Biological Assay of Rabies Vaccine

2. Introduction:
Rabies:
 An acute neurological disease caused by virus
(Rhabdoviridae) that attacks the CNS
 Causes an alternating symptoms of depression and
agitation and almost invariably leads to extreme type
of violent behavior and Death.
 Zoonotic (animals to human).
 Immunefluorescent Antibody Test is best method for
rapid diagnosis test for Rabies.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

3. Rabies Vaccine:
Developed by Louis Pasteur in 1885 .
It is a freeze dried or liquid preparation of a suitable
approved strains of fixed rabies virus grown in an
approved cell culture/embroys of duck/chicken and
inactivated by a validation method.
The freeze dried vaccine is reconstituted immediately
before use as stated in the label to give a clear or slightly
opalescent solution/suspension.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

4. Biological Assay of Rabies Vaccine:
 The potency of Rabies vaccine is determined by
comparing the dose necessary to protect mice from the
lethal effect of dose of rabies virus administered
intracerebrally with the dose of standard preparation of
Rabies vaccine necessary to give the same protection.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

5. Special Reagent Required:
10% Acid-digested casein hydrolysate pH 7.2:
(1) Casein hydrolysate solution— Dissolve 100.0 g of acid-
digested casein hydrolysate in sufficient distilled water to
make 500 ml, adjust the pH to 7.2 with 10 M NaOH and
sterilise by heating at 121o for 20 minutes.
(2) Sucrose solution— Dissolve 50.0 g of sucrose in
sufficient distilled water to make 500 ml, adjust the pH to
7.2 with 10 M NaOH and sterilise by filtration.
Mix equal volumes of solutions (1) and (2) aseptically
before use.
5Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

6. Standard Preparation and Test Animals:
 Standard Preparation:
The standard preparation is the freeze dried preparation,
the potency of which has been determined in relation to
the International reference preparation.
It may be obtained from Central Research Laboratory.
 Test Animals:
Mice 11-15 g, 3-4 weeks old.
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7. Mice are divided for 2
Test
Potency Test
Six Groups of 16 mice
Challenge Virus Test
Four Groups of 10 Mice
Throughout the test all the mice that die before
the 5th day after challenge virus injection are
excluded from the test.
And all the mice that die with signs of rabies between 5th
and 14th day after challenge and those that are alive but
shows sign of rabies after 14th day of challenge are
counted as failing to resist the challenge.
The strain of mice suitable for the test is such that when
0.03ml containing 5-50 LD50 of standard challenge virus
susp injected per mouse will produce 100% mortality.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

8. Standard Challenge Virus Suspension Preparation:
Challenge virus strain is prepared by injecting
intracerebrally 0.03ml of 10 fold dilution of Rabies
virus in 2%w/v sterile inactivated normal horse serum
in water for injection into a suitable no of test animal.
The animals when morbound, after showing
characteristics symptoms of rabies are sacrificed and
their brain harvested aspectically. Washed in chilled
physiological saline to remove blood clots.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

9. SCV Suspn Prepn contd…………..
 10 % suspension of brain is made in 10% acid
digested casein hydrolysate at pH 7.2 and throughly
homogenized.
 After centrifuging lightly the supernatant liquid is
distributed into sterile ampoules and freeze dried. And
stored at 4ºC.
 In the lyophilized state the virus titre is maintained for
at least 3 years.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

10. Virus Titre of Challenge Virus
Test:
 Prepare 10 fold serial dilution of standard challenge
virus suspension.
 Using 4 groups of 10 mice each, inject 0.03ml of virus
suspension intracerebrally into each mouse using
different group for each suspension.
 Observe the mice for 14 days.
 Calculate the virus titre of standard challenge virus
suspension in LD50 per doses of 0.03ml by standard
statstical method from the no of mice in each group that
survive the challenge.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

11. Determination of Potency of Vaccine:
Reconstitute the standard preparation with
a suitable diluent.
Prepare at least three 5 fold serial dilution
of solution of standard preparation. And
three 5 fold dilution of vaccine under
examination.
For both standard and sample the serial
dilution should be prepared in such a way
that the lowest dilution protects more than
50% of injected mice.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

12. Determination of Potency contd……
 Allocate 1 dilution to each of six groups of 16
mice each.
 Inject intraperitoneally each mouse in each group
with dilution of vaccine and standard preparation.
 After 7 days, prepare similar dilution of vaccine
and reference (std.) preparation and repeat the
injections.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

13. Determination of Potency contd……
 After a further 7 days, inject each vaccinated
mouse intracerebrally with 0.03ml of standard
challenge virus suspension such that on the
basis of preliminary titration, 0.03ml
comparison between 5 to 50 LD50.
 Observe the mice for 14 days and record the
no. of mice surviving the challenge in each
group.
13Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

14. Determination of Potency contd……
 Calculate potency of preparation under examination
by standard statistical method.
 The vaccine complies with test if the estimated
potency is NLT 2.5 IU per single human dose.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

15. Interpretation:
The test is valid only if:
a) for both the preparation under examination and
Standard preparation, the 50% protective doses lies
between the largest and smallest dose given to mice
b) There is no deviation from linearity or parallelism of
the dose response lines, the confidence limit
(P=0.95) are not less than 25 % and NMT 400 % of
estimated potency.
c) The virus titre of standard challenge virus
suspension lies between 5 to 50 LD50.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

16. Reference:
 Indian Pharmacopoeia 2007, Volume 3, The Indian
Pharmacopoeia Commission, Ghaziabad, Vaccine:
General Requirements; 1355-1356.
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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)

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Rabies Vaccine, Rishi Ram Parajuli M.Pharm (PA&QA)