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.

 Susceptibility is defined as the lack of such ability to resist infection caused by

pathogenic micro-organisms is called susceptibility.

Factors responsible for immunity

 Phagocytosis is defined as ingestion of bacteria by certain cells of the body.

 Antibody formation ANTIBODY FORMATION

 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

1. Bacteria producing exotoxin

 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 neutralizing the exotoxin is called antitoxin.
2. Bacteria producing endotoxin
 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 agglutination of bacteria.

Precipitin- Antibodies which precipitate the endotoxins.

Bacteriolysin- Antibodies which prepare bacteria for lysis.

Natural Immunity

 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.

Factors involved in natural immunity

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

Acquired Immunity

 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

a. Active Immunity

The body takes an active part in the formation of antibodies to develop resistance against disease.

Types of active immunity-

i. 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.
ii. 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.
b. Passive Immunity

Immunity which is produced by ready-made antibodies to the body against a disease is called
passive immunity.

Types of passive immunity-

i. 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
ii. 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.

IMMUNOLOGICAL PRODUCTS
  These are the biological or proteinous products meant for prevention of diseases or
diagnostic purposes.

Classification of Immunological Products

The immunological products can be classified into three categories on the basis of their use.

1. Immunological products for active immunization: These in-clued:

 Bacterial vaccines
 Viral and rickettsial vaccines
 Toxoids.

2. Immunological products for passive immunization: It includes preparations containing

antitoxins and antiserum to produce passive immunity in human beings.

3. Immunological products as diagnostic agents: These are preparations containing toxins or

preparations used for tuberculin and shick test.

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.

Storage of immunological products

 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.
  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.
 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.

Classification of immunological products

 Immunological Products for Active Immunization

*It includes bacterial vaccines; viral and rickettsial vaccines and toxoids.

1. 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.
 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.
 Vaccines containing living bacteria may be prepared from stains which are a virulent 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

Example of Bacterial Vaccines-

1) BCG Vaccines

 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.

Preparation-

 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 filtration in the form of a cake
 Cake is than homogenized in a grinding flask 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.

Storage

 Store in hermetically sealed light resistant glass containers at a temperature between 2

and 8 degrees 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.

2) Cholera Vaccine

 Cholera vaccine is a colorless, whitish or slightly colored opalescent liquid.

 It is a sterile suspension of killed Cholera vibrio’s (Vibrio cholera) of a strains selected
for high antigenic property and purity.

Preparation-

 It is prepared from equal portions of suspensions of Cholera 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 of Cholera vibrios is grown separately on solid medium for 24-48 hours.
 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 standardized so that 1ml of the vaccine contains not less than
12,000 million 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 immunization 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, dose,1ml after an interval of 4 to 6 weeks.

3) Pertussis Vaccine (Whooping Cough Vaccine)

 It is available as more or less turbid, whitish liquid nearly odorless or having faint odor
due to antimicrobial agent.
 It is a sterile bacterial suspension of killed Pertussis bacilli (Bordetella pertussis) of a
strain or strains selected for high antigenic efficiency.

Preparation-

 It is prepared by culturing Bordetella 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 standardized. The vaccine may show abnormal toxicity in animal tests
and this is removed by cold storage for up to 3 months.

Storage

 Store at a temperature between 2-8°C

Uses

 It is used for active immunization 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 at
least 4 weeks apart.

4) Typhoid Vaccine

 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.

Preparation-

 Salmonella typhi organisms are grown on a suitable culture media.

 The bacteria are killed by heat or bactericide such as phenol, formaldehyde or chemicals
like acetone
 It is then standardized, 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.

Storage

 Store at a temperature between 2 and 8°C. The vaccine must not be frozen.

Uses

 It is used for the immunization 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.

Viral and Rickettsial Vaccines

 These are the suspensions of viruses or rickettsial.

 They are prepared from infected tissue of blood obtained from artificially 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.

Examples of Viral and Rickettsial Vaccines-

1) Small pox Vaccine (Freeze-Dried)

  Small pox (Freeze dried) is almost white powder which reconstitutes to yield a viscid,
straw colored liquid.
 Small pox vaccine contains living attenuated vaccinia virus.
 The vaccine is prepared by 2 methods-
A. By using animals
B. By using eggs
A. Preparation of smallpox vaccine using animals
 Small pox vaccines are prepared by using calves or sheep.
 The method of preparation can be divided into the following steps-

a) 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.

b) Preparation of animal for scarification-

 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
scarified.
 The sacrificed 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.
 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-

a) By using brilliant green

b) 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

e) 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.

3) Measles Vaccine Live

 The vaccine is white or nearly white friable mass.

 Measles vaccine live is a bacterially 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.
  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 stabilizer 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-

 Pediatric, by subcutaneous injection, 0.5 ml of reconstituted vaccine.

4) poliomyelitis vaccine (oral)

 It is clear liquid which may have a reddish color 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.

Preparation-

 It is prepared by using 3 strains of poliomyelitis virus type-l,Il,

 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 than Penicillin and Streptomycin may be used in small
concentration.
 The temperature of growth medium should not be more than 37°C.
  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 bactericide.

Storage-

 The vaccine is stored in single or multiple dose containers in frozen state at -28°C.

Uses-

 It is used as active immunization 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.

5) Rabies Vaccine

 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 intracerebral with rabies virus.

Preparation-

 It is prepared by injecting sheep, rabbit, rat, mice or other animal intracerebral with rabies
virus.
 Animals that show typical paralysis are killed. The brain is 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.
 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 maintaining
between 7 to 7.2.
 It is then transferred into sterilized 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.

6) Typhus Vaccine

 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.
 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.

TOXOIDS

 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 immunization 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.

Examples of Toxoids-

1) Diphtheria Toxoid

 It is a modified form of exotoxin of Corynbacterium diphtheriae.

Preparation-

 A strain of Cory bacterium diphtheria 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.

Types of Diphtheria Toxoids

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 Diphtheria 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.

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 Aluminum Phosphate (PTAP) - It is prepared by treating formol toxoid

with hydrated aluminum phosphate.

2) Tetanus Toxoid

 This is prepared from the exotoxin of Clostridium 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) 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.

3) Diphtheria and Tetanus Vaccine (adsorbed)

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 aluminum hydroxide or aluminum
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.
 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 immunization against diphtheria 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 intramuscular 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.

4) Diphtheria, Tetanus and Pertussis Vaccine (absorbed)

It is a white sterile suspension prepared by adsorbing formaldehyde treated diphtheria toxoid and
tetanus toxoid on a mineral carrier such as aluminum hydroxide or aluminum phosphate and
adding a suspension of killedrdetella pertussis.

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 aluminum phosphate in a normal saline solution.
 It is then mixed with a quantity of suspension of killed Bordetella pertussis so that the
final product contains not more than 20 × 10° bacilli in each dose.
 A suitable preservative other than phenol or cresol group is added.
 The vaccine must comply with the test of sterility and toxicity.
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 immunization against diphtheria, tetanus and
pertussis 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 intramuscular 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.

IMMUNOLOGICAL PRODUCTS FOR PASSIVE IMMUNISATION

 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 immunized either by natural or artificial
means, the blood contains a large number of antibodies.
 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 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-

a) Diphtheria anti-toxins.

b) Tetanus anti-toxins.
 c) Gas gangrene anti- toxins.

1) Diphtheria Anti-toxins

 It is almost colorless, very faintly yellow or a slightly opalescent liquid.

*Preparations-

 The method of preparation of diphtheria anti-toxin is divided into following stages-

1) Preparation of toxin for active immunization of the horse- A pure culture of Corynebacterium
diphtheria 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 a
crude toxin. It is converted into toxoid.

2) Selection of the Horse-Horses are selected because-

a) They are easy to handle.

b) They readily produce antitoxins because a horse resembles man in having a natural
immunity against diphtheria.

c) R.B.C of the horse blood settles quickly and packs tightly. This property helps in
the separation of the

serum.

d) 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
immunized against tetanus by injecting tetanus toxoid.

3) Active immunization of the horse-

 To the selected Horses diphtheria toxoid is given for active immunization. The toxoid is
given by IV/ 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 liters 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 liters of blood is collected in the 3 batches of 8 liters 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 antitoxin 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 ammonium
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.

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.

i) Prophylactic- 500 to 2000 international units.

ii) Therapeutic- not less than 10,000 international units.

2) Tetanus Antitoxin

 It is almost colorless 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 immunization of the horse.

Storage

 Same as diphtheria antitoxin.

Dose

 It is administered by s/c or i/m injection in the following dose schedule.

i) Prophylactic- 1500 international units.

 ii) Therapeutic- not less than 50,000 international units.

Antiviral Serum

 These preparations are antiviral and produce passive immunity.

Example- 1) Rabies Antiserum

 It is almost colorless 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.

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.

Antibacterial Serum

 This type of serum is used to provide passive immunity to diseases caused by endotoxin
producing bacteria, eg- Pneumonia, meningitis 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.