BIOASSAYS

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 Bioassay is defined as the estimation of the potency of an active
principle in a unit quantity of preparation or
detection and measurement of the concentration of the substance in
a preparation using biological methods
(i.e. observation of pharmacological effects on living tissues,
microorganisms or immune cells or animal).

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 Importance of Bioassay
Bioassays, as compared to other methods of assays (e.g. chemical or
physical assay) are less accurate, less elaborate, more laborious, more
troublesome and more expensive.
However, bioassay is the only method of assay if
(1) Active principle of drug is unknown or cannot be isolated, e.g. insulin,
posterior pituitary extract etc.
(2) Chemical method is either not available or if available, it is too
complex and insensitive or requires higher dose e.g. insulin,
acetylcholine.
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(3) Chemical composition is not known, e.g. long acting thyroid stimulants.
(4) Chemical composition of drug differs but have the same
pharmacological action and vice-versa, e.g. cardiac glycosides,
catecholamines etc.
(5) When the substance gets inactivated by interacting with chemicals as in
the case of hormones.
(6) When the quantity of the sample is too small.
(7) To estimate the concentration of active principles, present in the tissue
extracts, the endogenous mediators like acetylcholine, 5-HT, prostaglandins
etc...
(8) To measure the pharmacological activity of new or chemically
unidentified substances.

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 (9) To measure drug toxicity.
(10) When the bioassay is more sensitive than the chemical assay e.g.
insulin,acetylcholine.
(11) Standardize the preparation so that each contains the uniform
specified pharmacological activity

Moreover, even if chemical methods are available and the results of

bioassay conflict with those of the chemical assay, the bioassay is relied
upon and not the chemical assay, since it is the assessment on living
organism.

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 • The basic principle of bioassay is to compare the test substance with the
International Standard preparation of the same and to find out how
much test substance is required to produce the same biological effect,
as produced by the standard.
• The standards are internationally accepted samples of drugs
maintained and recommended by the Expert Committee of the
Biological Standardization of W.H.O. They represent the fixed units of
activity (definite weight of preparation) for drugs. In India, standard
drugs are maintained in Government institutions like Central Drug
Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc.
• The problem of biological variation must be minimized as far as
possible. For that one should keep uniform experimental conditions and
assure the reproducibility of the responses.
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 Precaution to be taken to minimize error due to biological variation:
1. Experimental conditions must be kept constant.
2. Biological response should be sensitive to the drug.
3. Biological response should be giving constant and reproducible results.
4. Biological response should be insensitive to other drugs.
5. Standard drug must be available to compare the response.
6. Number of experiments should be sufficiently large and the assay
design be optimized to minimize biological variation.
7. Animals should be of same species and strain.

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 Applications of bioassay:
1. Standardization of drugs of natural origin.
2. Estimation of biologically active substances (Ach, 5HT, Adr.).
3. Screening of new compounds for biological activity-even synthetic
compound.
4. To assay drugs with complex mixtures of substances e.g. Digitalis.
5. For diagnosis and research e.g. to find out concentration of
gonadotropins in the blood.
6. Estimation of the dose of the drug required to produce a therapeutic
or toxic response e.g. ED50 or LD50.

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 Methods of Bioassay for Agonists
An agonist may produce graded response or quantal response.
• Graded response means that the response is proportional to the dose
e.g. contraction of smooth muscle preparation for assaying histamine or
the study of blood pressure response in case of adrenaline and the
response may lie between no response and the maximum response.
• By quantal, it is meant that the response is in the form of "all or none",
i.e. either no response or maximum response. e.g. insulin-induced
hypoglycaemic convulsive reaction or cardiac arrest caused by digitalis.
In both these cases the end point is an all or none response i.e. either
convulsions or no convulsions similarly the cardiac arrest.

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 The drugs producing quantal effect can be bio assayed by end point method.
• The drugs producing quantal effect can be bio assayed by end point method.
• The drugs producing graded responses can be bio assayed by
a. Matching bioassay or bracketing bioassay.
b. Graphical method (Interpolation).
c. Multiple point bioassay.

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 End Point Method
Here the threshold dose producing a positive effect is measured on each
animal and the comparison between the average results of two groups of
animals (one receiving standard and other the test) is done.
e.g. bioassay of digitalis in cats.

Here the cat is anaesthetized with chloralose and its blood pressure is
recorded. The drug is slowly infused into the animal and the moment the
heart stops beating and blood pressure falls to zero, the volume of fluid
infused is noted down. Two series of such experiments one using standard
digitalis and the other using test preparation of digitalis is done and then
potency is calculated as follows:
Conc. of Unknown = Threshold dose of the Standard X Conc. of Std.
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Threshold dose of the Test
 2. Matching or bracketing Method:
In this method a constant dose of the test is bracketed by varying doses
of standard till the exact match is obtained between test dose and the
standard dose.
Initially, two responses of the standard are taken. The doses are
adjusted such that one is giving response of approximately 20% and
other 70% of the maximum. The response of unknown which lies between
two responses of standard dose is taken.
The panel is repeated by increasing or decreasing the doses of
standard till all three equal responses are obtained. The dose of test
sample is kept constant. At the end, a response of the double dose of
the standard and test which match each other are taken. These should
give equal responses.
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 Concentration of the test sample can be determined as follows:
Conc. of Unknown = Threshold dose of the Standard X Conc. of Std.
Threshold dose of the Test

This method has following limitations:

1. It occupies a larger area of the drum as far as tracings are
concerned.
2. The match is purely subjective, so chances of error are there and one
cannot determine them.
3. It does not give any idea of dose-response relationship. However, this
method is particularly useful if the sensitivity of the preparation is not
stable.
Bioassay of histamine, on guinea pig ileum is preferably carried out by
18 this method.
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 3. Graphical method:
This method is based on the assumption of the dose-response
relationship.
Log dose-response curve is plotted and the dose of standard producing
the same response as produced by the test sample is directly read from
the graph. In simpler design, 5-6 responses of the graded doses of the
standard are taken and then two equiactive responses of the test
sample are taken. The height of contraction is measured and plotted
against the log-dose. The dose of standard producing the same
response as produced by the test is read directly from the graph and
the concentration of test sample is determined by the same formula as
mentioned before.

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 Concentration of unknown is extrapolated from a standard plot of a
log dose response
curve of at least 4 sub maximal concentrations.
The characteristic of log-dose response curve is that it is linear in the
middle (20-80%).
Thus, the comparison should be done within this range only. In other
words, the
response of test sample must lie within this range.
Advantage of this method is that, it is a simple method and chances of
errors are less if the sensitivity of the preparation is not changed

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 Multiple point assay: Other methods based on the dose-response relationship include
3 point, 4 point, 5 point and 6 point methods. In these 4 methods, the responses are
repeated several times and the mean of each is taken. Thus, chances of error are
minimized in these methods.
Three-point bioassay: In 3-point assay method 2 doses of the standard and one dose of
the test are used.

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 The mean responses are calculated and plotted against log-dose and amount of standard
producing the same response as produced by the test is determined graphically as well
as mathematically:

Where,
n1 = Lower Standard dose,
n2 = Higher Standard dose,
t = Test dose,
S1 =Response of n1,
S2 = Response of n2,
T = Response of test (t),
Cs = Concentration of standard.

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 Four-point bioassay: In 4-point method 2 doses of standard and 2 doses of the
test are
used, amount of standard producing the same response as produced by the test
can be
determined by graphical method. It is determined mathematically:
Conc. Of unknown=

Where, t1 = lower dose of test; t2 = higher dose of test; T1 = response of t1;

T2 =response of t2.
In 6-point method 3 doses of standard and 3 doses of the test are used. The
sequence of responses is followed as per the Latin square method of
randomization in order to avoid any bias.

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 Bioassay of Antagonists
Commonly used method for the bioassay of antagonist is simple graphical method. The
responses are determined in the form of the percentage inhibition of the fixed dose of
agonist. These are then plotted against the log dose of the antagonist and the
concentration of unknown is determined by finding out the amount of standard producing
the same effect as produced by the test.
In this method, two responses of the same dose of agonist (sub maximal giving
approximately 80% of the maximum response) are taken. The minimum dose of standard
antagonist is added in the bath and then the response of the same dose of agonist is taken
in presence of antagonist. The responses of agonist are repeated every ten min till
recovery is obtained. The higher dose of standard antagonist is added and responses are
taken as before. Three to four doses of the standard antagonist are used and then one to
two doses of test sample of the antagonist is used similarly.
The percentage inhibition is calculated, plotted against log dose of antagonist and the
concentration of unknown is determined as usual.
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 1. Bioassay of D-Tubocurarine
a) Rabbit Head-drop Method :
• Principle: Standard/ test sample of d-Tubocurarine hydrochloride is injected into the
marginal vein of a rabbit’s ear till the rabbit’s neck muscles are relaxed such that the
animal cannot hold its head up. The total amount of test sample required to produce
the endpoint is compared with the total amount of the standard sample required to
produce similar endpoint.
• Selection of Rabbits: Rabbits weighing 2 kg. are used. Animals should be free from
disease, obtained from a healthy colony and should be accustomed with the
experimental procedure.
• Experimental Procedure:
• Minimum 8 rabbits are used. They are divided into two groups each containing 4
rabbits. First group will receive standard sample and the second group will receive
the sample under test. d-Tubocurarine solution is injected at a constant speed by
infusion apparatus through the marginal ear vein.
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 • Injection should be given at a rate of 0.4 ml/min and should take about 10 min.
• Infusion is continued till the rabbit will not be in a position to hold its head erect or
there will be no response by focussing light on the eyes and the neck gets elongated
and toneless.

Suitable dose of d-tubocurarine is 0.012% w/v in saline. Rabbits recover immediately

from the effect of curarization. During the expt. there is a possibility or respiratory
embarrassment which is treated by injecting neostigmine methyl sulphate (0.05 mg.) and
atropine sulphate immediately through the marginal ear vein.

• Cross-over test is carried out to minimise biological error due to animal variation. Those
rabbits which received the standard sample on the first day will be given test sample
on the second day of expt. and vice versa.
• Mean dose which produces head drop of the test sample is compared with the mean
dose of standard preparation.
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 2. Frog’s Rectus Abdominis muscle Preparation:

• A frog is pithed and laid on its back on a cork covered board to which it is pinned.
The skin covering the abdomen is cut away and the rectus abdominis muscle of one
side is dissected from the pelvic girdle to its insertion in the cartilage of the pectoral
girdle.
• The muscle is then pinned to the cork by four pins to keep its normal length while a
thread is sewn through each end. It is then mounted in the organ bath containing frog’s
Ringer solution.
• Oxygenation is carried out to keep the tissue alive. The muscle is stabilized for 30-45
min. in order to get critical quantitative response. The responses are recorded using
isotonic frontal writing lever with 1 G. tension.
• Two similar contractions with the same concentration of acetylcholine are obtained.
• Three doses of the standard sample and one intermediate dose of the test sample
are selected and the reduction in height of contraction induced by acetylcholine is
32 noted down.
 • Acetylcholine contraction is recorded on slow moving drum for 90 sec.
• d-Tubocurarine is allowed to act for 30 sec. The percentage reduction at each dose
levels is calculated and log dose response curve of the standard drug is plotted. A
linear response will be obtained.
• The potency of test sample is calculated from the standard curve.

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 Bioassay of digitalis

• Principle: Potency of the test sample is compared with that of the standard
preparation by determining the action on the cardiac muscle.
• Standard Preparation and Units: The standard preparation is a mixture of dried and
powdered digitalis leaves (1 unit = 76 mg.)

• Preparation of Extracts: Exact amount of the powder is extracted with dehydrated

alcohol in a continuous extraction apparatus for six hours. The final extract should
contain 10 ml. (5 ml. alcohol + 5 ml. water) per 10 g. of digitalis powder. It should be
stored in between 5 °C and –5 °C.

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 1. Guinea–pig Method (End point method) :
• Standard and test sample extracts are diluted with normal saline in such a way that 1 g of
digitalis powder is diluted to 80 ml.
• A guinea pig is anaesthetized with a suitable anaesthetic. It is dissected on the operation
table. The jugular vein is traced out by removing adhering tissues and cannulated by means
of venous cannula.
• A pin is inserted in the heart, such that it gets inserted in the apex of the heart. In this way,
we can observe the heart beats by up and down movements of the pin.
• The injection is continued through venous cannula until the heart is arrested in systole.
• The amount of extract required to produce this effect is taken as the lethal dose of the
extract.
• Another set of 19 animals of the same species are used for this experiment and the
average lethal dose is determined. It is not necessary to determine the lethal dose of the
std. during each time of the experiment. But it should be occasionally checked.
• The lethal dose of the test sample is determined in a similar way using minimum 6 guinea–
35 pigs of the same strain.
 • The potency of the test sample is calculated in relation to that of the std. preparation by
dividing the average lethal dose of the standard to the test and expressed as units per
gram.
2. Pigeon Method:
• Minimum 6 pigeons are used for testing each sample. They should be free from gross
evidence of disease. The weight of the heaviest pigeon should not exceed twice the weight
of the lightest pigeon.
• Food is withheld 16-28 hours before the experiment. Pigeons are divided on the basis of
their sex, weight and breed, into two groups. They are anaesthetized with anaesthetic
ether.
• One side of the wing is dissected and the alar vein is cannulated by means of a venous
cannula. Dilutions are made with normal saline.
• Average lethal dose of each sample is determined; results are tabulated and calculated as
per guinea pig method. The lethal dose per kg. of body weight is determined for each
pigeon.
• The potency of the test sample is determined by dividing the mean lethal dose of standard
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by the mean lethal dose of the test sample.
 In pigeons, stoppage of heart is associated with a characteristic vomiting response called
‘emesis’. The milk from the crop sac of pigeons is being ejected out. This may be taken as the
end point response of digitalis.
Bioassay of insulin
Principle: Potency of the test sample is compared with that of the standard preparation.
Standard preparation and unit: It is pure, dry and crystalline insulin. One unit contains 0.04082
mg.
Preparation of standard solution: Accurately weigh 20 units of insulin and dissolve it in normal
saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative. Add 1.4% to 1.8%
glycerin. Final volume should contain 20 units/ml. Store the solution in a cool place and use it
within six months.
Preparation of test sample solution: The solution of the test sample is prepared in the same way
as the standard solution described above.

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 1. Rabbit Method:
Selection of rabbits: They should be healthy, weighing about 1800-3000 gms. They should
then be maintained on uniform diet but are fasted for 18 hrs. before assay. Water is
withdrawn during the experiment.
Standard and Sample Dilutions: These are freshly prepared by diluting with normal NaCl
solution so as to contain 1 unit/ml. and 2 units/ml.
Doses: The dose which can produce suitable fall in blood sugar level is calculated for the
standard.
Principle: The potency of a test sample is estimated by comparing the hypoglycemic effect of
the sample with that of the std. preparation of insulin. Any other suitable method can also be
used.

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 Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each. The rabbits
are then put into an animal holder. They should be handled with care to avoid excitement.
First part of the Test: A sample of blood is taken from the marginal ear vein of each
rabbit. Presence of reducing sugar is estimated per 100 ml. of blood by a suitable
chemical method. This concentration is called ‘Initial Blood Sugar Level’.

The four groups of rabbits are then given sc. injections of insulin as follows:
12 RABBITS

3 3 3 3
Standard Standard Test Sample Test Sample Dilution
Dilution (I) Dilution (II) Dilution (I) (II)

From each rabbit, a sample of blood is withdrawn up to 5 hrs. at the interval of 1 hr. each.
Blood sugar is determined again. This is known as ‘Final Blood Sugar Level’.
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 Second part of the test (Cross over test) : The same animals are used for the second part.
The experiment can be carried out after one week.
Again they are fasted and initial blood sugar is determined. The grouping is reversed, that
is to say, those animals which received the standard are given the test and those which
received the test are now given the standard. Those animals which received the less dose
of the standard are given the higher dose of the test sample and vice-versa. This test is
known as ‘Twin Cross Over Test’.
Mean percentage decrease in blood sugar of the first and second part is calculated.
2. Mouse Method: Mice show characteristic convulsions after s.c. inj. of insulin at elevated
temperatures. The percentage convulsions produced by the test and standard preparations
are compared.
Experimental procedure: Minimum 100 mice weighing between 18-22 gms. of the same
strain are used. They should be maintained on constant diet. They should be fasted 18 hrs.
prior to the experiment.

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 Standard and sample dilutions: Dilutions are prepared with sterile saline solution, so as to
contain 0.064 units/ml. (std dilution I) and 0.096 untis/ml. (std. dilution II). Similarly, test
sample solutions are also prepared.
Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as
follows:
100 MICE

25 25 25 25
Standard Standard Test Sample Test Sample Dilution II
Dilution I Dilution II Dilution I
(0.064 units/ml) (0.096 units/ml)

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 Mice are put in an air incubator at 33ºC and observed for one and a half hr. An air
incubator with a glass front provided with six shelves is used. The temperature is
thermostatically controlled. Two mice are kept in each of the boxes made up of
perforated sheets of metal.
The mice which convulse or die are taken out of the incubator and observed. These mice
usually convulse severely but failure of the animal to upright itself when placed on its
back, should as well be considered as convulsion. Convulsive mice may be saved by an inj.
of 0.5 ml. of 5% dextrose solution.
Percentage convulsions produced by the test sample are compared with those of the
standard sample. Those animals which survive may be used again for another expt. after
an interval of one week.
3. Rat diaphragm method: In this method increase in glycogen content of the muscle or
increase in glucose uptake by muscle in response to insulin is taken as the index of potency
of insulin.
4. Rat epididymal fat-pad method: Here, the ability of insulin to increase CO2
production by the fat-pad is taken as the parameter for the measurement of potency of
42the insulin preparation.
 5. Radioimmunoassay: It is the estimation of the concentration of the substance in a unit
quantity of preparation using radiolabelled antigens. A number of drugs are estimated
now days by radioimmunoassy methods because these methods are highly specific and
highly sensitive.
The radioimmunoassay of insulin is based on the ability of human insulin (unlabelled) to
displace beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies). The
method involves the following steps:
I. Bovine insulin is injected into the sheep. After a week the serum containing antibodies
produced against bovine insulin is collected form the blood of the sheep.
II. The serum containing antibodies is exposed to radiolabelled insulin and the bound vs
free ratio is determined.
III. The mixture of labelled antigen-antibodies is then added in different test-tubes
labelled as standard and test. About 6 concentrations of the standards are taken. They
are then added to different tubes and the bound vs free ratio is again determined
using gamma-counter.
IV. Standard curves are determined and the concentration of test insulin is determined
43 using this standard curve.
 Biological Assay of Heparin Sodium
Principle: The potency of heparin sodium is determined by comparing the concentration of
test necessary to prevent the clotting of sheep or goat or human plasma with the
concentration of the Standard Preparation of heparin sodium necessary to give the same
effect under the conditions of the following method of assay.
Standard Preparation and Unit: The Standard Preparation is the freeze-dried sodium salt of
the purified active principle from bovine intestinal mucous membranes or any other suitable
preparation, the potency of which has been determined in relation to the International
Standard. The Unit is the specific activity contained in 7.7 μ g of the Standard Preparation
and is the same as the International Unit; 1 mg contains 130 Units.
Special Reagents:
Prepared Plasma: Collect blood from sheep or goats or human volunteers directly into a
vessel containing 8% w/v solution of sodium citrate in the proportion of 1 volume to each 19
volumes of blood to be collected. Mix immediately by gentle agitation and inversion of the
vessel. Immediately centrifuge and pool the separated plasma. To 1 ml of the pooled
plasma in a clean test-tube add 0.2 ml of a 1% w/v solution of calcium chloride and mix.
44The plasma is suitable if a solid clot forms within 5 minutes.
Solution of standard preparation: Determine by preliminary trial, if necessary, approximately
the minimum quantity of the Standard Preparation of heparin sodium which, when added in
0.8 ml of saline solution, maintains fluidity in 1 ml of prepared plasma for 1 hour after the
addition of 0.2 ml of a 1% w/v solution of calcium chloride. On the day of the assay prepare
a solution of the Standard Preparation such that it contains in each 0.8 ml of saline solution the
above-determined quantity of the Standard Preparation.
Test solution: Weigh accurately about 25 mg of the preparation being examined and dissolve
in sufficient saline solution to give a concentration of 1 mg per ml and dilute to a concentration
estimated to correspond to that of the solution of the Standard Preparation.
Method:
• To very clean test-tubes add graded amounts of the solution of standard preparation, (the
largest dose does not exceed 0.8 ml and each step is approximately 5% greater than the
next lower).
• To each tube add sufficient saline solution to make the total volume 0.8 ml.
• Add 1.0 ml of prepared plasma to each tube.

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 • Then add 0.2 ml of a 1% w/v solution of calcium chloride, note the time, immediately
stopper each tube with a suitable stopper and mix the contents by inverting three times
in such a way that the entire inner surface of the tube is wet.
• In the same manner set up a series using the test solution, completing the entire process
of preparing and mixing the tubes of both the solution of standard preparation and the
test solution within 20 minutes after the addition of the prepared plasma.
• Exactly one hour after the addition of the calcium chloride solution, determine the extent
of clotting in each tube, recognizing three grades between zero and full clotting .

Calculate the estimated potency of the preparation being examined by combining the
results of these assays by standard statistical methods.

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Biological Assay of Oxytocin
Principle: The potency of oxytocin is determined by comparing its activity with that of the
Standard Preparation of oxytocin under the conditions of a suitable method of assay.
Standard Preparation: The Standard Preparation consist of freeze-dried synthetic oxytocin
peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units)
Suggested methods
Method A: By depression of the blood pressure in chicken —
• Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that
will maintain a prolonged and constant high blood pressure.
• Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal
artery and crural vein.
• Cannulate the popliteal artery and record the blood pressure on a suitable recorder.
• Cannulate the crural or brachial vein.
Immediately before use prepare a solution of the Standard Preparation in saline solution so
that the volume to be injected is between 0.1 ml and 0.5 ml.
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 • Record the blood pressure responses to the injection of two doses of this solution into the
cannulated vein; the required doses normally lie between 20 and 100 milliUnits.
• The interval between injections should be constant and lie between 3 and 10 minutes
depending on the rate at which the blood pressure returns to normal.
• Immediately before use dilute the preparation being examined with saline solution so as
to obtain responses similar to those obtained with the Standard Preparation. The ratio
between the two doses of the preparation being examined should be the same as that
between the two doses of the Standard Preparation and this ratio should be kept
constant throughout the assay.
• The two doses of the Standard Preparation and the two doses of the preparation being
examined should be given according to a randomised block or a Latin square design and
at least six responses to each should be recorded.
If the animal rapidly becomes insensitive to the repeated injections of the solutions another
animal must be used. Measure all the responses and calculate the result of the assay by
standard statistical methods.

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 Method B: By contraction of the rat uterus —
Inject 100 mcg of estradiol benzoate intramuscularly into a female rat weighing 120 to 200
g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear
that the rat is in estrus or proestrus. Kill the rat and suspend one horn of the uterus in a bath
containing a solution of the following composition:

Composition(%w/v)
Sodium chloride 0.662
Potassium chloride 0.045
Calcium chloride 0.007
Sodium bicarbonate 0.256
Disodium hydrogen phosphate 0.029
Sodium dihydrogen phosphate 0.003
Magnesium chloride 0.010
Dextrose 0.050
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• Maintain the bath at a temperature of 32o or at some other suitable temperature at
which spontaneous contractions of the uterus are abolished and the preparation
maintains its sensitivity.
• Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon dioxide
and record the contractions of the muscle using a suitable instrument giving a linear
response (for example an isotonic lever with a load not exceeding 2 g).
• Record the contractions produced by the addition to the bath of two doses of the
Standard oxytocin Preparation suitably diluted with the above solution. The required
doses normally lie between 10 and 50 micro Units per ml of bath liquid.
• When maximal contraction has been reached, replace the bath liquid by a fresh
solution. The doses should be added at regular intervals of 3 to 5 minutes depending
upon the rate of recovery of the muscle.
• Dilute the test preparation so as to obtain responses on the addition of two doses similar
to those obtained with the Standard Preparation. The ratio between the two doses of
the preparation being examined should be the same as that between the two doses of
the Standard Preparation and this ratio should be kept constant throughout the assay.
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 The two doses of Standard Preparation and the two doses of the preparation being
examined should be given according to a randomized block or a Latin square design and
at least six responses to each should be recorded.
Measure all the responses and calculate the result of the assay by standard statistical
methods.
Method C: By measurement of milk-ejection pressure in a lactating rat —
• Select a lactating rat, in the third to twenty-first day after parturition and weighing
about 300 g, separate it from the litter
• 30 to 60 minutes later anaesthetize (for example, by the intraperitoneal injection of a
solution of Pentobarbitone Sodium).
• Tie the rat to an operating table, maintained at 37o, by its hind legs leaving the front
legs free.
• Cannulate the trachea with a short polyethylene tube in such a manner so as to ensure a
free airway; apply artificial respiration only if necessary.
• Cannulate an external jugular or femoral vein with a polyethylene tube which is filled
with saline solution and closed with a pin.
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 • Shave the skin surrounding the inguinal and abdominal teats (nipple) and excise the tip
of one teat, preferably the lower inguinal teat.
• Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter
about 0.6 mm, to a depth 3 to 10 mm , into the primary teat duct which opens onto the
cut surface and tie firmly in place with a ligature.
• Connect this cannula with a suitable strain gauge transducer (such as that used for
recording arterial blood pressure in the rat) and fill the whole system with a 3.8% w/v
solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml
to prevent clotting of milk.
• After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct
through the transducer to clear the milk from the tip of the cannula. (This procedure may
be repeated during the assay should obstruction arise from milk ejected into the
cannula).
• Clamp the strain gauge so that a slight tension is applied to the teat and its natural
alignment is preserved and connect the gauge to a potentiometric recorder adjusted to
give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa.
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 • Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01
ml and wash them in with 0.2 ml of saline solution.
• Prepare a solution of the Standard Preparation and a solution of the preparation
being examined in saline solution so that the volume to be injected is between 0.1 ml
and 0.4 ml.
• Inject the four doses (two doses of the Standard Preparation and two doses of the
preparation being examined) at intervals of 3 to 5 minutes.
• The two doses of Standard Preparation and the two doses of the preparation being
examined should be given according to a randomized block or a Latin square design
and at least four responses to each should be recorded.
• Measure all the responses and calculate the result of the assay by standard statistical
methods.

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 Biological Assay for Vasopressin
Principle: The vasopressor activity is estimated by comparing the activity of the preparation
being examined with that of the Standard Preparation of arginine vasopressin under the
conditions of a suitable method of assay.
Standard Preparation:
It is a dried acetone extract of posterior lobes of pituitary gland of oxen or any other
suitable preparation.Standard unit: Specific pressor activity corresponding to that yielded
by gm of standard preparation (20units/ml).

Suggested Method:
• Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a
suitable alpha-adrenoceptor blocking agent,
• After 18 hours, anaesthetize the rat with an anaesthetic that will maintain a prolonged
and uniform blood pressure.
• After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs.
• Cannulate the trachea with a short polyethylene tube of external diameter about 2.5
54 mm and dissect a carotid artery ready for cannulation.
 • Then cannulate the femoral vein close to the inguinal ligament.
• dissect the femoral vein towards the inguinal ligament from the corresponding artery.
• When dissecting, a deep branch reaching the femoral vein must be found and tied off
to prevent bleeding during cannulation.
• Tie a short polyethylene cannula into the femoral vein by two ligatures and join by a
short piece of flexible tubing to a 1-ml burette with an attached thistle funnel
containing saline solution at about 37o.
• Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and
cannula.
• At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline
solution, per 100 g of body weight.
• Then tie in a carotid cannula of external diameter about 1 mm and connect by a
column of saline solution containing heparin with a suitable pressure measuring device
such as a mercury manometer of internal diameter about 2 to 3 mm

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 Taking care that no air is injected, inject all solutions through the venous cannula by means
of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the
burette.
Dilute the extract of the Standard Preparation and the preparation being examined with
saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two
doses of the Standard Preparation such that the elevation of the blood pressure is about 4
kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the
ratio of low to high dose being determined by the response and usually being 3 to 5
Inject doses at intervals of 10 to 15 minutes. The two doses of the Standard Preparation
and the two doses of the preparation being examined should be given in a randomised
block or a Latin square design and four to five responses to each should be recorded.
Measure all the responses and calculate the result of the assay by standard statistical
methods

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 • METHOD 2: Rise in B.P in cats
• Anaesthetize healthy cat with volatile anaesthetic agent
• Insert a tracheal tube for artificial respiration
• Expose spinal cord from behind by removing second cervical vertebrae
• Destroy brain by passing suitable instrument through foramen magnum
• Start artificial respiration through tracheal tube & leave animal for an hour to remove
anaesthetic effect
• Cannulate carotid artery for B.P. measurement & femoral vein for injection of drug
solutions
• Maintain normal B.P.at 50 torr
• Select 2 doses of test & standard,
• inject units at 30 min. interval
• Record maximum rise in B.P. in response to each dose

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 Biological Assay of Diphtheria Antitoxin
The potency of diphtheria antitoxin is determined by comparing the dose necessary to
protect guinea-pigs or rabbits against the erythrogenic effects of a fixed dose of the
Standard Preparation of diphtheria antitoxin necessary to give the same protection. For
this purpose, a suitable preparation of diphtheria toxin is required to be used as a test
toxin. The test dose of the toxin is determined in relation to the Standard Preparation. The
potency of the preparation being examined is then determined in relation to the
Standard Preparation using the test toxin.
Standard Preparation: The Standard Preparation consisting of the dried hyperimmune
horse serum and glycerin, or another suitable preparation the potency of which has been
determined in relation to the International Standard.

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 Method
Test toxin: Prepare diphtheria toxin by filtering through bacteria-proof filter the medium in
which a toxigenic strain of C. diphtheriae has grown. Store at a temperature of 2o and 8o.
Selection of test toxin: In selecting a toxin for use as the test toxin determine the following:
Lr/100 dose — This is the smallest quantity of the toxin which, when mixed with 0.01 Unit of
antitoxin and injected intracutaneously into guinea-pigs or rabbits causes a characteristic
reaction at the site of the injection within 48 hours.
Minimal reacting dose — This is the smallest quantity of toxin which, when injected
intracutaneously into guinea-pigs or rabbits, causes a characteristic reaction at the site of
injection within 48 hours.
A suitable toxin is one which contains at least 200 minimal reacting doses in the Lr/100
dose.
The test toxin is allowed to stand for some months before being used for the assay of
samples of antitoxin. During this time its toxicity declines and the Lr/100 dose may be
slightly increased. When experiment shows that the Lr/100 dose is constant, the test toxin is
ready for use and may be used for a long period. Determine the minimal reacting dose
60 and the Lr/100 dose at frequent intervals.
 Store the test toxin in the dark at a temperature between 0o and 5o. Maintain its sterility
by the addition of toluene or other antimicrobial preservative which does not cause a rapid
decline in specific toxicity
Determination of test dose of toxin (Lr/100 dose):
Prepare a solution of the Standard Preparation with saline solution such that 1 ml contains
0.1 Unit. Prepare mixtures such that 2.0 ml of each mixture contains 1.0 ml of the dilution
of the Standard Preparation (0.1 Unit) and one of a series of graded volumes of the test
toxin. Dilute each mixture with saline solution to the same final volume (2.0 ml). Allow the
mixtures to stand at room temperature, protected from light, for 15 to 60 minutes and
inject intracutaneously 0.2 ml of each mixture at suitably spaced sites into the shaven or
depilated flanks of two animals. Observe the animals for 48 hours.
The test dose (Lr/100) of the toxin is the amount present in 0.2 ml of that mixture which
causes at the site of injection a small, characteristic reaction in the skin of the guinea-pig or
rabbit. Mixtures containing larger amounts of toxin cause larger reaction and necrosis and
mixtures containing smaller amount of toxin cause no reaction.

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 Determination of potency of the antitoxin: Dilute the test toxin with saline solution so that
1.0 ml contains 10 times the test dose. Prepare mixtures such that 2.0 ml of each mixture
contains 1.0 ml of the dilution of the toxin and one of a series of graded volumes of the
preparation being examined. Prepare further mixtures such that 2.0 ml of each contains
1.0 ml of the solution of the test toxin and 0.1 Unit of antitoxin. Dilute each mixture with
saline solution to the same final volume (2.0 ml). Allow the mixtures to stand at room
temperature, protected from light, for 15 to 60 minutes. Inject a dose of 0.2 ml of each
mixture into the animals under the conditions described in the determination of the Lr/100
dose of the toxin.
The mixture of the preparation being examined that contains 0.01 Unit of antitoxin in 0.2
ml is the mixture that produces the same degree of local reaction as that produced by the
injection into the same animals of the mixture of the Standard Preparation that contains in
0.2 ml the test dose (Lr/100) of the toxin and 0.01 Unit of antitoxin.
When at least four distinct tests are carried out by this method, the limits of error have
been estimated to be between 90% and 111%.

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 Bioassay of Histamine
(i) cat's blood pressure,
(ii) (ii) guinea pig's uterus or
(iii) (iii) the guinea pig's ileum
Contractions of isolated guinea pig ileum
Bioassay of histamine on isolated guinea pig ileum can be determined by
Matching bioassay
Interpolation bioassay
Bracketing assay
Multiple point assays

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 1. Gunea pig ileum:
• A piece of guinea pig ileum is suspended in Tyrode’s solution in a small glass
chamber which is mounted in a constant temperature bath.
• The piece of ileum is anchored firmly to the glass chamber on the lower end and is
suspended at the upper end by a thread which is tied to one end of a lever arm.
• Contractions of the ileum strip are recorded on the kymograph.
• Theoretically, the amplitude of contraction is proportional to the concentration of
histamine in the solution in which the ileum is suspended.
2. Cat blood pressure method
For assay by the Cat blood pressure method, the cat is anesthetized and Set up for
recording of the carotid arterial pressure. The histamine solution usually is injected into
the femoral vein. The drop in blood pressure, recorded on the kymograph, should be
proportional, to the amount of histamine injected-within certain limits.

64
 • Experiments were performed on cats of either sex, body weight 1.4–2.5 kg
• anaesthetized by an intraperitoneal injection of sodium pentobarbitone, 60 mg/kg,
and
• on beagle dogs of either sex, body weight 9.5–12 kg, anaesthetized by an
intravenous injection of sodium pentobarbitone, 30 mg/kg.
• Supplementary doses of sodium pentobarbitone were given as necessary to
maintain anaesthesia.
• In the cats the trachea was cannulated and the dogs were intubated with a cuffed
endotracheal tube.
• The right femoral artery was cannulated to measure systemic blood pressure with a
blood pressure transducer. Blood pressure was monitored on a Electronic recorder.
• Drugs were administered via cannulae inserted into peripheral veins, usually the
right femoral vein and right brachial vein

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 • Histamine was administered by injection, at intervals of 5 min, in a volume of 0.1 ml/kg
and the venous cannula washed with 0.5 ml of saline.
• Dose-response curves were made by injection of increasing doses of histamine.
Results
• Anaesthetized dogs
Histamine administered intravenously caused dose-dependent falls in systemic blood
pressure the threshold dose was about 1 × 10−10 mol/kg.
• Anaesthetized cats
Histamine administered intravenously caused dose-dependent falls in blood pressure over
the dose-range 1 × 10−10 to 1 × 10−7 mol/kg

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 Bioassay of ACTH
PRINCIPLE
Administration of ACTH decreases ascorbic acid present in adrenals. This relationship is
used for bioassay of ACTH.
Procedure
• Male Wistar rat (100-200 g) are hypophysectomized (pituitary gland removed by
surgery) one day prior to the test.
• For one test with 3 dose of test preparation and standard
• Number of hypophysectomized rats required: at least 36 (preferably 60)
Solution:
• 5 units of test or standard dissolved in 0.25 ml of 0.5% phenol solution and diluted with
8.1 ml of 15% gelatin solution (Now 0.5 ml contain 300 mU ACTH). (Solution A)
• 3ml of solution A diluted with 6 ml gelatin solution. Now concentration reduced to 100
mU ACTH/ 0.5 ml (solution B)
• Again 3 ml of solution B diluted with 6 ml of gelatin solution, the resulting solution
67 contains 33 mU ACTH/ 0.5 ml
• The hypophysectomized rats are randomly distributed in to six groups.
 • Three hours after injection, the animals are anesthetized and both adrenals removed,
freed from extraneous tissue and weighed.
• The rats are sacrificed and the scull opened to verify completeness of
hypophysectomy.
• The adrenals are homogenized in glass tubes contains 200 mg pure sand and 8.0 ml
of 4% trichloroacetic acid and the ascorbic acid determined.
• The potency ratio including confidence limits is calculated with the 3 + 3 point assay.

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 Biological Assay of Rabies Vaccine:
Principle
The potency of Rabies vaccine is determined by comparing the dose of test preparation
necessary to protect mice from the lethal effect of dose of rabies virus administered
intracerebrally with the dose of standard preparation of Rabies vaccine necessary to give
the same protection.
Special Reagent Required: 10% Acid-digested casein hydrolysate pH 7.2: (1) Casein
hydrolysate solution— Dissolve 100.0 g of acid digested casein hydrolysate in sufficient
distilled water to make 500 ml, adjust the pH to 7.2 with 10 M NaOH and sterilise by
heating at 121o for 20 minutes. (2) Sucrose solution— Dissolve 50.0 g of sucrose in sufficient
distilled water to make 500 ml, adjust the pH to 7.2 with 10 M NaOH and sterilise by
filtration
Mix equal volumes of solutions (1) and (2) aseptically before use.
Standard Preparation: The standard preparation is the freeze dried preparation, the
potency of which has been determined in relation to the International reference preparation.
It may be obtained from Central Research Laboratory.
73 Test Animals: Mice 11-15 g, 3-4 weeks old.
 Mice are divided for 2 Test

Potency Test Challenge Virus Test

Six Groups of 16 mice Four Groups of 10 Mice

Throughout the test all the mice that die before the 5 th day after challenge virus injection are
excluded from the test.

And all the mice that die with signs of rabies between 5 th and 14th day after challenge and
those that are alive but shows sign of rabies after 14th day of challenge are counted as failing
to resist the challenge.

The strain of mice suitable for the test is such that when 0.03ml containing 5-50 LD50 of
standard challenge virus susp injected per mouse will produce 100% mortality.
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 Standard Challenge Virus Suspension Preparation:
• Challenge virus strain is prepared by injecting intracerebrally 0.03ml of 10 fold
dilution of Rabies virus in 2%w/v sterile inactivated normal horse serum in water for
injection into a suitable no of test animal
• The animals when moribund (at the point of death), after showing characteristics
symptoms of rabies are sacrificed and their brain harvested aseptically and washed in
chilled physiological saline to remove blood clots.
• 10 % suspension of brain is made in 10% acid digested casein hydrolysate at pH 7.2
and thoroughly homogenized.
• After centrifuging lightly the supernatant liquid is distributed into sterile ampoules and
freeze dried. And stored at 4ºC.
• In the lyophilized state the virus titre is maintained for at least 3 years.
Virus Titre of Challenge Virus Test:
• Prepare 10 fold serial dilution of standard challenge virus suspension.
• Using 4 groups of 10 mice each, inject 0.03ml of virus suspension intracerebrally into
each mouse using different group for each suspension.
75 • Observe the mice for 14 days.
 • Calculate the virus titre of standard challenge virus suspension in LD50 per doses of
0.03ml by standard statistical method from the no of mice in each group that survive
the challenge
Determination of Potency of Vaccine:
• Reconstitute the standard preparation with a suitable diluent.
• Prepare at least three 5 fold serial dilution of solution of standard preparation. And
three 5 fold dilution of vaccine under examination.
• For both standard and sample the serial dilution should be prepared in such a way
that the lowest dilution protects more than 50% of injected mice.
• Allocate 1 dilution to each of six groups of 16 mice each.
• Inject intraperitoneally each mouse in each group with dilution of vaccine and standard
preparation.
• After 7 days, prepare similar dilution of vaccine and reference (std.) preparation and
repeat the injections.
• After a further 7 days, inject each vaccinated mouse intracerebrally with 0.03ml of
standard challenge virus suspension.
• Observe the mice for 14 days and record the no. of mice surviving the challenge in
76 each group.
 • Calculate potency of preparation under examination by standard statistical method.
• The vaccine complies with test if the estimated potency is NLT 2.5 IU per single human
dose.

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