antibody binds with high affinity to Fc
receptors on the surface of tissue mast cells and blood basophils.

Mast cells and basophils coated by IgE are said to be sensitized. A later exposure to the
same allergen cross-links the membrane-bound IgE on sensitized mast cells and
basophils, causing degranulation of these cells.
The pharmacologically active mediators released from the granules act on the
surrounding tissues. The principal effectsvasodilation and smooth-muscle
contractionmay be either systemic or localized, depending on the extent of mediator
release.
The clinical manifestations of type I reactions can range from life-threatening conditions,
such as systemic anaphylaxis and asthma, to hay fever and eczema, which are merely
annoying
113

General mechanism underlying a type I hypersensitive reaction. Exposure to an allergen activates B cells to form IgE
secreting plasma cells. The secreted IgE molecules bind to IgE specific Fc receptors on mast cells and blood basophils.
(Many molecules of IgE with various specificities can bind to the IgE-Fc receptor.) Second exposure to the allergen leads
to crosslinking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from
mast cells and basophils. The mediators cause smooth-muscle contraction, increased vascular permeability, and
vasodilation.
114

2. Antibody Mediated cytotoxic Hypersensitivity

Type II hypersensitive reactions involve antibody-mediated destruction of cells.
Antibody can activate the complement system, creating pores in the membrane
of a foreign cell, or it can mediate cell destruction by antibody dependent cell-

mediated cytotoxicity (ADCC). In this process, cytotoxic cells with Fc receptors

bind to the Fc region of antibodies on target cells and promote killing of the
cells .Antibody bound to a foreign cell also can serve as an opsonin, enabling
phagocytic cells with Fc or C3b receptors to bind and phagocytose the
antibody-coated cell.
Examples : The various examples are as stated below :
(i) Transfusion reactions i.e., when blood groups are not matched properly,
(ii) Haemolytic disease concerning the newly born babies via Rhesus
incompatibility,
(iii) Graft destruction or rejection i.e., antibody-mediated graft destruction or
rejection.
(iv) Autoimmune reactions usually directed against the formed elements of the
blood, and the kidney glomerular basement membrances, etc.

115

Example 1: Transfusion Reactions Are Type II Reactions

A large number of proteins and glycoproteins on the membrane of red blood cells

are encoded by different genes, each of which has a number of alternative alleles. An
individual possessing one allelic form of a blood-group antigen can recognize other
allelic forms on transfused blood as foreign and mount an antibody response. In
some cases, the antibodies have already been induced by natural exposure to similar
antigenic determinants on a variety of microorganisms present in the normal flora
of the gut. This is the case with the ABO blood-group antigens.
Antibodies to the A, B, and O antigens, called isohemagglutinins, are usually of the
IgM class. An individual with blood type A, for example, recognizes B-like epitopes
on intestinal microorganisms and produces isohemagglutinins to the B-like
epitopes.
If a type A individual is transfused with blood containing type B cells, a transfusion
reaction occurs in which the anti-B iso-hemagglutinins bind to the B blood cells
and mediate their destruction by means of complement-mediated lysis. Antibodies
to other blood-group antigens may result from repeated blood transfusions because
minor allelic differences in these antigens can stimulate antibody production. These
antibodies are usually of the IgG class.
The clinical manifestations of transfusion reactions result from massive
intravascular hemolysis of the transfused red blood cells by antibody plus
complement. These manifestations may be either immediate or delayed.
116

117

Hemolytic Disease of the Newborn Is Caused by Type II Reactions

Hemolytic disease of the newborn develops when maternal IgG antibodies
specific for fetal blood-group antigens cross the placenta and destroy fetal red
blood cells. The consequences of such transfer can be minor, serious, or lethal.
Severe hemolytic disease of the newborn, called erythroblastosis fetalis, most
commonly develops when an Rh+ fetus expresses an Rh antigen on its blood
cells that the Rh mother does not express.
This most commonly happens when a woman with Rh negative blood becomes
pregnant by a man with Rh positive blood and conceives a baby with Rh
positive blood.
Red blood cells from the baby can leak across the placenta into the woman's
bloodstream during pregnancy or delivery. This causes the mother's body to
make antibodies against the Rh factor.
If the mother becomes pregnant again with an Rh-positive baby, it is possible
for her antibodies to cross the placenta and attack the baby's red blood cells.
After birth, an affected newborn may develop kernicterus. This happens when
bile pigments are deposited in the cells of the brain and spinal cord and nerve
cells are degenerated.
Incompatibilities between ABO blood types can also cause this condition.
These are less common than those of the Rh factor and tend to be less severe.

118

3. Immune ComplexMediated (Type III) Hypersensitivity

The reaction of antibody with antigen generates immune
complexes. Generally this complexing of antigen with antibody
facilitates the clearance of antigen by phagocytic cells.
In some cases, however, large amounts of immune complexes can
lead to tissue-damaging type III hypersensitive reactions.
The magnitude of the reaction depends on the quantity of
immune complexes as well as their distribution within the body.
When the complexes are deposited in tissue very near the site of
antigen entry, a localized reaction develops.
When the complexes are formed in the blood, a reaction can
develop wherever the complexes are deposited.
In particular, complex deposition is frequently observed on
blood-vessel walls, in the synovial membrane of joints, on the
glomerular basement membrane of the kidney, and on the
choroid plexus of the brain. The deposition of these complexes
initiates a reaction that results in the recruitment of neutrophils
to the site. The tissue there is injured as a consequence of
granular release of lytic enzymes from the neutrophil.

119

 Type III hypersensitive reactions develop when immune complexes activate the
complement systems array of immune effector molecules. The C3a, C4a, and
C5a complement split products are anaphylatoxins that cause localized mastcell degranulation and consequent increase in local vascular permeability. C3a,
C5a, and C5b67 are also chemotactic factors for neutrophils, which can
accumulate in large numbers at the site of immune-complex deposition. Larger
immune complexes are deposited on the basement membrane of blood vessel
walls or kidney glomeruli, whereas smaller complexes may pass through the
basement membrane and be deposited in the subepithelium. The type of
lesion that results depends on the site of deposition of the complexes.
Much of the tissue damage in type III reactions stems from release of lytic
enzymes by neutrophils as they attempt to phagocytose immune complexes.
The C3b complement component acts as an opsonin, coating immune
complexes.
A neutrophil binds to a C3b-coated immune complex by means of the type I
complement receptor, which is specific for C3b. Because the complex is
deposited on the basement- membrane surface, phagocytosis is impeded, so
that lytic enzymes are released during the unsuccessful attempts of the
neutrophil to ingest the adhering immune complex. Further activation of the
membrane-attack mechanism of the complement system can also contribute to
the destruction of tissue. In addition, the activation of complement can induce
aggregation of platelets, and the resulting release of clotting factors can lead to
formation of microthrombi.

120

121

Type IV or Delayed-Type Hypersensitivity (DTH)

When some subpopulations of activated TH cells encounter certain types

of antigens, they secrete cytokines that induce a localized inflammatory

reaction called delayed-type hyper- sensitivity (DTH).
The reaction is characterized by large influxes of nonspecific inflammatory
cells, in particular, macrophages.
This type of reaction was first described in 1890 by Robert Koch, who
observed that individuals infected with Mycobacterium tuberculosis
developed a localized inflammatory response when injected intradermally
with a filtrate derived from a mycobacterial culture.
He called this localized skin reaction a tuberculin reaction.
The characteristic of a type IV reaction are the delay in time required for
the reaction to develop and the recruitment of macrophages as opposed to
neutrophils, as found in a type III reaction.
In this type, sensitized TH1 cells release cytokines that activate
macrophages or TC cells which mediate direct cellular damage.
Macrophages are the major component of the infiltrate that surrounds the
site of inflammation.

122

123

124

125

126

127

Antibody Production (Immunogen Preparation)

The production of specific antibody probes is a

relatively
straightforward
process
involving
immunization of animals and reliance upon their
immune systems to raise responses that result in
biosynthesis of antibodies against the injected
molecule. Even so, several factors affect the probability
of inducing an immunized animal to produce useful
amounts of target-specific antibodies. Antigens must
be prepared and delivered in a form and manner that
maximizes production of a specific immune response
by the animal. This is called immunogen preparation.
128

 Antibody production is conceptually simple. However, because it

depends upon such a complex biological system (immunity of a living

organism), results are not entirely predictable. Individual animals
even those that are genetically identical will respond uniquely to the
same immunization scheme, generating different suites of specific
antibodies against an injected antigen. Even so, equipped with a basic
understanding of how the immune system responds to injection of a
foreign substance and a knowledge of available tools for preparing a
sample for injection, researchers can greatly increase the probability of
obtaining a useful antibody product.
For example, small compounds (drugs or peptides) are not sufficiently
complex by themselves to induce an immune response or be processed
in a manner that elicits production of specific antibodies. For antibody
production to be successful with small antigens, they must be
chemically conjugated with immunogenic carrier proteins such as
keyhole limpet hemocyanin (KLH). Adjuvants can be mixed and
injected with an immunogen to increase the intensity of the immune
response.
129