TETANUS ANTITOXIN AND ITS BIOASSAY METHODS

1. TETANUS ANTITOXIN
BIOASSAY
RVS Chaitanya Koppala
Assistant professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. INTRODUCTION
Tetanus antitoxin is a liquid preparation of purified hyperimmune
serum of horses or other appropriate animals.
It contains specific antitoxin globulins or their derivatives that
possess the capability of neutralizing toxins that are especially
produced by clostridium tetani.
A suitable preservative may also be included in the preparation to
prevent the growth of microbes.
It has a strength of not less than 1000units/ml and not less than
3000units/ml for prophylactic and therapeutic use respectively

3. Principle
 The potency of tetanus antitoxin is generally determined by comparing the dose
required to save mice from the prophylactic effects of a fixed dose of tetanus
toxin with that of the standard preparation of tetanus toxin.
 which provides the same protection. Therefore this assay requires suitable
preparation of toxin which will be considered as the test toxin and standard
preparation of the tetanus antitoxin.
 Initially the test dose of this test toxin is determined with respect to the standard
preparation of antitoxin. Finally, the potency of the preparation under
examination is determined with respect to the standard preparation by using the
test toxin

4. Test animal:
healthy mice weighing between 17-22g belonging to the
same stock should be used.
Standard preparation:
It is the 2nd international standard for tetanus antitoxin which
contains freeze dried hyperimmune serum of horse or any
other suitable preparation whose strength has been
determined according to the international standard. It is
available in the form of ampoules containing 1400units.

5. Test toxin:
Tetanus toxin is prepared by using a sterile filtrate consisting of 8-10day
culture of Tetani
Method I :in this method, one volume of sterile filtrate is added to one or
two volumes of glycerin and the resultant solution is stored below 0c
Method II: In this method, the sterile filtrate is saturated with ammonium
sulphate and the resulting precipitate is dried over phosphorus pentoxide at
a pressure of 0.22-0.36 psi.
The dried residue obtained is crushed to a fine powder and preserved in
dry condition at low temperature in sealed ampoules.

6. Estimation of Test dose of Toxin (Lp/10dose)
 Lp/10 or limes paralyticum dose refers to the minimum amount of toxin required to cause
tetanic paralysis in mice within 4 days. When it has been administered with 0.1 U of
standard preparation.
 The procedure involved in the estimation of Lp/10 dose have been outlined below
1. Standard solution of tetanus antitoxin is prepared using a suitable solvent such that each
ml contains 0.5units of standard preparation.
2. Accurately weighed amount of test toxin is diluted with or dissolved in a suitable solvent
and different mixtures are prepared with the resulting solution.each mixture should
contain 2ml of standard prearation one of the graded volumes of toxin solution and
suitable solvent sufficient to make up the volume upto 5ml.

7. The prepared mixtures are maintained at room temperature
for 1hour without exposing them to light
Then 0.5ml of each mixture is administered subcutaneously
(using 6mice for each mixture) and the mice are observed for
4 days
Test dose Lp/10 of toxin: the amount of toxin present in
0.5ml of that mixture which is sufficient to cause tetanic
paralysis in all the 6mice within 4 days in the test dose of the
toxin.

8. Estimation of potency of the
antitoxin
 A standard solution of tetanus antitoxin is prepared using a suitable solvent such
that each ml contains 0.5units of standard preparation.
 Accurately weighed amount of test toxin is diluted with or dissolved in a suitable
solvent such that each ml contains 5 times the amount of Lp/10 DOSE.
 Different mixture are prepared using the diluted solution such that each mixture
contains 2ml of test toxin solution. One of the series of graded volumes of the
preparation under test and suitable solvent sufficient to make up the volume upto
5ml
 Mixtures containing 2ml of test toxin and 2 ml of one of the series of graded
volumes of the standard preparation containing 1Unit are also prepared.

9.  These mixtures are allowed to stand for 1hour at room temperature protected
from light
 0.5ml of each mixture is administered subcutaneously to each mouse and kept
under observation for 4 days. 6 mice should be used for each mixture.
 The mixture contains the maximum volume f the preparation under test which
fails to save the mice from paralysis contains 1 unit of antitoxin
 The test is valid only if all the mice which have been injected with 2ml or less of
the solution of standard preparation experience paralysis and all those mice have
been injected with larger volumes of the solution do not exhibit paralysis
 Finally the strength of the preparation under examination is determined in
units/ml

10. THANK YOU