Biological test and assay of Absorbed Diphtheria vaccine

1. Presented by,
Usha Khanal
M.Pharm 1st year (Pharmaceutical analysis
& quality assurance)
Himalayan Pharmacy Institute

2. vaccine
 A vaccine is a substance containing
a harmless form of the germs that cause a
particular disease.
 It is given to people, usually by injection,
to prevent them getting that disease.

3. Classification of vaccine
• Live or attenuated vaccines
 Dead or inactivated vaccines
 Conjugate vaccines
 Recombinant vaccines
 Toxoid vaccines
 DNA vaccines
 The two main classifications of vaccines are: live and
dead vaccines.

4. Live or attenuated vaccines
 They are derived from disease-causing “wild”
bacteria or viruses.
 These bacteria and viruses are weakened or
“attenuated” by culturing them in a laboratory.
 After being injected, live attenuated vaccines grow
and replicate inside the vaccinated person’s body
to produce an immune response.

5. Dead or inactivated vaccines
 These vaccines consist of killed or inactivated forms of
the pathogen, killed with either heat, chemicals like
formalin, or radiation.
 These vaccines function when the immune system
responds to the introduction of the virus, bacteria, or
toxin produced by the germ.

6. Adsorbed Diphtheria Vaccines
 Diphtheria vaccine(absorbed) is a sterile suspension prepared
by mixing purified diphtheria formol toxoid + Mineral
carries[which is hydrated aluminum hydroxide [or] calcium Po 4
] , in saline solution or other suitable solution isotonic with
blood.
 The formal toxoid is prepared from the toxin, produced from the
Corynebacterium diphtheriae.
 It is an active immunizing agent.

7. Dose
 Dose of 0.5ml by intramuscular injection at 2 months.
 2nd dose of 0.5ml after 4 weeks.
 3rd dose of 0.5ml after another 4 weeks.
 A reinforcing dose of 0.5ml is recommended at school
entry age(4 to 6 yrs) preferably after an interval of at
least 3yrs after completing basic course.

8. BIOLOGICAL ASSAY OF
ADSORBED DIPHTHERIA VACCINE
1.Intradermal challenge method (POTENCY
DETERMINATION)
 The potency of ADV is determined by comparing the dose
necessary to protect guinea-pigs against effects of a range
of intradermal injections of diphtheria toxin with the dose
of Standard Preparation of adsorbed diphtheria toxoid
necessary to give the same protection.
 For this, the Standard Preparation of adsorbed diphtheria
toxoid and a suitable preparation of diphtheria toxin, for
use as a challenge toxin, are required.

9.  Standard preparation:Consist of toxoid absorbed on
aluminum hydroxide with polygeline.
 TEST ANIMAL : White guinea pig weight between
250g-350g

10. SELECTION OF CHALLENGE
TOXOIN:
select a preparation of diphtheria toxin containing 67-
133 Lime Reactionis/100 in limes flocculation and
25000-50000 minimal reacting doses for guinea pig
skin in 1 LF(limes flocculation )

11. PREPARATION OF CHALLENGE
TOXOIN SOLUTION
 Dilute the challenge toxin with a suitable diluents to
obtain a challenge toxin solution containing about
512×10 -4 LF in 0.2ml.

12. DETERMINATION OF POTENCY OF
VACCINE:-
Vaccine diluite with saline solution
↓
Inject guinea pig subcutaneously (5 numbers)
↓
After 28 days shave both flanks of each guinea pig
↓
0.2ml challenge toxin solution inject guinea pig-intradermally
↓
after 4 hours , record the incidence of diptheria erythema .
↓
assay limit falls between 50% and 200% of the estimated potency.

13. 2)LETHAL CHALLENGE METHOD
TEST ANIMAL: guinea pig weight between 250g-350g
divide them into 6 groups of 16animals and 4groups
containg-5 animals.
• All guinea pigs should have same sex.
CHALLENGE TOXIN: Diphtheria toxin containing not
less than 100 LD 50 in 1.0ml.

14. PREPARATION OF CHALLENGE TOXIN SOLUTION
• Challenge toxin + phaosphate buffer saline soln
(pH7.4).
• Dilute the challenge toxin soln to 2LD 50 , 1LD 50 ,
1/2LD 50 in the same solution

15. 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 subcuatneously into guinea
pigs.
 This should (intermediate conc) Protect 50% animal
from lethal effect of subcutaneous injection.

16. Allocate 6 dilutions, one to each of the 6 groups of
16guinea 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.

17.  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.
 The test is not valid unless vaccine under examination
and std preparation, the 50% protective doses lie
between the largest and smallest doses.
 4 groups of 5 guinea pigs ingested challenge toxin
should produce 100 LD 50
 Mortality increases when increases the toxin dose
level.

18. Reference
 INDIAN PHARMACOPOEIA- VOLUME I (Pg; 262-
264).