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Botulism Antitoxin

Category Passive immunizing agent.

          Botulinum Antitoxin is a sterile liquid or freezedried preparation containing antitoxic globulins that have the power of specifically neutralizing the toxins formed by Clostridium botulinum type A, type B or type E, or any mixture of these types. It is obtained by fractionation from serum or plasma of horses, or other mammals, that have been immunized against Clostridium botulinum type A, type B and type E toxins.

          The antitoxin complies with the requirements stated under Antisera, with the following modifications.

Description Transparent or slightly opalescent liquid, practically colourless, and practically odourless or having an odour because of the antimicrobial agent.

          Freeze-dried antitoxin, when reconstituted, becomes colourless or slightly yellowish brown, clear or slightly whitish turbid.

Strengths available One ml contains 500 to 750 IU of type A, 500 to 550 IU of type B, and/or 50 to 850 IU of type E antibodies.

Dose Intramuscular or intravenous infusion as directed by the physician.

Contra-indication Botulinum antitoxin prepared from horse serum is contra-indicated in individuals with history of hypersensitivity or anaphylaxis to equinederived serums.

Warning Tachycardia, cardiovascular collapse, flushing, cyanosis, sinus tachycardia, serum sickness, photophobia, bronchospasm, apnea, and anaphylaxis may occur.

Additional information

          1. For intravenous infusion, dilute 1 volume of antitoxin with 10 volumes of sodium chloride injection (0.9 per cent) and give slowly over at least 30 minutes.

          2. Patients should be kept under medical observation after the administration of full dose.

          3. The relative proportions of types A and B present in Botulinum Antitoxin Bivalent (Equine) Types A and B are believed to be adequate to establish a level of neutralizing capacity in the bloodstream, against the homologous toxins, of several times the level shown to be clinically efficacious for type E antitoxin.

          4. The type E Botulinum Antitoxin is administered consecutively with Botulinum Antitoxin Bivalent, types A and B, only when type E botulinum (usually associated with consumption of contaminated fish products) is suspected.

Expiration date The expiration date for Antitoxin containing a 20 per cent excess of potency is not later than 5 years after the date of the last satisfactory test for potency.

Labelling Complies with the “General Information for Antisera”, p. 225. In addition the label states the types of Clostridium botulinum toxin neutralized by the preparation as stated in the accompanying leaflets.

Identification It specifically neutralizes the types of Clostridium botulinum toxin stated on the label, rendering them harmless to susceptible animals.

Assay Not less than the value stated on the label for each of types A, B and E.

          The potency of botulinum antitoxin is determined by comparing the dose necessary to protect mice against the lethal effects of a fixed dose of botulinum toxin with the quantity or the standard preparation of botulinum antitoxin necessary to give the same protection. For this comparison a reference preparation of each type of botulinum antitoxin, calibrated in International Units, and suitable preparations of botulinum toxins, for use as test toxins, are required. The potency of each test toxin is determined in relation to the specific reference preparation; the potency of the botulinum antitoxin to be examined is determined in relation to the potency of the test toxins by the same method.

          International Units of the antitoxin are the specific neutralizing activity for botulinum toxin type A, type B and type E contained in stated amounts of the International Standards which consist of dried immune horse sera of types A, B and E. The equivalence in International Units of the International Standard is stated from time to time by the World Health Organization.

          Selection of animals Use mice having body masses such that the difference between the lightest and the heaviest does not exceed 5 g.

          Preparation of test toxins

          Prepare type A, B and E toxins from sterile filtrates of approximately 7-day cultures in liquid medium of Clostridium botulinum types A, B and E. To the filtrate, add 2 volumes of glycerol, concentrate, if necessary, by dialysis against glycerol and store at or slightly below 0º.

          Selection of test toxins Select toxins of each type for use as test toxins by determining for mice the L+/10 dose and the LD₅₀, the observation period being 96 hours. The test toxins contain at least 1000 LD₅₀ in an L+/10 dose.

          Determination of test doses of the toxins (L+/10 dose) Prepare solutions of the reference preparations in a suitable liquid such that each contains 0.25 IU of antitoxin per ml. Using each solution in turn, determine the test dose of the corresponding test toxin.

          Prepare mixtures of the solution of the reference preparation and the test toxin such that each contains 2.0 ml of the solution of the reference preparation, one of a graded series of volumes of the test toxin and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Using four mice for each mixture, inject a dose of 1.0 ml intraperitoneally into each mouse. Observe the mice for 96 hours.

          The test dose of toxin is the quantity in 1.0 ml of the mixture made with the smallest amount of toxin capable of causing, despite partial neutralization by the reference preparation, the death of all four mice injected with the mixture within the observation period.

          Determination of potency of the antitoxin Prepare solutions of each reference preparation in a suitable liquid such that each contains 0.25 IU of antitoxin per ml.

          Prepare solutions of each test toxin in a suitable liquid such that each contains 2.5 test doses per ml.

          Using each toxin solution and the corresponding reference preparation in turn, determine the potency of the antitoxin. Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that each contains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be examined, and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also prepare mixtures of the solution of the test toxin and the solution of the reference preparation such that each contains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution of the reference preparation centred on that volume (2.0 ml) that contains 0.5 IU, and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Using fourmice for each mixture, inject a dose of 1.0 ml intraperitoneally into each mouse. Observe the mice for 96 hours.

          The mixture that contains the largest volume of antitoxin that fails to protect the mice from death contains 0.5 IU. This quantity is used to calculate the potency of the antitoxin in International Units per ml.

          The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of the solution of the reference preparation die and all those injected with mixtures containing more survive.