Bioassay and biological test of tetanus antitoxin

1. Presented by
Trisha Das
M Pharm 1st year
PharmaceuticalAnalysis &
QualityAssurance

2.  Tetanus is a bacterial
disease marked by
rigidity and spasms of
the voluntary muscles
 Also known as
“Lockjaw”

3.  Tetanus is cased by the Clostrium tetani
bacterium.
 Clostrium tetani spores are able to survive for a
long period of time outside the body. They are
most commonly found in animal manure and
contaminated soil, but may exists virually
anywhere.
 When Clostridium tetani enter the body, they
multiply repidly and release tetonospasmin, a
neurotoxin. When tetanospasmin enters he
bloodstream, it rapidly spreads around the body,
causing tetanus symptoms.
 Clostridium tetani enters the body mainly through
skin cut or puncture wounds. Thoroughly
cleaning any cuts helps prevent an infection from
developing.

4.  Stiffness in neck and other muscle
 Difficulty in swallowing
 Irritability
 Spasms in jaw and neck muscle
 High blood pressure
 A fast heart rate

5. Tetanus antitoxin is a sterile,
non pyrogenic solution of
refined and concentrated
proteins chiefly globulin
containing antitoxin
antibodies obtained from
the blood plasma of horse
that have been immunized
against tetanus toxin.

6.  It has a potency of not less than 400 Antitoxin units
per ml based on the US standard tetanus antitoxin
and US control tetanus test toxin is tested in guinea
pig.
 Tetanus antitoxin neutralizes the toxin produced by
Clostridium tetani.

7. PRINCIPLE
The potency of tetanus antitoxin is determined
by comparing the dose necessary to protect the
mice against the paralytic effects with that of
the standard anti toxin preparation.

8. STANDARD PREPARATION
1. 2nd INTERNATIONAL STD. For Tetanus antitoxin
established in 1969 consisting freeze dried horse serum.
2. 1 ampoule= 1400 units
ANIMAL MODEL
Mice weighing between 17-22gm

9. Extract C. Tetani from 8-10 culture
1 volume toxin+ 2 volume glycerine. Saturate the
filtrate with ammonium sulphate.
Collect the ppt and dry over phosphorus Pentoxide
Dried powder preserve in ampoules 0oC
over phosphorus pentaoxide.

10.  Determination of lp/10 dose
 Lp/10 dose: the smallest quantity of toxin which when mixed
with 0.1 unit of standard antitoxin 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 animal in the group should get paralyzed.

11. STANDARD ANTITOXIN
2ml std. Toxin+ 1ml serial
dilution of test antitoxin
Make upto5ml with saline
Store in dark for 60 mins
Inject 0.5ml s/c in 6 rats
Observe for 4 days
TEST ANTITOXIN
2ml std.toxin + 1ml of
serial dilution of test
antitoxin
Make up to 5ml with saline
Store in dark for 60 min
Inject 0.5 ml s/c in 6 rats
Observe for 4 days

13.  Tetanus Antitoxin, INDIAN
PHARMACOPOEIA 1996, Volume-
II(P-Z), Page no. 744-745, Published
by the controller of publications, Delhi.