BIOASSAY OF TITANUS ANTI TIOXIN AND DIPTHERIA VACCINE MPAHRM IST SEM

1. UNDER THE GUIDANCE OF
Dr.C.SREEDHAR
HEAD OF DEPARTMENT OF ANALYSIS
PRESENTATION BY,
ASMITA SINGH
M.PHARM 1st SEM
DEPARTMENT OF ANALYSIS

2. Tetanus Anti-toxin
Adsorbed Diptheria vaccine

3. Principle
Tetanus Antitoxin is a preparation containing the
specific antitoxic globulins or their derivatives
obtained by purification of hyperimmune serum
of horses or other suitable animals and have the
specific activity of neutralising the toxin formed
by Clostridium tetani. The liquid preparation
may contain a suitable antimicrobial
preservative.
Category: Passive immunising agent.

4.  The genus clostridium consists of gram
positive,anaerobic,spore forming,splindle shaped
and highly pleomorphic bacilli.
 Tetanus results from the contamination of wound
by clostridium tetani.
 The spores are found in soil.Germination and
multiplication occur if certain factors like
necrotic tissue,ionisable calcium salts and lactic
acid are present.
 Infection of wound with pyrogenic organisms
increases the risk of tetanus.Toxin is probably
absorbed from the area of infection and through
motor nerve endings reach anterior horn cell.

5.  The potency of tetanus antitoxin is
determined by comparing the dose necessary
to protect mice against the paralytic effects
of a fixed dose of tetanus toxin with the dose
of the Standard Preparation of tetanus
antitoxin necessary to give the same
protection.
 For this purpose the Standard Preparation of
tetanus antitoxin and a suitable preparation
of toxin, for use as a test toxin, are required.

6.  The Standard Preparation is the 2nd
International Standard for Tetanus antitoxin,
established in 1969, consisting of freeze-
dried hyper immune horse serum (supplied in
ampoules containing 1400 Units), or another
suitable preparation the potency of which
has been determined in relation to the
International Standard.

7. Test animals: Use healthy mice, weighing
between 17 and 22 g, from the same stock.
Preparation of test toxin:
Prepare tetanus toxin from a sterile filtrate of
an 8- to 10- day culture of Clostridium tetani.
 Test toxin may be prepared by adding this
filtrate to glycerin in the proportion of 1
volume of the filtrate to 1 or 2 volumes of
glycerin

8. This solution of test toxin is stored and test toxin
may also be prepared in stable form by saturating
the filtrate with ammonium sulphate.
Collecting the resulting precipitate, drying it over
phosphorus pentoxide at a pressure of 1.5 to 2.5
kPa and reducing it to a fine powder.
The powder so obtained is preserved in the dry
condition at a low temperature either in sealed
ampoules or over phosphorus pentoxide at a
pressure of 1.5 to 2.5 kPa.

9. First determine the Limes paralyticum/10
(Lp/10) dose of the test toxin. This is the smallest
quantity of the toxin which when mixed with 0.1
Unit of the Standard Preparation and injected
subcutaneously into mice causes tetanic paralysis
within 4 days.
Prepare a solution of the Standard Preparation in
a suitable liquid such that 1 ml contains 0.5 Unit.
Accurately measure or weigh a quantity of the
test toxin and dilute it with, or dissolve it in, a
suitable liquid.

10.  Prepare mixtures such that each contains 2.0
ml of the solution of the Standard Preparation
(1 Unit), one of a series of graded volumes of
the solution of the toxin, and sufficient
suitable liquid to give a final volume of 5.0 ml.
 Allow the mixtures to stand at room
temperature, protected from light, for 60
minutes and then inject 0.5 ml of each mixture
subcutaneously into mice, six mice being used
for each mixture, and observe the mice for 4
days.

11.  The test (Lp/10) dose of the toxin is the
amount present in 0.5 ml of that mixture
that contains the smallest amount of toxin
sufficient to cause tetanic paralysis in all six
mice injected with it within 4 days.

13. Adsorbed Diphtheria Vaccine (ADV) is an anti-
toxin preparation containing antitoxic globulins
that have the power of specifically neutralizing
the toxin formed by Corynebacterium diphtheria.
It is obtained by fraction of horse or other
mammal serum, that have been immunized
against Diphtheria toxin.
Diphtheria vaccine is generally available in the
combination with the Tetanus And Pertusis
vaccine.
The formal Toxoid is prepared from the toxin

14. It is generally prepared by combining Diphtheria
formal toxoid with mineral carrier i.e. hydrated
Aluminum hydroxide, Ammonium hydroxide or
Phosphates ,Calcium Phosphates etc.

16. The potency of Diphtheria antitoxin is determined
by comparing the dose necessary to protect the
guinea pig or rabbit against the erythrogenic effect
of a fixed dose of the standard preparation of
Diphtheria toxin required necessary to give same
protection.

17. Selection of the challenge toxin is based upon
reacting dose (Lr).
The preparation containing 67-133 Lr/mL in Lime
flocculation's (lf) of Diphtheria Toxin (DT) is selected
or the preparation with 25000 to 50000 minimal
reacting doses for guinea pig skin in 1lf.
The toxin are stored in the refrigeration 0-5˚C and
Toluene is added as antimicrobial preservatives
which does not cause rapid decline in toxicity.

18. It is prepared in a saline solution having
1mL of preparation having 0.1 IU of toxin
followed by:
1mL of standard preparation along with
0.2 IU test toxin.
1mL of standard preparation along with
0.3IU test toxin.
Store the preparation away from light.

19. Challenge toxin / preparation to be assayed is
diluted with phosphate buffer solution (PBS) of
pH 7.4.
The challenge toxin solution is diluted in the
same solution upto the following level:
2LD50
LD50
½LD50

20. Test animal such as guinea Pig, Horses, Rabbits are
selected for this.
If guinea pig then it should be of 250-350 gm.
The animal are grouped into 6 groups of 16
animals.
0.2mL of each mixture should be injected
subcutaneously or intradermally into the animal.
The animals are observed after 48 hours for
specific Diphtheria erythema.
Mixture containing larger amount of toxin will give
severe reaction and vice versa.

21. The 3 dilutions of sample and standard vaccine
are prepared in saline solution.
Inject into guinea pigs.
This should protect 50% animals from lethal
effect of subcutaneous injection.
Calculate the potency of the vaccine relative
to the potency of potency of standard
preparation on the basis of the number of
animals survived in each group of 16 animal.

22. Vaccine under examination and standard
preparation , the 50% protective doses lies between
the largest and the smallest dose of the preparation
given to guinea pigs.
Mortality increases when increases in toxin dose
level is there the minimum amount of toxin which
when combined should cause a local skin reaction
that is just visible and indicates the presence of
toxin.

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