Saturday 12 May 2012

Biological Assay of Diphtheria Antitoxin

Biological Assay of Diphtheria Antitoxin

The potency of diphtheria antitoxin is determined by comparing the dose necessary to
protect guinea-pigs or rabbits against the  erythrogenic effects of a fixed dose of the
Standard Preparation of diphtheria antitoxin necessary to give the same protection. For
this purpose, a suitable preparation of diphtheria toxin is required to be used as a test
toxin. The test dose of the toxin is determined in relation to the Standard Preparation. The
potency of the preparation being examined is then determined in relation to the Standard
Preparation using the test toxin.


Standard Preparation:  The Standard Preparation is the Ist International Standard for
Diphtheria antitoxin, equine, established in 1934, consisting of the dried hyperimmune
horse serum and glycerin, or another suitable preparation the potency of which has been
determined in relation to the International Standard.
Method
Test toxin: Prepare diphtheria toxin by filtering through bacteria-proof filter the medium
in which a toxigenic strain of C. diphtheriae has grown. Store at a temperature of 2o
 and
8o
.
Selection of test toxin: In selecting a toxin for use as the test toxin determine the
following:
  25Lr/100 dose — This is the smallest quantity of  the toxin which, when mixed with 0.01
Unit of antitoxin and injected  intracutaneously into guinea-pigs or rabbits causes a
characteristic reaction at the site of the injection within 48 hours.
Minimal reacting dose — This is the smallest quantity  of toxin which, when injected
intracutaneously into guinea-pigs or rabbits, causes a characteristic reaction at the site of
injection within 48 hours.

A suitable toxin is one which contains at least 200 minimal reacting doses in the Lr/100
dose. The test toxin is allowed to stand for some months before being used for the assay
of samples of antitoxin. During this time its toxicity declines and the Lr/100 dose may be
slightly increased. When experiment shows that the Lr/100 dose is constant, the test toxin
is ready for use and may be used for a long period. Determine the minimal reacting dose
and the Lr/100 dose at frequent intervals. Store the test toxin in the dark at a temperature
between 0o
 and 5o
. Maintain its sterility by the addition of toluene or other antimicrobial
preservative which does not cause a rapid decline in specific toxicity.
Determination of test dose of toxin (Lr/100 dose): Prepare a solution of the Standard
Preparation with saline solution such that 1 ml contains 0.1 Unit. Prepare mixtures such
that 2.0 ml of each mixture contains 1.0 ml of the dilution of the Standard Preparation
(0.1 Unit) and one of a series of graded volumes of the test toxin. Dilute each mixture
with  saline solution to the same final volume (2.0 ml). Allow the mixtures to stand at
room temperature, protected from light, for 15 to 60 minutes and inject intracutaneously
0.2 ml of each mixture at suitably spaced sites into the shaven or depilated flanks of two
animals. Observe the animals for 48 hours.

The test dose (Lr/100) of the toxin is the amount present in 0.2 ml of that mixture which
causes at the site of injection a small, characteristic reaction in the skin of the guinea-pig
or rabbit. Mixtures containing larger amounts of toxin cause larger reaction and necrosis
and mixtures containing smaller amount of toxin cause no reaction.
Determination of potency of the antitoxin: Dilute the test toxin with saline solution so
that 1.0 ml contains 10 times the test dose. Prepare mixtures such that 2.0 ml of each
mixture contains 1.0 ml of the dilution of the toxin and one of a series of graded volumes
of the preparation being examined. Prepare  further mixtures such that 2.0 ml of each
contains 1.0 ml of the solution of the test toxin and 0.1 Unit of antitoxin. Dilute each
mixture with  saline  solution to the same final volume (2.0 ml). Allow the mixtures to
stand at room temperature, protected from light, for 15 to 60 minutes. Inject a dose of 0.2
ml of each mixture into the animals under the conditions described in the determination
of the Lr/100 dose of the toxin.

The mixture of the preparation being examined that contains 0.01 Unit of antitoxin in 0.2
ml is the mixture that produces the same degree of local reaction as that produced by the
injection into the same animals of the mixture of the Standard Preparation that contains in
0.2 ml the test dose (Lr/100) of the toxin and 0.01 Unit of antitoxin.

When at least four distinct tests are carried out by this method, the limits of error have
been estimated to be between 90% and 111%.
  26

20 comments:

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