Bioassay of titanus antitoxin as per Indian Pharmacopoea

1. Presented by: Mukul S. Tambe
M. Pharm Sem. I
Pharmacology
Roll. No. 8
Submitted to: Dr. Padmavati
BIOASSAY OF TETANUS
ANTITOXIN
10/10/20171

2. TETANUS:
 Also known as “Lockjaw”
 Characterized by severe muscle
spasm
 Other symptoms:
 fever,
 sweating,
 headache,
 trouble swallowing,
 high blood pressure,
 a fast heart rate.
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3. TETANUS ANTI-TOXIN:
 Tetanus anti-toxin is a
preparation containing
antitoxic globulins that
have the power of
specifically neutralizing
the toxin formed by
Clostridium tetani.
 It is obtained by
fractionation from horse
serum or other mammals
that have been immunized
against tetanus toxin.
10/10/20173

4. BIOASSAY OF TT ANTI-TOXIN:
 PRINCIPLE:
 The potency of tetanus anti-toxin is determined by comparing
the dose necessary to protect the mice against the paralytic
effects with that of the standard anti-toxin preparation.
 Procedure:
 Standard Preparation:
 2nd International std. for TT anti-toxin, established in
1969, consisting freeze dried horse serum
 1ampoule = 1400 units.
 Animal model:
 Mice weighing between 17 to 22gm. 10/10/20174

5. PREPARATION OF TEST TOXIN:
Extract Clostridium tetani from 8-10 days culture
1 volume toxin + 2 volume glycerin
Saturate the filtrate with Ammonium sulphate
Collect the ppt and dry over Phosphorus pentoxide
Dried powder preserve in ampoules at 0°C
over Phosphorus pentoxide
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6. DETERMINATION OF TEST DOSE:
 Determination of Lp/10 dose
 Lp/10 Dose: The smallest quantity of toxin which when
mixed with 0.1 unit of standard anti-toxin preparation,
injected subcutaneously in mice, causes tetanic
paralysis within 4 days.
 The test (Lp/10) dose of toxin is the amount present in
0.5 ml of toxin efficient to cause titanic paralysis in all 6
mice injected with it within 4 days.
 All the 6 animals in group should get paralyzed.
10/10/20176

7. DETERMINATION OF POTENCY:
STANDARD ANTI-TOXIN TEST ANTI-TOXIN
2ml Std. Toxin (0.5unit/ml) (5 Lp/10
doses)
1ml of serial dilution of test Antitoxin
Make up to 5ml with saline
Store in dark for 60 min
Inject 0.5ml s/c in 6 rats
Observe for 4 days
2ml Std. Toxin (0.5unit/ml) (5 Lp/10
doses)
1ml of serial dilution of test Antitoxin
Make up to 5ml with saline
Store in dark for 60 min
Inject 0.5ml s/c in 6 rats
Observe for 4 days10/10/20177

8. FAILUER OF THE TEST:
Criteria for test failure:
 If the Largest concentration of test preparation fails to
protect the animal from paralysis.
 If the smallest concentration of standard preparation fails to
protect the animal from paralysis.
 Then calculate the potency of test anti-toxin by comparing
with standard anti-toxin.
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9. REFERENCES:
1. Indian Pharmacopoeia 1996.
2. http://www.authorstream.com/Presentation/chan
dusvcp-1138381-vaccines/
10/10/20179

10. 10/10/201710