สารบัญ

15.2 ANTISERA

15.2.7 Biological Assay of Cobra Antivenin

            The potency of Cobra Antivenin is determined by estimating the dose necessary to protect mice against the lethal effect of a fixed dose of the cobra venom.

SUGGESTED METHOD

            Preparation of cobra test venoms

            Use venoms that have the normal physico-chemical, toxicological and immunological characteristics of venoms from the particular species of cobra (Naja kaouthia). They are preferably freeze-dried and stored in the dark at 2º to 8º.

            Selection of test venom Select a venom for use as a test venom by determining the following quantity.

            LD₅₀ The smallest quantity of venom that, when injected into mice, causes the death within 48 hours of one half of the mice injected.

            Determination of the dose of test venom Prepare graded dilutions of the reconstituted venom in saline TS or other isotonic diluent in such a manner that the middle dilution contains in 0.25 ml the dose expected to be the LD₅₀. Dilute with an equal volume of the same diluent. Using at least four mice, each weighing 18 to 20 g, for each dilution inject 0.5 ml intravenously into each mouse. Observe the mice for 48 hours and record the number of deaths. Calculate the LD₅₀ by standard statistical methods.

            Determination of the potency of the antivenin Dilute the reconstituted test venom so that 0.25 ml contains the test dose of 5 LD₅₀. Prepare 1.5- to 2.5-fold serial dilutions of the antiserum being examined in saline TS or other isotonic diluent. Use a sufficient number and range of dilutions to enable a mortality curve between 20 and 80 per cent mortality to be established and to permit an estimation of the statistical variation.

            Prepare mixtures such that 5 ml of each mixture contains 2.5 ml of one of the dilutions of the antiserum being examined and 2.5 ml of the dilution of the test venom. Allow the mixtures to stand in a water-bath at 37º for 30 minutes. Using not less than six mice, each weighing 18 to 20 g, for each mixture inject 0.5 ml intravenously into each mouse. Observe the mice for 48 hours and record the number of deaths. Calculate the ED₅₀ by standard statistical methods. At the same time verify the number of LD₅₀ in the test dose of venom, using the method described above. The potency is expressed in terms of the number of LD₅₀ or the quantity of test venom in mg that is neutralized by 1 ml or some other defined volumes of the antivenin.