RAGHAV DOGRA

M .PHARMA(PHARMACEUTICAL ANALYSIS)

2ND SEMESTER

Assay of Adsorbed Diptheria Vaccine

and Adsorbed Tetanus Vaccine

TABLE OF CONTENT INTRODUCTION TO ADV.

PRINCIPLE OF ASSAY.

LETHAL CHALLENGE METHOD.

INTRODUCTION TO ATV.

PRINCIPLE OF ASSAY.

ASSAY METHOD OF ATV.

METHOD A. CHALLENGE TOXN IN GUINEA PIG.

METHOD B. CHALLENGE TOXIN IN MICE.

METHOD C. DETERMINATION OF ANTIBODIES IN GUINEA PIG

GUIDELINES

INTRODUCTION OF ADV: Adsorbed Diphtheria Vaccine (ADV) is an anti-toxin

preparation containing antitoxic globulins that havethe power of specifically neutralizing the toxinformed by Corynebacterium diphtheria.

It is obtained by fraction of horse or other mammalserum, that have been immunized againstDiphtheria toxin.

Diphtheria vaccine is generally available in thecombination with the Tetanus And Pertusis vaccineor DPT.

The formal Toxoid is prepared from the toxinproduced from the bacterium Corynebacterium

Cont… It is generally prepared by combining Diphtheria

formal toxoid with mineral carrier i.e. hydrated

Aluminum hydroxide, Ammonium hydroxide or

Phosphates ,Calcium Phosphates etc.

Diphtheria formal

Toxoid

Mineral Carrier such

as Aluminum,

Ammonium Hydroxide

etc

Adsorbed Diphtheria Toxin(

ADV)

PRINCIPLE OF ASSAY:

The potency of Diphtheria antitoxin is

determined by comparing the dose necessary

to protect the guinea pig or rabbit against the

erythrogenic effect of a fixed dose of the

standard preparation of Diphtheria toxin

required necessary to give same protection.

SELECTION OF CHALLENGE TOXIN:

Selection of the challenge toxin is based upon reacting

dose (Lr).

The preparation containing 67-133 Lr/mL in Lime

flocculation's (lf) of Diphtheria Toxin (DT) is selected or

the preparation with 25000 to 50000 minimal reacting

doses for guinea pig skin in 1lf.

The toxin are stored in the refrigeration 0-5˚C and

Toluene is added as antimicrobial preservatives which

does not cause rapid decline in toxicity.

PREPARATION OF STANDARD TOXIN:

It is prepared in a saline solution having 1mL of

preparation having 0.1 IU of toxin followed by:

1mL of standard preparation along with 0.2 IU test

toxin.

1mL of standard preparation along with 0.3IU test

toxin.

Store the preparation away from light.

PREPARATION OF CHALLENGE

TOXIN:

Challenge toxin / preparation to be assayed is diluted

with phosphate buffer solution (PBS) of pH 7.4.

The challenge toxin solution is diluted in the same

solution upto the following level:

2LD50

LD50

½LD50

LETHAL CHALLENGE METHOD:

Test animal such as guinea Pig, Horses, Rabbits are

selected for this.

If guinea pig then it should be of 250-350 gm.

The animal are grouped into 6 groups of 16 animals

each having same sex.

0.2 mL of each mixture should be injected

subcutaneously or intradermally into the animal.

The animals are observed after 48 hours for specific

Diphtheria erythema.

Mixture containing larger amount of toxin will give

severe reaction and vice versa.

DETERMINATION OF POTENCY OF

THE VACCINE:

The 3 dilutions of sample and standard vaccine are

prepared in saline solution.

Inject into guinea pigs.

This should protect 50% animals from lethal effect of

subcutaneous injection.

Calculate the potency of the vaccine relative to the

potency of potency of standard preparation on the

basis of the number of animals survived in each

group of 16 animals.

VALIDITY OF TEST:

Vaccine under examination and standard

preparation , the 50% protective doses lies between

the largest and the smallest dose of the preparation

given to guinea pigs.

Mortality increases when increases in toxin dose

level is there the minimum amount of toxin which

when combined should cause a local skin reaction

that is just visible and indicates the presence of

toxin.

INTRODUCTION TO ATV:

Tetanus vaccine, also known as tetanus toxoid (TT), is

an inactive vaccine used to prevent tetanus.

During childhood five doses are recommended, followed by

additional doses every ten years. After three doses almost

everyone is immune. In those who are not up to date on

their tetanus immunization a booster should be given within

48 hours of an injury.

In those with high risk injuries who are not fully immunized

tetanus antitoxin may also be recommended. Making sure

women who are pregnant are up to date on their tetanus

immunization and, if not, immunizing them can

prevent neonatal tetanus.

Contd..

Tetanus Toxoid Adsorbed USP, for intramuscular use, isa sterile suspension of alum-precipitated (aluminumpotassium sulfate) toxoid in anisotonic sodium chloride solution containingsodium phosphate buffer to control pH.

Clostridium tetani culture is grown in a peptone-basedmedium and detoxified with formaldehyde. The detoxifiedmaterial is then purified by serial ammonium sulfatefractionation, followed by sterile filtration, and the toxoid isadsorbed to aluminum potassium sulfate (alum). Theadsorbed toxoid is diluted with physiological saline solution0.85%.

Each 0.5 mL dose is formulated to contain 5 Lf(flocculation units) of tetanus toxoid and not more than

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.

ASSAY METHODS OF ATV:

ASSAY OF ADSORBED

TETANUS VACCINE

METHOD B. CHALLENGE

TEST IN MICE.

METHOD A. CHALLENGE

TEST IN GUINEA-PIGS.

METHOD C. DETERMINATION OF

ANTIBODIES IN

GUINEA-PIGS

METHOD 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.

Contd…SELECTION OF CHALLENGE TOXIN:

Preparation of tetanus toxin containing not less than 50times the 50 per cent paralytic dose per millilitre isselected.

If the challenge toxin preparation has been shown to bestable, it is not necessary to verify the paralytic dose forevery assay.

PREPARATION OF CHALLENGE TOXIN SOLUTION:

The challenge toxin is immediately diluted to 50 times the50 per cent paralytic dose per millilitre with the, peptonebuffered saline solution pH 7.4 etc to obtain stablechallenge toxin.

It necessary then use the challenge toxin diluted with same

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.

Contd..

Contd…

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 per cent paralytic dose.

It is then not necessary to repeat the determination for

Contd..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

Contd..

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 per cent paralytic dose,

the confidence limits (P = 0.95) are not less than 50 per

cent 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 dose

response line.

METHOD 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

TOXIN:

Contd..

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 guinea-pigs,

using the usual statistical methods.

VALIDATE ASSAY:

METHOD C. DETERMINATION OF

ANTBODIES IN GUINEA PIG SELECTION AND DISTRIBUTION OF TEST ANIMALS:

Healthy guinea pigs from the same stock should be selected ofweight 250-350 gm.

Animals are distributed in not less than 6 equal groupscontaining no. of animals sufficient to obtain results.

Animals should be of same sex if both sex are included then theshould be equally distributed among groups.

Use a further group of non-vaccinated guinea-pigs of the sameorigin to provide a negative serum control. If test consistencyhas been demonstrated, a reference negative serum controlmay be used.

Contd… REFERENCE PREPARATION:

A suitable reference preparation such as tetanusvaccine(adsorbed) BRP or a batch of vaccine shown to beeffective in clinical studies, or a batch representative thereof,and which has been calibrated in International Units withreference to tetanus vaccine (adsorbed) BRP or theInternational Standard for tetanus toxoid (adsorbed) are used.

DILUTION OF THE TEST AND REFERENCE SOLUTION:

The vaccine to be examined and the reference preparation arediluted with the 9g/l solution of NaCl.

The dilution forms series which do not differ the by 2.5 to 5 foldsfrom the alternating previous and next dilutions. Use not fewerthan 3 dilutions within the range for example 0.5-16 IU/ml foreach series. Use dilutions for immunization preferably within 1 hof preparation. Allocate 1 dilution to each group of guinea-pigs.

Contd…

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.

Contd…

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 (for example 5.3).

NOTE: International Units of potency refer to the

reference vaccine and not to the International Units of

antitoxin of the reference guinea-pig serum.

Contd… Requirements for a valid assay. The test is not valid

unless :

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 dose-

response 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.

GUIDELINES…

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. The tetanus

grades in guinea-pigs and mice are characterized by

the following signs

Contd..

T1: slight stiffness of 1 forelimb, but difficult to observe

T2: paresis 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 ;

D:death.

Contd… METHOD C. DETERMINATION OF ANTIBODIES IN

GUINEA-PIGS

PREPARATION OF SERUM SAMPLES:

For preparation of serum samples, the following technique

has been found suitable. Invert the tubes containing blood

samples 6 times and allow to stand at 37 °C for 2 h, then at

4 °C for 2 h. Centrifuge at room temperature at 800 g for

20 min. Transfer the serum to sterile tubes and store at a

temperature below −20 °C. At least 40 per cent yield of

serum is obtained by this procedure.

DETERMINATION OF ANTIBODY TITRE:

The ELISA and ToBI tests shown below are given as

examples of immunochemical methods that have been

found suitable for the determination of antibody titre.