1. MICRO BIOLOGICALASSAY OF
VACCINES
Under the guidence of
J. Venkateswara rao
(M. pharm)
Associate proffesor
Presented
by
K.SIVA GANESH
I/II M.Pharm
Pharmaceutical Analysis
3. INTRODUCTION :
Vaccines:
A preparations of killed
micro organisms that are
attenuated/living fully virulent
organisms that is administered to
produce immunity to a paticular
disease.
5. Biological Assay
A . INTRADERMAL CHALLENGE METHOD
Principle:
The potency of adsorbed diphtheria vaccine is
determined by comparing the dose is necessary to protect
guinea pig against the erythrogenic effect of range of
intradermal injection of diphtheria toxin with dose of the
standard preparation of adsorbed diphtheria toxin
necessary to give the same protection.
6. Procedure :
Standard preparation:
Standard preparation consisting of toxoid adsorbed
on a aluminium hydroxide with polygeline.
Test animal:
White guinea pig
Weight between 250-350gm
7. Selection of challenge toxin:
Selection of preparation of diphtheria toxin
containing 67 –133Lr / 100 in limes flocculation (Lf) and 25000
– 50000minimal reacting doses for guinea pig skin in 1Lf
(limesflocculation).
Preparation of challenge toxin solution:
Dilute challenge toxin with a suitable diluents to
obtain achallenge toxin solution containing about 512 × 10-4Lf
in0.2ml.
DETERMINATION OF POTENCY OF THE VACCINE
Vaccine is dilute with the saline solution
Inject guinea pig subcutaneously (5 numbers)
After 28 days shave the both flanks of each guinea pig
0.2ml challenge toxin solution is inject to guinea pig intradermally
After 48 hrs, record the incidence of specific diphtheria erythema
Assay limit falls between 50% and 200% of the estimated potency.
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8. B.LETHAL CHALLENGE METHOD :
TEST ANIMAL:
• Guinea pig.
• Weight between 250g – 350g.
• Divide them in to 6 groups of 16 animals.
• A group containing 5 animals.
• All guinea pigs have same sex.
9. Challenge toxin :
Diphtheria toxin containing NLT100 LD50 in 1ml.
Preparation of challenge toxin solution:
• Challenge toxin + Phosphate buffer saline solution (PH 7.4).
• Dilute the challenge toxin solution to 2LD50 , 1LD 50 , 1/2LD 50
in the same solution.
10. Determination of potency of the vaccine:
• 3 Dilutions of sample and standard vaccine prepared in saline
solution each dilution difference by 2.5 fold.
• Intermediate conc. inject subcutaneously into guinea pigs.
• This should (intermediate conc) protect 50% animal from
lethal effect of subcutaneous injection.
11. Allocate 6 dilutions , one to each of the 6 groups of 16 guinea
pigs(1ml)
After 28 days
Test challenge toxin dilution
1ml inject to 4 groups of 5 - guinea pigs - subcutaneously
After 4 days count the number of survival animals
Calculate the potency of the vaccine relative to the potency
Of standard preparation on the basis of number of animals
survived in each group of 16 animals.
12. Test validation :
• Vaccine under examination and standard preparation, the 50%
protective doses lies between the largest and smallest doses of
the preparation given to the guinea-pigs
13. RABIES VACCINE
DEFINITION :
• Rabies vaccine is a suspension of a suitable
strain of fixed rabies virus grown in suitable
approved cell culture and inactivated by a
suitable method.
• The vaccine is prepared immediately before use
by reconstitution from the dried vaccine with a
suitable sterile liquid.
Dried vaccine + sterile liquid Suspension.
14. BIOLOGICALASSAY OF RABIES VACCINE
Principle:
The potency of rabies vaccine is determined by
comparing a lethal intracerebral dose of a rabies virus with the
dose of the standard preparation of rabies necessary to give for
same protection.
Standard preparation: Freeze – dried preparation.
15. Test animals:
• White mice weight 11gm –15gm.
• 6 groups of 16 animals , 4 groups of
10 animals.
• These two groups are used for
titration of LD50 challenge
suspension.
• Injected 0.03ml/mice intracerebrally.
16. STANDARD CHALLENGE VIRUS SUSPENSION
Standard challenge virus suspension is prepared by injecting
Intracerebrally 0.03ml of 10 fold dilution standard
strain horse serum to the test animal.
Showing characteristic symptoms of encephalitis are
sacrified
Harvest their brains aseptically
Wash it with saline solution to remove blood clot
Homogenized brain with 10% digested casein hydrolysate (PH 7.2)
Centrifuge and supernated liquid is distributed into sterile vials and
stored at 2 – 80. 21
17. DETERMINATION OF CHALLENGE VIRUS
(Determination of virus titre of the challenge virus)
Prepare 3-10 fold dilution of standard challenge virus suspension.
0.03 ml inject intra cerebrally to a groups of 10 mice.
Observe for 14 days
Count the number of mice surviving in each group
Calculate the virus titre of standard challenge virus
suspension by statistical method.
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18. DETERMINATION OF POTENCY OF THE VACCINE
Prepare 3-5 fold serial dilution of standard and test solution of vaccines
Separate mice in 6 groups of 16 each
Inject 0.03 ml intra peritoneally and 7 days
prepare same solution and
inject
Both standard and test should prepared in such away
After 7 days inject 0.03 ml standar virus suspension to vaccinated mice
Observe if for 14 days and calculate its potency by statistical method.
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19. • Lowest dilution should protect the 50% of the animal
• Highest dilution protect less than 50% of the animal
20. ADSORBED TETANUS VACCINE
• Tetanus Toxoid Adsorbed USP, for intramuscular use, is a
sterile suspension of alum-precipitated (aluminum potassium
sulfate) toxoid in an isotonic sodium chloride solution
containing sodium phosphate buffer to control pH.
• Clostridium tetani culture is grown in a peptone-based
medium and detoxified with formaldehyde. The detoxified
material is then purified by serial ammonium sulfate
fractionation, followed by sterile filtration, and the toxoid is
adsorbed to aluminum potassium sulfate (alum). The adsorbed
toxoid is diluted with physiological saline solution 0.85%.
• Each 0.5 mL dose is formulated to contain 5 Lf
21. Biological Assay Of Tetanus Vaccine
Principle :
• The potency of tetanus vaccine is determined by
administration of the vaccine to animals i.e. guinea-pigs or
mice followed administration of either challenge with tetanus
toxin or by determining of the titre of antibodies against
tetanus Toxoid in the serum of the guinea-pigs.
• In both cases the potency of the vaccine is calculated by
comparison with a reference vaccine, calibrated in
International Units.
• Tetanus vaccine (adsorbed) BRP is calibrated in International
Units with reference to the International Standard.
22.
23. A. CHALLENGE TOXIN IN GUINEA PIG
SELECTION AND DISTRIBUTION OF TEST ANIMALS:
• Healthy guinea pigs from the same stock should be selected of
weight 250-350 gm.
• Animals are distributed in not less than 6 equal groups containing
no. of animals sufficient to obtain results
• If the validity of the challenge toxin is to be determined 3 further
groups of the 5 animals each should be taken which are used as
unvaccinated control.
• Animals should be of same sex if both sex are included then the
should be equally distributed among groups.
24. SELECTION OF CHALLENGE TOXIN:
• Preparation of tetanus toxin containing not less than 50 times
the 50 percent paralytic dose per millilitre is selected.
• If the challenge toxin preparation has been shown to be stable,
it is not necessary to verify the paralytic dose for every assay.
PREPARATION OF CHALLENGE TOXIN SOLUTION:
• The challenge toxin is immediately diluted to 50 times the 50
percent paralytic dose per millilitre with the, peptone buffered
saline solution pH 7.4 etc to obtain stable challenge toxin.
25. DILUTION OF THE TEST AND REFERENCE
SOLUTION:
• The vaccine to be examined and the reference preparation are
diluted with the 9g/l solution of NaCl.
• The dilution forms series which do not differ the by 2.5 folds
from the alternating previous and next dilutions.
• When injected subcutaneously with dose of 1.0 mL per guinea
pig should protect approximately 50% of animals from the
paralytic effects of tetanus toxin prescribed for test.
IMMUNIZATION CHALLENGE AND CHALLENGE:
• Allocate the dilutions to the groups.
• Then Inject subcutaneously 1 mL allocated dilution into the
guinea pig of respective groups.
26. DETERMINATION OF ACTIVITY OF THE CHALLENGE
TOXIN:
• 3 dilutions made from the challenge toxin and each dilution
were allocated to 1 group of 5 guinea pigs i.e. 3 groups if
necessary.
• Subcutaneously inject 1mL of allocated solution into each
guinea pig of the group.
• The activity and stability of the challenge toxin are determined
by carrying out a suitable number of determinations of the 50
percent paralytic dose.
27. REQUIREMENTS FOR A VALID ASSAY:
• The test is not valid unless, for both the vaccine to be
examined and the reference preparation the 50 per cent
protective dose lies between the largest and smallest doses of
the preparations given to the guinea-pigs,
• If applicable, the number of paralyzed animals in the 3 groups
of 5 injected with the dilutions of the challenge toxin solution
indicates that the challenge was approximately 50 times the 50
percent paralytic dose.
• The confidence limits (P = 0.95) are not less than 50 per cent
and not more than 200 percent of the estimated potency,
• The statistical analysis shows significant slope and no
deviation from linearity and parallelism of the dose.
28. READING AND INTERPRETATION OF
RESULTS:
• The guinea-pigs are examined twice daily. Remove and
humanely kill all animals showing definite signs of tetanus
paralysis.
• The number of guinea-pigs without paralysis 5 days after
injection of the challenge toxin are counted.
• Calculate the potency of the vaccine to be examined relative to
the potency of the reference preparation on the basis of the
proportion of challenged animals without paralysis in each of
the groups of vaccinated guinea-pigs, using the usual statistical
methods
29. B.CHALLENGE TOXIN IN MICE:
SELECTION AND DISTRIBUTION OF TEST ANIMALS:
• Use in the test healthy mice from the same stock, about 5
weeks old and from a strain shown to be suitable. Rest
same as guinea pig.
SELECTION OF CHALLENGE TOXIN:
• Preparation of tetanus toxin containing not less than 100
times the 50 per cent paralytic dose per millilitre is
selected.
• PREPARATION OF CHALLENGE TOXIN SOLUTION:
• DILUTION OF THE TEST AND REFERENCE
SOLUTION:
• IMMUNIZATION CHALLENGE AND CHALLENGE:
• DETERMINATION OF ACTIVITY OF THE
CHALLENGE:
30. READING AND INTERPRETATION OF
RESULTS:
• The mice are examined twice daily. Remove and humanely kill
all animals showing definite signs of tetanus paralysis.
• The number of mice without paralysis 4 days after injection of
the challenge toxin are counted.
• Calculate the potency of the vaccine to be examined relative to
the potency of the reference preparation on the basis of the
proportion of challenged animals without paralysis in each of
the groups of vaccinated mices, using the usual statistical
methods.
31. C. DETERMINATION OF ANTBODIES IN GUINEA PIG
SELECTION AND DISTRIBUTION OF TEST ANIMALS:
• Healthy guinea pigs from the same stock should be
selected of weight 250-350 gm.
• Animals are distributed in not less than 6 equal groups
containing no. of animals sufficient to obtain results.
• Animals should be of same sex if both sex are included
then the should be equally distributed among groups.
• Use a further group of non-vaccinated guinea-pigs of the
same origin to provide a negative serum control. If test
consistency has been demonstrated, a reference negative
serum control may be used.
32. REFERENCE PREPARATION:
• A suitable reference preparation such as tetanus
vaccine(adsorbed) BRP or a batch of vaccine shown to be
effective in clinical studies, or a batch representative thereof,
and which has been calibrated in International Units with
reference to tetanus vaccine (adsorbed) BRP or the International
Standard for tetanus toxoid (adsorbed) are used.
DILUTION OF THE TEST AND REFERENCE SOLUTION:
• The vaccine to be examined and the reference preparation are
diluted with the 9g/l solution of NaCl.
• The dilution forms series which do not differ the by 2.5 to 5
folds from the alternating previous and next dilutions. Use not
fewer than 3 dilutions within the range for example 0.5-16
IU/ml for each series. Use dilutions for immunization preferably
within 1 h of preparation. Allocate 1 dilution to each group of
guinea-pigs.
33. IMMUNISATION:
• Inject subcutaneously in the nape of each guinea-pig 1.0 ml of
the dilution allocated to its group.
BLOOD SAMPLING:
• 35-42 days after immunization, take a blood sample from each
vaccinated and control guinea-pig using a suitable method.
PREPARATION OF SERUM SAMPLES:
• Avoid frequent freezing and thawing of serum samples. To
avoid microbial contamination, it is preferable to carry out
manipulations in a laminar-flow cabinet.
34. DETERMINATION OF ANTIBODY TITRE:
• Determine the relative antibody titre or score of each
serum sample by a suitable immunochemical method .
The methods shown below (enzyme-linked
immunosorbent assay (ELISA) and toxin-binding
inhibition (ToBI)) have been found suitable.
CALCULATION OF POTENCY:
• Calculate the potency of the vaccine to be examined in
International Units relative to the reference preparation,
using the usual statistical methods.
NOTE: International Units of potency refer to the reference
vaccine and not to the International Units of antitoxin of the
reference guinea-pig serum.
35. Requirements For A Valid Assay :
• The confidence limits (P = 0.95) are not less than 50 percent
and not more than 200 per cent of the estimated potency.
• The statistical analysis shows significant slope and no
deviation from linearity and parallelism of the doseresponse
lines .
• The test may be repeated but when more than 1 test is
performed the results of all valid tests must be combined in the
estimate of potency.
36. READING AND INTERPRETATION OF RESULTS:
• In order to minimize suffering in the test animals, it is
recommended to note the degree of paralysis on a scale such as
that shown below.
• The scale gives typical signs when injection of the challenge toxin
is made mid-ventrally directly behind the sternum with the needle
pointing towards the neck of the guinea-pig mice and .
• Grade T3 is taken as the end-point, but with experience grade T2
can be used instead.
• Tetanus toxin produces in at least 1 of the forelimbs paralysis that
can be recognized at an early stage.
37. The tetanus grades in guinea-pigs and mice are characterized by
• T1: slight stiffness of 1 forelimb, but difficult to observe
• T2: paralysis of 1 forelimb which still can function.
• T3: paralysis of 1 forelimb. The animal moves reluctantly, the
body is often slightly banana-shaped owing to scoliosis.
• T4 : the forelimb is completely stiff and the toes are
immovable. The muscular contraction of the forelimb is very
pronounced and usually scoliosis is observed;
• T5: tetanus seizures, continuous tonic spasm of muscles .
