Study of immunological products

Study of
Immunological
Products
D-PHARM -1st Year
PRESENTED BY-
Sandhya Punetha

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Immunity
The term immunity is derived from Latin
term- “Immunis” means exempt.
It is deﬁned as the power of the body to
resist the effects of invasion of micro-
organisms.

It is deﬁned as the lack of such ability
to resist infection caused by
pathogenic micro-organisms is called
suspectibility.
Susceptibility

Factors responsible for immunity
Phagocytosis Antibody formation
Factors responsible for immunity

 Phagocytosis is deﬁned as ingestion of bacteria by certain
cells of the body.
PHAGOCYTOSIS
Cells involved in
phagocytosis
Reticulo-
endothelial
cells
These cells
are present
in liver and
spleen,
they digest
the
bacteria
and make
them
harmless.
WBCs
These cells
help in
destruction
of bacteria .

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Antibodies may be deﬁned as substances formed
in the body in response to the presence of foreign
proteins and certain other materials in the tissues.
The nature of the antibodies depends upon the
manner in which pathogenic micro-organisms
produce their harmful effects.
For this purpose pathogenic micro-organisms are
divided as-
a) Bacteria producing exotoxin
b) Bacteria producing endotoxin
ANTIBODY FORMATION

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During the growth, some pathogenic bacteria form
poisonous substances which diffuse through the
bacterial cell wall into the medium in which they grow ;
hence called exotoxin
These toxins are carried to different parts of the body
and thus producing harmful effects.
In response to these endotoxins human body produces
antibody to neutralizes its effect.
The antibodies which are capable of neutralising the
exotoxin are called antitoxin.
1) Bacteria producing exotoxin

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Toxin produced by some pathogenic micro-organisms which
do not diffuses into the cell wall but remains in the cell of the
bacteria are called endotoxin.
Endotoxins are liberated only when bacteria are disintegrated.
In such the antibody produced are very effective against such
bacteria.
These antibodies are named according to their mode of action.
Eg- Opsonins- Antibodies which make bacteria more susceptible
to phagocytosis
Agglutinins- Antibodies which causes aagglutination of bacteria.
Precipitin- Antibodies which precipitate the endotoxins.
Bacteriolysin- Antibodies which prepare bacteria for lysis.
2) Bacteria producing endotoxin

TYPES OF IMMUNITY
Natural Immunity Acquired Immunity
TYPES OF IMMUNITY

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It is also known as innate or non-speciﬁc
immunity.
Immunity which is present since birth is called as
natural immunity.
First line of defense against foreign pathogens or
micro-organisms or infections.
Occur in all types of organisms.
It has no memory.
Natural Immunity

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Age- Large no. of children in the age of 2-5 years
are susceptible to Diptheria disease, whereas
adults are immune to it.
Race- Negroes have a high resistance to yellow
fever, while white races are very susceptible to it.
Species- Male rats are susceptible to typhoid fever
while female mice are immune to it.
Individuals- Some persons have more resistance
against cold and skin diseases than others.
Factors involved in natural immunity

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Immunity which is acquired by an individual
during his life time by producing antibody in the
body is called acquired immunity.
Types of acquired immunity are-
Acquired Immunity

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The body takes an active part in the formation of
antibodies to develop resistance against disease.
Types of active immunity-
Naturally acquired active Immunity- Immunity which is
acquired as a result of infection caused by pathogens or foreign
material. In this the body resist the infection and disease does
not occur.
Example- Small pox; Pneumonia.
Artiﬁcial acquired active Immunity- Immunity which is
provided by antigenic substances like vaccines to stimulate
antibodies inside the body such type of immunity is known as
artiﬁcial acquired active immunity and the process of injecting
living micro-organisms; dead bacteria and bacterial derivatives
is called as immunization.
Active Immunity

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Immunity which is produced by ready-made antibodies
to the body against a disease is called passive
immunity.
Types of passive immunity-
Naturally acquired passive Immunity- Immunity which is
acquired by an individual after birth either naturally or artificially
is called naturally acquired active immunity.
Example- Small pox; Pneumonia
Artificial acquired passive Immunity- Immunity which is
provided by ready-made antibodies by injecting certain
preparation like antiserum or sera into the body are called
artificial acquired passive immunity and it last for short time.
Passive Immunity

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Define immunity and susceptibility
Write the factors responsible for immunity
Define- opsonins; agglutinins; precipitin;
bacteriolysin
What is immunity? Classify different types of
immunity?
What is the mechanism of producing immunity of
cells of reticuloendothelial system?
Explain how WBCs help in fighting against a disease.
Questions-

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These are the biological or proteinous products
meant for prevention of diseases or diagnostic
purposes.
Eg- Vaccines; Sera etc.
Almost all these preparations are administered
by parental routes except Poliomyelitis vaccines
which is administered orally.
This is because the immunological products
become inactive when administered by oral route.
IMMUNOLOGICAL PRODUCTS

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Immunological products lose their potency on storage.
The preservation of potency of immunological
products involves maintaining the viability of living
cells or preventing the denaturation of protein i.e.
antigens or antibodies.
The reduction of potency occur due to the chemical
changes which are directly proportional to the
temperature of storage.
Therefore, all immunological products are required to
be stored below room temperature. For this we store
all immunological products in a refrigerator to ensure a
reasonable life.
Storage of immunological products

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Majority of immunological product should be
stored at dark at temperature between 2 -8
degree Celsius.
The viral vaccines and oral poliomyelitis are more
stable at or below its freezing point.
The bacterial vaccines and antitoxins get
deteriorated if they are allowed to freeze.
The required storage condition of immunological
products are always ﬁxed on the label of the
container.

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Light also cause the deterioration of
immunological product so they must be
protected from light.
The light resistant glass are not recommended
for storage of immunological products because
in that case it becomes diﬃcult to detect the
changes in the product.

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What do you mean by Immunological Products?
What is Active Immunity?
What is Passive Immunity?
Write in brief about storage of immunological
products.
Discuss natural immunity in brief.
Write in brief about Phagocytosis.
Write a note on Artiﬁcial immunity.
Questions-

Classiﬁcation of Immunological
Products

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It includes bacterial vaccines; viral and rickettsial
vaccines and toxoids.
Bacterial Vaccines
Bacterial vaccines are preparations containing
antigens which stimulate the body to produce
antibodies.
These antibodies make the animal or person immune
to that disease for which the vaccine is given.
Immunological Products For Active Immunisation

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Bacterial vaccines are either sterile suspension
of live or killed bacteria or sterile extracts of
derivatives of bacteria.
They may be simple vaccines prepared from one
species or may be mixed vaccines prepared by
mixing two or more simple vaccines from
different species or vaccines.
These vaccines are prepared from cultures
grown on suitable solid or liquid media.
The bacteria are then suspended in normal
solution or freeze dried.

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Vaccines containing living bacteria may be prepared
from stains which are avirulent for man but which can
stimulate the production of antibodies against
pathogenic strains of the same species.
Bacterial vaccines are free from any substance that
are known to cause toxic, allergic or other undesirable
immunological reactions in man. Example- BCG
vaccines [ Bacillus Calmette- Guerin].
Vaccines containing killed organisms may be
prepared by killing the organism by chemical or
physical means provided the antigenic potency of the
vaccine is preserved. Example- Cholera vaccine;
Typhoid vaccine

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BCG vaccine is the form of white pellet which
when reconstituted yields an opalescent
suspension.
Its full form is Bacillus Calmette- Guerin
It is a freeze dried preparation containing live
culture of the bacillus of calmette and guerin
strain of
Mycobacterium bovis.
Example of Bacterial Vaccines-
1) BCG Vaccines

 The bacilli are grown on suitable culture media
until 1mg when plated out on suitable solid culture
media, shows not less than 20 million colonies.
The growth period should not be more than 14
days in
any case.
After a suitable growth, they are separated by
ﬁlteration in the form of a cake
Cake is than homogenised in a grinding ﬂask
Preparation-

And suspended in a suitable medium designed to
preserve the antigenicity and viability of the
vaccine.
The solution is transferred into the ﬁnal sterile
containers and freeze dried.
The containers are than sealed to prevent
deterioration or contamination of vaccine.
The vaccine contains no anti-microbial agent.

 Store in hermetically sealed light resistant glass
containers at a temperature between 2 and 8 degree
Celsius.
Uses-
B.C.G vaccines are used as an immunizing agent
which provides protection against tuberculosis.
Dose-
Prophylactic, 0.1ml as a single dose by
intracutaneous injection.
Storage-

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Cholera vaccine is a colourless, whitish or slightly
coloured opalescent liquid.
It is a sterile suspension of killedCholera vibrios (
Vibrio cholerae) of a strains selected for high
antigenic property and purity.
2) Cholera Vaccine

 It is prepared from equal portions of suspensions
of
Chloera vibrios of
Vibrio cholera.
Inaba and Ogawa strains selected for high antigenic
eﬃciency.
Either a single strain or several strains of each type
may be used.
Each strain ofChloera vibrios is grown separately on
solid medium for 24-48 hours.
Preparation-

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Bacteria are then washed with normal saline solution.
The suspension of bacteria which is obtained is killed
either by heating at 56˚C for one hour or adding
alcohol or other bactericide, such as phenol or
formaldehyde.
All precautions are taken to ensure its antigenic
property.
This preparation is than standaridised so that 1ml of
the vaccine contains not less than 12,000 millions
bacteria.
After adding suitable preservative, it is transferred into
ﬁnal containers and then sealed.

 The vaccine must comply with the tests for sterility and
also the test for undue toxicity of vaccine.
Storage
Store at a temperature between 2- 8˚C
Uses
It is used for immunisation against Cholera but it has a
limited use as it has only about 50% effectiveness for a
period of 3-6 months. The vaccine does not prevent
transmission of the disease.
Dose
Prophylactic, initial dose 0.5ml ; second dose ,1ml after
an interval of 4 to 6 weeks.

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It is available as more or less turbid, whitish liquid
nearly odourless or having faint odour due to
antimicrobial agent.
It is a sterile bacterial suspension of killedPertussis
bacilli (
Bordetella pertussis) of a strain or strains
selected for high antigenic eﬃciency.
3) Pertussis Vaccine (Whooping Cough Vaccine)

 It is prepared by culturingBordetella pertussis in a
suitable culture media.
It is then separated, washed and suspended in normal
saline solution.
The bacteria are killed either by heating or by adding
some chemicals.
The suspension is standardised. The vaccine may
show abnormal toxicity in animal tests and this is
removed by cold storage for upto 3 months.
Preparation-

Storage
Store at a temperature between 2-8˚C
Uses
It is used for active immunisation of children against
whooping cough especially when Diphtheria and
Tetanus toxoids and pertussis vaccine (DPT) causes
untoward reactions .
Dose
It is administered by subcutaneous injection in three
doses of 0.5ml ,1ml and 1.5ml and atleast 4 weeks
apart.

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Typhoid vaccine is white or creamy white turbid
liquid free from clumps.
It is a sterile suspension prepared from one or more
strains of
Salmonella typhi that are smooth and
have the full components of O, H and VI antigens.
4) Typhoid Vaccine

Salmonella typhi organisms are grown on a suitable
culture media.
The bacteria are killed by heat or bacteriocide such
as phenol, formaldehyde or chemicals like acteone.
It is then standardised, so that 1 ml of typhoid
vaccine contains not less than 1000 million bacteria.
The vaccine must comply with tests for sterility and
toxicity of vaccine.
Preparation-

Storage
Store at a temperature between 2 and 8˚C. The
vaccine must not be frozen.
Uses
It is used for the immunisation against infections
caused by typhoid bacilli.
Dose
Prophylactic, initial dose 0.5 ml followed by second
dose of 1 ml by subcutaneous injection after an
interval of 4 to 6 weeks.

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Viral and Rickettsial Vaccines
These are the suspensions of viruses or rickettsial.
They are prepared from infected tissue of blood
obtained from artiﬁcally infected animals, from
cultures in fertile eggs or from cell of tissue cultures.
Viral vaccines may be live or killed. Live vaccines are
usually prepared from attenuated strains of speciﬁc
organism.
Killed vaccine may be inactivated by suitable
chemical or physical means,These vaccines may be
freeze dried.

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Small pox (Freeze dried ) is almost white powder
which reconstitutes to yield a viscid, straw coloured
liquid.
Small pox vaccine contains living attenuated
vaccinia virus.
The vaccine is prepared by 2 methods-
a) By using animals
b) By using eggs
Examples of Viral and Rickettsial Vaccines-
1) Small pox Vaccine ( Freeze-Dried)

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a)
Small pox vaccines are prepared by using calves or sheep.
The method of preparation can be divided into the following
steps-
Selection of animal free from disease-
Healthy animals are used for the production of vaccines.
Animals are kept for 10-14 days in an isolated area under
observation.
They are given thorough examination to exclude
communicable diseases.
Preparation of smallpox vaccine using animals

b) Preparation of animal for sacrification-
The abdomen and flanks are thoroughly scrubbed,
washed and disinfected.
For this the animal is taken into special room where
abdomen and flanks are shaved, scrubbed and then
thoroughly disinfected.
c) Inoculation-
Light incisions are made in the cleared skin without
drawing blood with the help of scarifier.
The sacrified area is then rubbed with some of seed
vaccine of known potency

d)Incubation-
During next 7-9 days vesicles or pustules form along the
line of scariﬁcation.
During the incubation period, every precaution is taken to
keep the animal as clean and aseptic as possible.
Any animal showing any sign of disease is rejected.
e) Collection of viruses-
Animal is taken to the operation table and killed.
A post mortem is done to detect the presence or
absence of disease
Abdomen and ﬂanks are washed with sterile water.

a)
b)
The material in the pustules is withdrawn with the
help of a sharp edged spoon under aseptic conditions.
f) Purification-
The contents of pustules are mixed with equal
volume of glycerin , cooled and then finely ground to
form homogeneous mixtures.
It is then stored for a long time at -10˚C to remove
impurities. This is the old method of purification.
The effective methods that are used nowadays are-
By using brilliant green
By using Trichlorofluoro ethane

c) By adding 0.4% phenol and then incubated at 22˚C
for 2 days.
d) By treating with a mixture of glycerin and peptone
and then storing it at -10 ˚C
g) Filling; sealing and storage-
It is ﬁlled into the ﬁnal container under aseptic
conditions and freeze dried.
The containers are sealed to exclude the possibility of
any contamination.

 Eggs of hens are examine and selected for fertility.
Eggs are incubated for 12 days.
On the following day a small portion of the shell of the
egg is removed and the chorio-allantoic membrane is
inoculated with seed vaccine is known potency.
The portion of the shell is replaced and sealed in
position with a little melted oaraﬃn wax.
Preparation of Small pox vaccine by using eggs

The eggs are incubated for 72 hours.
Using aseptic precautions the shell is removed and
chorio-allantoic memberane is separated.
It is placed in sterile solution at 0˚C. Maintaining a low
tempersture,50% glycerine is added.
The material is grounded to produce a homogenous
suspension. It is then transferred into ﬁnal containers,
freeze dried and sealed.

 Store at a temperature between 2-8 degree Celsius.
Uses
It is used for immunisation against small pox.
Dose
For prophylaxis of smallpox, about 0,02ml is applied
to the skin and inoculated by scariﬁcation or pressure.
Storage-

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It is white, slightly yellow or lightbrown scale or
powder. The vaccine is prepared before use by
reconstitution of the dried vaccine with normal
saline solution.
Yellow fever vaccine is an aqueous suspension of
chick embryo tissue infected with the strains of
yellow fever virus which is virulent for mice, but
although avirulent of man ,still retains its
immunising eﬃciency.
2) Yellow Fever Vaccine

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The virus is injected into the embryo of fertile eggs
which have been incubated for 7-8 days.
After incubation for further 3 to 4 days , the embryos
are removed,pooled in batches,ground and then
extracted with puriﬁed water.
Suspension is then centrifuged and the supernatant
liquid is separated, to which a suitable preservative
may be added
Preparations-

 It is then transferred into sterile glass ampoules and
freeze dried.
The air is removed from ampoules or replaced by
oxygen-free nitrogen before these are sealed.
Storage
It is stored in dark at temperature 0 degree Celsius
and the vaccine loses its potency within a few days.

Uses
It is used to stimulate the production of antibodies
against yellow fever. It develop immunity within 7
days and last for several years. It is recommended for
the age group of 6 months or older.
Dose
Prophylactic, by subcutaneous injection not less than
1000 LD50 doses.

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The vaccine is white or nearly white friable mass.
Measle vaccine live is a bacterically sterile aqueous
suspension of a suitable live, attenuated strain of
measles virus grown on cultures of chick embryo
cells.
Preparation-
Strain of attenuated measles virus used in the
manufacture of live vaccine is tested on the monkeys
for freedom from neuro virulence.
The strain is grown with aseptic precautions in
cultures of chick embryo cells.
3) Measles Vaccine Live

The chick embryos are derived from healthy and
pathogens free ﬂocks.
Only primary cell cultures are used in the
manufacturing of vaccine.
The growth of virus is done within 14 days of
inoculation. The virus suspension is than evaluated for
identity, sterility and anti-viral agents.
The cultures which pass these tests are pooled and
clariﬁed to remove intact tissue cells. A suitable
stabiliser is added and it is distributed into sterile
containers, freeze dried and sealed.

Storage -
Vaccine is stored in a light resistant container at a
temperature 2 to 8 degree Celsius.
Uses-
It produces active immunity against measles in
approximately 99% of recepients of a single dose and
this immunity lasts for many years.
Dose-
Paediatric, by subcutaneous injection, 0.5 ml of
reconstituted vaccine.

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It is clear liquid which may have a reddish colour if
phenol red has been used in its preparation.
Poliomyelitis vaccine (oral) is an aqueous
suspension of one or a combination of 2 or 3 types
of live attenuated strains of poliomyelitis virus
tested for neuro- virulence on monkeys.
4) Poliomyelitis Vaccine (Oral)

 It is prepared by using 3 strains of poliomyelitis virus
type-I,II,III. The virus of each type is grown in suitable
culture medium in aseptic precautions.
Tissue should be from extraneous micro-organisms
and adventitious agents.
Suitable antibiotics other thanPenicillin and
Streptomycin may be used in small concentration.
The temperature of growth medium should not be more
than 37˚C
Preparation-

The growth of bacteria is done within 4 days of
inoculation and the virus suspension is tested.
Final vaccine is prepared by mixing the dilution of 3
types of virus type and adding appropriate bacteriocide.
Storage-
The vaccine is stored in single or multiple dose
containers in frozen state at -28˚C.
Uses-
It is used as active immunisation against poliomyelitis.
Dose-
It is a monovalent vaccine, 6 to 8 weeks apart and fourth
reinforcing dose of the trivalent vaccine, 8 to 12 months
later.

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The vaccine is white, ﬂocculent suspension in a
clear liquid or white to brownish white turbid liquid.
Rabies vaccine is a sterile suspension in saline or
other appropriate solution, isotonic with blood, of a
suitable killed rabies virus in uncontaminated brain
tissues from animals previously injected
intracerebrally with rabies virus.
5) Rabies Vaccine

It is prepared by injecting sheep, rabbit,rat, mice or
other animal intracerebrally with rabies virus.
Animals that show typical paralysis are killed. The
brain are harvested under aseptic condition and then
tested for test of bacteria and ﬁnally suspended into
sodium chloride injection.
The suspension is inactivated by using phenol or beta
propiolactone or any other means until the virus will
no longer infect mice.
Preparation-

It is than diluted to produce a vaccine of required
strength.
The vaccine is preserved by adding 0.01% w/v
thiomersal and the pH is maintain between 7 to 7.2.
It is then transferred into sterilised containers and
sealed.
The vaccine must comply with the test of sterility and
toxicity.
Storage-
It is stored in a container protected from light at a
temperature of 2 to 8˚C.

Uses-
It is used as prophylactic against rabies.
Dose-
By subcutaneous injection 1 to 5 ml daily for 7 to 14
days according to the site and severity of the bite and
the risk of exposure to infection.

 Typhus vaccine is a sterile suspension of the killed rickettsia
organisms of a strain or strains of epidemic typhus rickettsia
( Rickettsia prowazeki) selected for antigenic eﬃciency.
Preparation-
It is prepared by injecting virulent rickettsia into the yolk sacs
of fertile eggs which have been incubated for 7 days for a
period of 9-13 days in aseptic conditions.
It is subjected to suitable treatment to yield maximum number
of rickettsia.
Material is suspended in normal saline solution.
6) Typhus Vaccine

Formaldehyde solution is added at a concentration is
between 0.2% - 0.5%
The suspension containing 10-15% of yolk sac tissue
is puriﬁed with the solvent ether.
The aqueous middle layer is collected and distributed
into sterile containers under aseptic precautions.
Vaccine should comply the test of sterility and toxicity.

Storage-
Store at a temperature between 2˚C and 8 ˚C.
Uses-
It is a prophylactic agent to protect against epidemic
typhus.
Dose-
Prophylactic, by subcutaneous injection, 0.25 to 1 ml.

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The pathogenic bacteria during their growth in a liquid
media release a toxic substance known as toxins.
These toxins are disease producing and are antigenic in
nature.
These toxins cannot be used for immunisation because
of their toxicity.
When these toxins are treated with some chemicals like-
formaldehyde their toxic properties are destroyed
without causing any signiﬁcant loss of antigenic
properties. These are called toxoids.
TOXOIDS

It is a modified form of exotoxin ofCorynbacterium
diphtheriae.
Preparation-
A strain ofCorynbacterium diphtheriae is grown in a
liquid media at 47˚C for 7-10 days until required
amount of toxin is obtained.
Phenol is now added to kill micro-organism and filtered
through bacteria proof filters
Now toxin is converted to toxoids having same
antigenic property.
Examples of Toxoids-
1) Diphtheria Toxoid

1) Formol Toxoid (F.T)- It is prepared by adding 1%
formaldehyde into toxin and incubated at 37˚C for
2-3 weeks. Due to undesirable reactions produced
with this vaccine, it is not used commonly used
these days.
2) Toxoid Antitoxin Floccules (TAF)- It is prepared by
adding Dipththeria antitoxin to formol toxoid and the
ﬂoccules or precipitates thus obtained are suspended
in normal saline solution. It has weaker antigenic
properties but is free from harmful antigenic reactions
due to which it is useful for sensitive persons.
Types of Diptheria Toxoids

3) Alum Precipitated Toxoid (APT)- It is prepared by
treating formal toxoid with an adequate quantity of
potash alum which precipitates the diphtheria toxoid.
The precipitates are separated , washed and
suspended in normal saline solution containing some
preservative.
4) Puriﬁed Toxoid Aluminium Phosphate ( PTAP)- It is
prepared by treating formol toxoid with hydrated
aluminium phosphate.

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This is prepared from the exotoxin ofClostridium
tetani, the speciﬁc toxicity of which been completely
removed by the action of chemical substances in
such a way that it retains its antigenic properties.
Tetanus toxoid are of following types-
1) Formol Toxoid (F.T)- It is prepared by treating the
sterile culture ﬁltrate of
Clostridium tetani with
formaldehyde solution.
2) Tetanus Toxoid

2) Alum Precipitated Tetanus Toxoid - It is prepared
by treating tetanus toxoid with an adequate quantity
of potash alum in simple solution. The precipitates
are separated , washed and suspended in normal
saline solution.

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It is white turbid liquid, and is a sterile suspension of
purified diphtheria vaccine and purified tetanus vaccine
absorbed on a mineral carrier such as aluminium
hydroxide or aluminium phosphate.
Preparation-
Vaccine is prepared by mixing purified diphtheria toxoid
containing not less than 1500 flocculation equivalents.
And purified tetanus toxoid containing toxoid not less
than 1000 flocculation equivalents in a normal saline
solution.
3) Diphtheria and Tetanus Vaccine (adsorbed)

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The vaccine contains a preservative other than any
of the phenol or cresol.
The vaccine must comply with the test of sterility
and toxicity.
Storage-
The vaccine is stored in a single dose or multi dose
container at a temperature between 2 to 8˚C.

 Uses-
The vaccine provides active immunisation against
diptheria and tetanus. The product should not be used
after 6 years because the diphtheria toxin present in the
vaccine may cause undesirable changes.
Dose-
The vaccine is administered in 3 doses by intramscular
injection. The ﬁrst dose is of 0.5ml followed by second
dose of 0.5 ml after 4 to 6 weeks, a third dose of 0.5ml
after 6 to 8 months later.

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It is a white sterile suspension prepared by
adsorbing formaldehyde treated diphtheria toxoid
and tetanus toxoid on a mineral carrier such as
aluminium hydroxide or aluminium phosphate and
adding a suspension of killed
Bordetella pertusis.
Preparation-
Diphtheria toxoid containing not less than 1500
ﬂocculation equivalent and tetanus containing not
less than 1000 ﬂocculation equivalent per mg of
protein nitrogen are added to suspension of hydrated
aluminium phosphate in a normal saline solution.
4) Diphtheria, Tetanus and Pertusis Vaccine
(absorbed)

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Storage-
It is stored in a single dose or multiple dose containers
at a temperature between 2 to 8 ˚C. The vaccine should
not be frozen.
Uses-
The vaccine is used for simultaneous immunisation
against diphtheria, tetanus and pertusis in infants and
small children. The preparation is not generally used
after the 6th year of age.
Dose- The vaccine is administered in 3 doses by
intramscular injection. The ﬁrst dose is of 0.5ml
followed by second dose of 0.5 ml after 4 to 6 weeks, a
third dose of 0.5ml after 6 to 8 months later.

1)
2)
3)
4)
5)
6)
7)
8)
9)
Deﬁne the term Vaccine?
Give some examples of bacterial vaccines.
Name the vaccines prepared from virus.
Deﬁne the term toxoids.
Discuss in brief about B.C.G Vaccine.
Give in method of preparation of smallpox vaccine.
Write in brief about tetanus toxoid.
What is the storage condition of oral poliomyielitis
vaccine?
What are the preparation which produce active
immunisation? Explain different types of vaccines with
suitable example. Write in brief about rabies vaccine.
QUESTIONS-

10) Describe brieﬂy- B.C.G Vaccine; Smallpox vaccine;
Measles vaccines; Rabies vaccine.
11) Differentiate between- Toxoin and toxoid
12) Write short note on Bacterial Vaccine.




These products contain antibodies which produce
passive immunity.
These preparations are mainly used for the
treatment of diseases.
When a person or animal has been actively
immunised either by natural or artiﬁcial means, the
blood contains a large number of antibodies.
IMMUNOLOGICAL PRODUCTS FOR PASSIVE
IMMUNISATION





If blood is withdrawn and allowed to clot , the
antibodies are found in the serum.
The serum is called antitoxin, if the antibodies it
contains are antitoxin.
The serum is called antiserum, if the antibodies in it
are antibacterial antibodies.
If the antibodies in it are antiviral, the serum is
known as antiviral serum.



a)
b)
c)
A majority of the serum preparations are made from
the serum of animals (Horse).
The parenteral administration of the products of
animal origin may lead to immediate or delayed
hypersensitivity reactions.
Antitoxins
The important antitoxins preparations are-
Diphtheria anti-toxins.
Tetanus anti-toxins.
Gas gangrene antitoxins.



1)
It is almost colourless, very faintly yellow or a slightly
opalescent liquid.
Preparations-
The method of preparation of diphtheria anti-toxin is
divided into following stages-
Preparation of toxin for active immunisation of the
horse- A pure culture of
Corynebacterium
diphtheriae is grown in a suitable culture media at
37˚C for 4-7 days. After incubation, 0.5% phenol is
added and the culture media is filtered through
bacteria proof filters. The filtrate is acrude toxin. It is
converted into toxoid.
1) Diphtheria Anti-toxins

a)
b)
c)
d)
2) Selection of the horse-
Horses are selected because-
They are easy to handle.
They readily produce antitoxins because a horse
resembles man in having a natural immunity
against diphtheria.
R.B.C of the horse blood settles quickly and packs
tightly. This property helps in the separation of the
serum.
Considerable volume of blood can be taken without
any ill effects.
The horses selected must be free from diseases and
are isolated for 7 days and are immunised against
tetnus by injecting tetanus toxoid.

3) Active immunisation of the horse-
To the selected horses diphtheria toxoid is given for
active immunisation. The toxoid is given byIV/ IM
routes in the gradually increasing doses.
The ﬁrst dose is of about 5ml, further injections are
given at a interval of 2-3 days, by doubling the volume
of injecting each time. In this way the volume of the
last dose is about 600 ml.

4) Separation of serum from the horse-
After a period of about 10 days from the ﬁrst dose, the
blood is collected under aseptic conditions to see if the
adequate antitoxin has been obtained.
Eight litres of blood is collected three times during a
period of 8 days. After a rest of 2 weeks, further
injections of diphtheria toxoids are given as per the
past schedule to stimulate the production of antibodies.
Again 24 litres of blood is collected in the 3 batches of
8 litres each. The process is again repeated, but it
should not be more than 4 or 5 times.



After the collection of the blood, it is allowed to clot
to separate the serum. The serum contains antitoxin
along with other proteins such as beta- globulins,
gamma globulins and albumins.
Antitoxins are largely associated with beta- globulin.
5) Concentration and reﬁnement- Horse serum
contains a high concentration of several other
proteins which may cause undesirable reactions such
as anaphylactic shock or serum sickness.

So these undesirable proteins are separated by the
following methods-
Concentration by fractional precipitation- The serum
is mixed with suﬃcient ammonium sulfate to produce
25-33% saturation.
The gamma globulin is precipitated which is
separated and discarded.

More ammonium sulfate is added to produce 50%
saturation.
The beta globulin along with anti9toxin is precipitated.
The serum is ﬁltered to remove liquid portion
containing albumin.
The ﬁltrate is rejected.

The precipitates are subjected to dialysis against
sterile water to separate ammounium sulphate.
The precipitates are passed into solution containing
0.3% tricresol or any other suitable preservative or the
preparation is freeze dried without any preservative.
The antitoxin activity of the original serum is increased
only about 4 times.
Concentration of fractional proteolytic digestion- The
serum is diluted three times its own volume with
normal saline solution. Pepsin is added and pH is
adjusted to 4. It is incubated for about 2 days at 37˚C.

Gamma globulin is precipitated which is precipitated
which is separated by filteration. The filtrate is subjected
to special ultra filter.
The transparent liquid is obtained which contains purified
concentrated antitoxin together with beta globulin.
In this way the antitoxin activity of the original serum is
increased about eight times.
Diphtheria antitoxin has a potency of not less than 1000
international units/ ml in the case of the antitoxins
obtained from horse serum and not less than 500
international units per ml for anti toxins obtained from
other animals.

i)
ii)
Storage-
It is stored in containers protected from light at a
temperature between 2 and 8˚C. It should not be
allowed not be allowed to freeze.
Dose-
It is administered by s/c or i/m injection in the
following dose schedule.
Prophylactic- 500 to 2000 international units.
Therapeutic- not less than 10,000 international
units.



i)
ii)
It is almost colourless or a very faintly yellow liquid.
Preparation-
It is prepared in the same way as diphtheria antitoxin. The
toxin is obtained from
Clostridium tetani which is used for
active immunisation of the horse.
Storage-
Same as diphtheria antitoxin.
Dose-It is administered by s/c or i/m injection in the
following dose schedule.
Prophylactic- 1500 international units.
Therapeutic- not less than 50,000 international units.
2) Tetanus Antitoxin

These preparations are antiviral and produce passive
immunity.
Example- 1) Rabies Antiserum
It is almost colourless or faintly yellow, slightly
opalescent liquid free from suspended liquid.
Preparation- Suspension of dead rabies viruses are
injected into healthy horses. After a speciﬁed time
period the blood is collected and by a suitable
method, the gamma globulin is separated which
contains anti-viral antibodies.
Antiviral Serum

Storage-
It is stored in a single dose or multi dose containers at
a temperature between 2 to 8˚C. It is protected from
light.It should not be allowed not be allowed to freeze.
Uses-
It is used along with vaccine for preventing rabies when
a person is bitten in an area.
Dose-
By i/m or s/c injection, 40 international units per kg of
body weight.

Antibacteial Serum
This type of serum is used to provide passive
immunity to diseases caused by endotoxin producing
bacteria, eg- Pneumonia, meningititis and typhoid.
The serum contains antibacterial antibodies.
The antibacterial serum or antiserum were used in the
past but have now been replaced by the
chemotherapy.

Preparation-
1) The horse is injected with a dead or living
suspension of the organism. The intravenous route is
used because reactions often follow the injection of a
large number of bacterial cell by i/m.
2) There are different methods of reﬁning the sera.




These preparations produce passive immunity. The
following are the two basic preparation-
Human normal immunisation.
Dried human immunisation.
Immune Blood Derivatives




It is transparent or slight opalescent liquid,
colourless or brownish in colour which on storage
may show a slight granular deposits.
It is a sterile solution containing antibodies derived
from human blood.
It contains gamma-G globulins together with
smaller amounts of other plasma proteins obtained
from blood, blood plasma or blood serum of healthy
donors.
1) Human Normal Immunoglobulin

 Preparations-
It is prepared from pooled material of a minimum
collection of 25L which is collected from 1500 donors.
The globulins are separated and are dissolved in
suitable vehicle by adding some preservative and
stabilising agent.It is then filtered and filled in the final
container.
Storage- Preparation is stored in a colourless glass
container at a temperature 2 to 10˚C.
Uses- Used for the prevention of measles in children,
infective hepatitis and rubella in pregnant women.

Dose-
By intramuscular injection, following amount of
protein should be administered-
1- For prevention of measles- 250 mg for infants
under one year of age, 750mg for children aged three
years and for attenuation of measles 250 mg.
2- For prevention of rubella in pregnant women-
750mg.
3- For prevention of infective hepatitis – 250mg upto
10 years of age and 750 mg over 10 years.





It is white or slight yellowish powder or solid which
is completely soluble in water for injection.
Preparation- It is prepared from pool of human
normal immunoglobulin by freeze drying. The ﬁnal
solution of human normal immunoglobulin is
distributed into its ﬁnal sterile containers. Freeze
dried and the containers are sealed.
Storage- It is stored in a cool place, protected from
light.
Dose- Same as human normal immunoglobulin.
2) Dried Human Normal Immunisation.





These preparations which contain bacterial toxins,
are used to test for-
Immunity or susceptibility to a particular infection.
The presence of a particular disease.
A proper degree of protection after immunisation.
Immunological product as diagnostic agent-



It is a clear, colourless liquid or very pale straw
coloured liquid, which is used in the shick test to
determine susceptibility to diphtheria.
Preparation- It is a sterile ﬁltrate, prepared from a
culture ofCorynebacterium diphtheriae grown on a
sterile liquid medium. The ﬁltrate contains diphtheria
exotoxin. It is then diluted so that the test dose is
contained in 0.1 ml to 0.2 ml with a diluent isotonic
with blood and containing suitable bactericides and
stabilisers. It is then transfer to the sterile containers.
Shick Test Toxin



Storage-
It is stored at a temperature between 2 and 10 ˚C.
Dose-
0.1ml by intradermal injection.
Shick control or schick test control
It is transparent liquid. Schick control is in fact a
schick test toxin, the toxin of it is destroyed by
heating at a temperature between 70 and 80 ˚C for 5
minutes. The schick control must be prepared from
the same batch of schick test toxin as that with which
it is to be used.


Storage-It is stored at a temperature between 2 and 10
˚C.
Dose-
0.1ml by intradermal injection.
Schick Test
It is performed to test the degree of immunity of an
individual against diphtheria.
Schick test toxin is injected intradermally into the left
forearm and at the same time an equal volume of the
Shick control is given in the right forearm.The arms are
exmined under 48 hours.

 Observations-
S.No. Left arm
(Schick test toxin)
Right arm
(Schick control)
Explanation Result
1) Large flushed
area
No reaction Toxin is not
neutralised
Not immune
2) No reaction No reaction Toxin is
neutralised
Immune
3) Large flushed
area
Smaller flushed
area
Not
neutralised
sensitivity to
broth
constituents
Not immune
4) Small flushed
area
Small flushed
area
Neutralised
sensitivity
Immune and a
pseudo-reactor



It is transparent, yellow to brown viscous liquid,
produced by the concentration of a ﬂuid medium on
which
Mycobacterium tuberculosis has been grown.
Preparation- Pure culture ofMycobacterium
tuberculosis has been grown on a liquid medium
containing 5% w/v of glycerin at about 37 ˚C for 6
weeks or more.
The growth should be rapid and abundant. The culture
is heated and ﬁltered under reduced pressure. A
suitable preservative is added like 0.5% w/v of phenol.
It is than diluted and transferred to the container.
Old Tuberculin




Storage-
It is stored in dark away from light at a temperature
between 2 and 10 ˚C.
Dose- 0.1ml by intradermal injection.
Tuberculin Puriﬁed Protein Derivative or Tubeculin (PPD)
It is a clear, colourless liquid or very pale straw
coloured powder or pellet, made from heat treated
products of growth and lysis of
Tubercle bacillus.

Preparation-
Mycobacterium tuberculosis has been grown in a
suitable medium which is used in the preparation of old
tuberculin.
The culture is filtered to remove bacteria and then
subjected to ultra filtration to remove the glycerin and
mineral salts.
The proteins are precipitated through ammonium
sulphate and purified and converted to freeze dried
powder or solution which is hypodermic.



Storage-
It is stored in dark away from light at a temperature
between 2 and 10 ˚C.
Dose- 0.1ml by intradermal injection containing 10
or 100 international units.

• 5 tuberculin units
old tuberculin dose
is injected after 48
to 72 hours
reaction of test is
observed.A
positive reactive
consists of a
raised indurated
area. Induration of
5 to 9mm is
regarded as
positive
Mantous Test
Tuberculin Test

• In this stainless steel disc
with 4 times 2 mm long
attached to a plastic
handle. The plastic are
dipped into old tuberculin
solution.
Tine test






Write short notes on- a) Old tuberculin; b) Schick test toxin
Write about exotoxin and endotoxin
Write about- rabies vaccine; diphteria antivaccine; measles
vaccines.
How are antitoxin differs from antiserum? Write in brief
about diphtheria antitoxin.
Explain the term Vaccine. What are different types of
vaccines? Write the method of preparation of smallpox
vaccine.
QUESTIONS-

THANK YOU
Presented By-
Sandhya Punetha
( Assistant Professor)

Study of immunological products

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