1. BIOLOGICAL ASSAY OF GAS GANGRENE ANTITOXIN

2. BIOLOGICAL ASSAY OF GAS GANGRENE ANTITOXIN (oedematiens)
Gas-Gangrene & clostridial myonecrosis are interchangeable terms used to describe an infection of muscle by clostridium.
Clostridium is an anaerobic, gram +ve , spore forming bacillus of genus clostridium.
G.G-3 members of the genus clostridium bacteria like C.welchii (perfringens) , C.Novyi (oedematiens) & C.septicum may produce G.G.

3. ASSAY
Potency of G.G.A (oedematiens) is determined by comparing dose necessary to protect mice or other suitable animals against toxic effects of G.G toxin (oedematiens) with dose of standard preparation of G.G.A (clostridium oedematiens) necessary to give same protection.

4. Requirements
Standard preparation of G.G.A
Test toxin –suitable preparation of G.G.toxin (oed.)
Test dose of toxin is determined in relation to standard preparation.
Potency of preparation being examined is then determined in relation to standard preparation using test toxin.

5. Standard preparation & unit
Standard preparation – Dried serum/ suitable preparation of G.G.A
Potency determined in relation to international standard.
Unit – specific neutralizing activity for G.G.Toxin contained in such an amount of standard preparation as Ministry of Health & Family welfare , Govt. of India.
May time to time indicate quantity exactly equivalent to unit accepted for International use.

6. Suggested Method Test Toxin
prepare G.G.Toxin from sterile filterate of C.O.
Filterate being prepared after 5 days growth of organism , by precipitation with ammonium sulphate , resulting precipitate which contains toxin is collected , dried in vacuo over phosphorus pentoxide , powdered & kept dry.

7. Selection of Test Toxin
Lt dose- This is smallest quantity of toxin which when mixed with 1 unit of anti-toxin & injected I.M into mice, causes death of animals with in 72 hrs.
LD50 – smallest quantity of toxin , which when injected I.M cause death within 72 hrs of one half of mice injected.
A suitable toxin is one which has an Lt dose in 0.5 mg/less & contains not less than 25 LD50 in an Lt dose.

8. Determination of potency of G.G.A
Preliminary test weigh accurately a quantity of test toxin & dissolve it in saline solution so that 1ml = 10 times test dose.
Prepare mixtures that 2ml of each mixture = 1ml of toxin +one of series of graded volume of preparation being examined.
Dilute each mixtures with saline solution to 2ml.
Allow mixtures to stand for 60min.
Inject 0.2 ml of each mixture into each of 3 mice under condition described for determination of test dose of toxin.
If none of mice is killed 0.2ml = more than 1 unit of anti toxin
If all mice are killed 0.2ml = <1 unit of anti toxin

9. Final test
prepare similar fresh mixtures such that 2ml of each mixture = 1ml of solution of test toxin & amount of preparation being examined intermediate b/w smallest volume which protects all of mice & largest volume which fails to protect any of mice , as determined by preliminary tests.
Prepare further mixture that 2ml = 1ml of solution of toxin +10 units of anti toxin
Dilute each mixture with saline solution up to 2ml

10. Allow mixture to stand for 60min
Inject 0.2ml of each mixture to each of 6 mice under conditions described in determination of test dose of toxin.
Mixture of preparation being examined that contains 1 unit in 0.2 ml is that mixture which, killing some but not all of mice , kills the same / most nearly same , number as reference mixture containing 1 unit of anti- toxin in 0.2 ml

11. Limits of error
Limits of error(p=0.99) attainable with test are b/w78&130percent.
When 2 animals per dose are used, b/w 83&120percent.
When 4 animals per dose are used &b/w 86&116 percent when 6 animals per dose are used.