In this slide contains definition and biological assay of Adsorbed Diphtheria Vaccine. Presented by: G.CHIRANJEEVI (Department of pharmaceutical analysis). RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 1
Adsorbed Diphtheria Vaccine
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presentedby
Mr G chiranjeevi
(Reg. No. L81S0706)
Under the guidance/Mentorship of
Mr S SHAKIR BASHA
Assistants Professor
Dept. of Pharmaceutical Analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction
• Diphtheria
• Adsorbed diphtheria vaccine
• Biological assay of adsorbed diphtheria vaccine
contents

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Vaccine:-
A substance used to stimulate the production of antibodies
and provide immunity against one or several diseases,
prepared from the causative agent of a disease, its products,
or a synthetic substitute, treated to act as an antigen without
inducing the disease.
• Examples:-
• Adsorbed tetanus vaccine
• Adsorbed diphtheria vaccine
• Rabies vaccine
Introduction

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 4
• Live or attenuated vaccines Ex:- BCG vaccine
• Whole killed or inactived vaccines Ex:- Rabies vaccine
• Recombinant vaccines Ex:- hepatitis B vaccine
• Toxoid vaccines Ex:- Adsorbed Diphtheria Vaccine
• Conjugate vaccines Ex:- Hib vaccine
• Nucleic acid vaccines
Classificationof vaccine

5. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Toxoid Vaccines
• Some bacteria release toxins (poisonous proteins) when
they attack the body, and it is the toxins rather than the
bacteria itself that we want to be protected against.
• The immune system recognises these toxins in the same
way that it recognises other antigens on the surface of the
bacteria, and is able to mount an immune response to
them.
• Some vaccines are made with inactivated versions
of these toxins.

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 6
• They are called ‘toxoids’ because they look like toxins but
are not poisonous.
• They trigger a strong immune response
Examples:
• Diphtheria vaccine
• Tetanus vaccine
• Pertussis (whooping cough) vaccine

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Diphtheria is a serious infection caused by strains of
bacteria called Corynebacterium diphtheriae that make
toxin (poison)
• A sheet of thick, grey matter covers the back of the
throat, making breathing hard.
• Symptoms:
• Difficulty breathing,
• Heart failure,
• Paralysis,
• Death in severe cases
Diphtheria

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 8
Diagnosis:
detection of tox gene by PCR, ELISA
Treatment :
Treatments include antibiotics(penicillin) and an antitoxin that
neutralises the diphtheria toxin. A vaccine is available.
History :
1826,Pierre Bretonneau gave the disease the name diphtheria
1883 Edwin klebs identify the bacteria named it klebs Loeffler
bacterium

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Diphtheria Vaccine (Adsorbed) is a preparation of
diphtheria formol toxoid with a mineral adsorbent
(hydrated aluminium hydroxide or aluminium
phosphate).
• The formol toxoid is prepared from the toxin produced
by the growth of Corynebacterium diphtheria.
• It is a toxoid type of vaccine.
Adsorbed Diphtheria Vaccine

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 10
• The immunization schedule for the DTaP vaccine in
children is a series of 5 shots at the following ages:
• 2 months
• 4 months
• 6 months
• 15 to 18 months
• 4 to 6 years
• Each dose contains 6.7 to 25Lf per 0.5 ml volume.
Dose

11. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 11
(a) Intradermal challenge method
• The potency of adsorbed diphtheria vaccine is
determined by comparing the dose necessary to
protect guinea-pigs against the erythrogenic effects of
a range of intradermal injections of diphtheria toxin
with the dose of the Standard preparation of adsorbed
diphtheria toxoid necessary to give the same
protection.
Biological assayof adsorbeddiphtheria
vaccine

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 12
• For this comparison, the Standard preparation of
adsorbed diphtheria toxoid and a suitable preparation of
diphtheria toxin, for use as a challenge toxin, are required.
• Standard preparation
The Standard preparation is International standard of
Diphtheria toxoid adsorbed
• Test animals
Use white guinea-pigs, weighing between 250 and
350 g

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 13
• Selection of the challenge toxin
Select a preparation of diphtheria toxin containing 67 to
133 Limes reactionis/100 (Lr/ 100) in Limes flocculationis
(Lf) and 25,000 to 50,000 minimal reacting doses for
guinea-pig skin in 1 Lf
• Preparation of the challenge toxin solutions
Immediately prior to use, dilute the challenge toxin with a
suitable diluent to obtain a challenge toxin solution
containing about 512x10-4 Lf in 0.2 ml.
Dilution portion give a series of Five 4 fold dilutions

14. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 14
Vaccine dilute with salinesolution
Inject guinea pig subcutaneously (1ml)
After 28 days shave both flanks of each guinea pig
0.2ml challenge toxin solution inject guinea pig-intradermally
After 48 hours , record the incidence of diphtheria erythema
Tabulate the intradermal score
Determinationof potency of vaccine

15. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 15
• Process is not valid unless
• Mean score obtained at lowest dose level is more than 3
• For toxin 4×10-5 give +ve erythema at least 80% of Guinea
pigs
• For toxin 2×10-5 give no reaction In at least 80% of Guinea
pigs
• Fiducial limits of assay between 50 to 200% of estimated
potency
• No deviations from linearity and parallelism

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 16
➢ Test animal :
• Guinea pig weight between 250g-350g divide them
into 6 groups of 16animals and 4groups containg-4
animals.
• All guinea pigs should have same sex.
➢ Challenge toxin:
• Diphtheria toxin containing not less than 100 LD50 in
1.0ml.
Lethal challenge method

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 17
➢ Preparation of challenge toxin solution
➢ Challenge toxin + phosphate buffer saline
solution (pH7.4).
➢ Dilute the challenge toxin solution to 2LD50 ,
1LD50 , 1/2LD50 in the same solution( 3 five
fold dilutions)

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 18
➢Determination of potency of the vaccine
• Three dilutions of sample and standard vaccine prepared
in saline solution each dilution difference by 2.5 fold.
• Intermediate conc. inject subcutaneously into guinea
pigs(1 ml) 6 groups of 16 guniea pigs
• This should (intermediate conc.) Protect 50% of
animals from lethal effect of subcutaneous injection.

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 19
After 28 days
Immunized guineig challenged with toxin (100 LD50)
Test challenge toxin dilution
1ml inject to 4 groups of 4-guinea pigs subcutaneously
After 5 days count the number of survival animals

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 20
• Calculate the potency of the vaccine on the basis of
number of animals survived .
• The 50% protective dose lie b/w the largest and smallest
doses.
• Survivors of 4 groups of guinea pigs indicate that the
challenge was approximately 100 LD50
• Mortality increases when increases in the toxin dose level.

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 21
• Indian pharmacopeia volume 3, 2007
• Manual for Quality Control of Diphtheria, Tetanus and
Pertussis Vaccines(WHO/IVB/11.11) by WHO April 2013
References

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.PalliCross, Chiyyedu, Anantapuramu, A. P- 515721 22