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Category Passive immunizing agent.

          Diphtheria Antitoxin is a sterile non-pyrogenic solution of the refined and concentrated proteins, chiefly globulins, containing antitoxic antibodies obtained from serum or plasma of horses, or other mammals, that have been immunized against diphtheria toxin or toxoid.

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

Description Transparent or slightly opalescent, practically colourless liquid; odour, practically odourless or of the antimicrobial agent.

Strength available 10,000 IU per container.

Dose Prophylaxis: Intramuscular, 5000 to 10,000 IU. Therapeutic----

          Mild or moderate: Intramuscular, 10,000 to 30,000 IU.

          Severe: Intramuscular, 40,000 to 100,000 IU. The dosage may be administered as prescribed by the physicians.

Warning

          1. It is to be administered only by or under the supervision of the physician or other health care professionals.

          2. It should be used with extreme caution in individuals with a history of allergic disorders and/or who have exhibited previous sensitivity to equine serum. Epinephrine and other supportive measures should be available for immediate management of an anaphylactic reaction if it occurs.

          3. A few days after the injection of a heterologous serum, serum sickness may also occur.

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

Labelling Complies with the “General Information for Antisera”, p. 225. In addition the label on the container states the recommended human dose for the various prophylactic and therapeutic uses.

Identification It specifically neutralizes the toxin formed by Corynebacterium diphtheriae rendering it harmless to susceptible animals. The potency test may serve as an identification test.

Assay Not less than 500 IU of antitoxin per ml. 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 diphtheria toxin with the quantity of the standard preparation of diphtheria antitoxin necessary to give the same protection. For this comparison a reference preparation of diphtheria antitoxin, calibrated in International Units, and a suitable preparation of diphtheria toxin, for use as a test toxin, are required.

          The potency of the test toxin is determined in relation to the reference preparation; the potency of the diphtheria antitoxin being examined is determined in relation to the potency of the test toxin by the same method.

          The International Unit of antitoxin is the specific neutralizing activity for diphtheria toxin contained in a stated amount of the International Standard, which consists of a quantity of dried immune horse serum.

          Preparation of test toxin Prepare diphtheria toxin from cultures of Corynebacterium diphtheriae in a liquid medium. Filter the culture to obtain a sterile toxic filtrate and store at 4º. Selection of test toxin Select a toxin for use as a test toxin by determining for guinea-pigs or rabbits the lr/100 dose and the minimal reacting dose, the observation period being 48 hours. The test toxin has at least 200 minimal reacting doses in the lr/100 dose.

          Minimal reacting dose This is the smallest quantity of toxin which, when injected intracutaneously into guinea-pigs or rabbits, causes a small, characteristic reaction at the site of injection within 48 hours.

          The test toxin is allowed to stand for some months before being used for the assay of antitoxin. During this time its toxicity declines and the lr/100 dose may be increased. Determine the minimal reacting dose and the lr/100 dose at frequent intervals. When the experiment shows that the lr/100 dose is constant, the test toxin is ready for use and may be used for a long period. Store the test toxin in the dark at 0º to 5º. Maintain its sterility by the addition of toluene or other antimicrobial preservatives that do not cause rapid decline in specific toxicity.

          Determination of test dose of toxin (lr/100 dose). Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.1 IU of antitoxin per ml.

          Prepare mixtures of the solution of the reference preparation and of the test toxin such that each contains 1.0 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 2.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 15 to 60 minutes. Using two animals for each mixture, inject a dose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal. Observe the animals for 48 hours.

          The test dose of toxin is the quantity in 0.2 ml of the mixture made with the smallest amount of toxin capable of causing, despite partial neutralization by the reference preparation, a small but characteristic erythematous lesion at the site of injection.

          Determination of potency of the antitoxin Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.125 IU of antitoxin per ml.

          Prepare a solution of the test toxin in a suitable liquid such that it contains 12.5 test doses per ml.

          Prepare mixtures of the solution of the test toxin and of the antitoxin being examined such that each contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin being examined and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Also prepare mixtures of the solution of the test toxin and the solution of the reference preparation such that each contains 0.8 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 (0.8 ml) that contains 0.1 IU and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 15 to 60 minutes. Using two animals for each mixture, inject a dose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal. Observe the animals for 48 hours.

          The mixture that contains the largest volume of antitoxin that fails to protect the guinea-pigs from the erythematous effects of the toxin contains 0.1 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 sites injected with mixtures containing 0.8 ml or less of the solution of the reference preparation show erythematous lesions and at all those injected with mixtures containing more there are no lesions.