Introduction to Absorbed Tatanus Vaccine Principle, Assay methods of ATV, Preparation, Symptoms, Causes, Risk factor, Complications Presnted by SHAIK GOUSE UL AZAM Department of Pharmaceuticals Analysis

1. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
ABSORBED TATANUS VACCINE
A Seminar as a part of curricular for Master of Pharmacy,
I Year - I semester
Presented by
SHAIK GOUSE UL AZAM
(20L81S0708)
PHARMACEUTICAL ANALYSIS
Under the guidance of
SHAIK SHAKIR BASHAM.Pharm,
Assistant Professor
Department of Pharmaceutical Analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents
• Introduction
• Principle
• Assay methods of ATV
• Preparation
• Symptoms
• Causes
• Risk factor
• Complications
• References
2

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Itroduction
Vaccine:
• A preparation of killed micro
organisms that are attenuated /living
fully virulent organisms that is
administration to produce immunity
to a particular disease
3

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Absorded Tatanus vaccine
• Tatanus is a serious disease caused by a bacterial toxin that affects
nervous system, leading to painful muscle contractions, particularly
of jaw and neck muscles.
• Tatanus can interfere with ability to breathe and can threaten life.
• Tatanus is commonly known as “lockjaw”
4

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Biological Assay Of T
etanus V
accine
• 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
5

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
A. CHALLENGE TOXIN IN GUINEAPIG
• SELECTIONAND DISTRIBUTION OFTESTANIMALS:
• Healthy guinea pigs from the same stock should be selected
• weight 250-350 gm.
• Animals are distributed in not less than 6 equal groups containing
number 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.
7

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
• 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.
8

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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 CHALLENGEAND CHALLENGE:
• Allocate the dilutions to the groups.
• Then Inject subcutaneously 1 ml allocated dilution into the
guinea pig of respective groups.
9

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
DETERMINATION OFACTIVITY OFTHE
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.
10

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
REQUIREMENTS FORAVALIDASSAY
• 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.
11

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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
12

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
B.CHALLENGE TOXIN IN MICE:
• SELECTIONAND DISTRIBUTION OFTESTANIMALS:
• 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 CHALLENGEAND CHALLENGE:
• DETERMINATION OFACTIVITY OF THE CHALLENGE:
13

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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.
14

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
C.DETERMINATION OF ANTBODIES IN GUINEA
PIG
• SELECTIONAND DISTRIBUTION OFTESTANIMALS:
• Healthy guinea pigs selected of weight 250-350 gm.
• Animals are distributed in not less than 6 equal groups containing
number 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.
15

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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 OFTHE TESTAND 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.
16

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
• 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.
17

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
DETERMINATION OFANTIBODY 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.
18

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Tatanus vaccine preparation
• A process for the preparation of tatanus toxoid, which process
comprises incubating purified tatanus toxin with 0.2 to 1%(v/v)
formaldehyde in the presence of 0.005 to 0.25M lysine for 24 to 32
days at a PH of 6.0 to 8.0 and temperature of 30 to 45 c
19

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Symptoms
• Common signs and symptoms of tatanus include:
• Spasms and stiffness in jaw muscles (trismus)
• Stiffness of neck muscles
• Difficulty swallowing
• Stiffness of abdominal muscles
• Fever
• Sweating
• Elevated blood pressure
• Rapid heart rate
20

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Causes
• Tatanus is caused by a toxin made by spores of bacteria, Clostridium
tetani, found in soil, dust and animal feces.
• When the spores enter a deep flesh wound, they grow into bacteria
that can produce a powerful toxin, tetanospasmin.
• Nearly all cases of tatanus occur in people who have been
vaccinated or in adult or in adults who have not kept up with their
10-years booster shots.
21

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Risk factor
• Tatanus cases have developed from the following
• Puncture wounds
• Gunshot wounds
• Burns
• Surgical wounds
• Injection foot ulcers
• Dental infections
• Infected umbilical stumps in newborns born of inadequately
vaccinated mothers
22

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Complications
Complications of tatanus infection may include
• Broken bones: The severity of spasms may causes the spine and
other bones to break.
• Blockage of lung artery (pulmonary embolism): A Blood clot that
travel from body can block the main artery of the lung or one of it
branches.
• Death: severe tatanus –induced muscle spasms can interfere with or
stop your breathing .lack of oxygen may also induce cardiac arrest
and death . Pneumonia is another cause of death.
23

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Prevention
• One can easily prevent tatanus by being vaccinated.
• The primary vaccine series
• The tatanus vaccine usually is given to childrens as part of the
diphtheria and tatanus toxoids and a cellular pertussis vaccine .
• This vaccination provides protection against three diseases throat
and respiratory infection(diphtheria),whooping cough etc.
• The Dtap vaccine is a series of five shots, typically given in the
arms or thigh to childrens at ages:2months,4months,6months,15 to
18months,4 to 6years
24

25. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Reference
• J Hamborsky, A Kroger, S Wolfe, Epidemiology and prevention of
vaccine-preventable diseases 2015.
• V Baldo, P Bonanni, M Castro “combined hexavalent diphtheria –
tatanus –acellular pertussis 2014.
• JH Brown Precision of potency assay of alum-precipitated tetanus
toxoid in mice: an interinstitutional study. Journal of Immunology,
1953.
• J Lyng , G Nyerges The second international standard for tetanus
toxoid (adsorbed). Journal of Biological Standardization, 1984.
25

26. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26