BIOASSAY

ASSAY

 Assay is an investigative (analytic) procedure for qualitatively

assessing or quantitatively measuring the presence or amount or
the functional activity of a target entity (the API).
TYPES OF ASSAY
Assay may be
 Chemical Assay: It is the study of the separation, identification, and
quantification of the chemical components of natural and artificial materials
 Immuno assay: A technique that makes use of the binding between an
antigen and its homologous antibody in order to identify and quantify the
specific antigen or antibody in a sample
 Bioassay or biological assay: Biological testing procedure for estimating
the concentration of a pharmaceutical drug substance in a formulated drug
product or bulk material. The specific dose of drug is given to animal or
human volunteers and then drug response is compared with the standards.
 BIOLOGICAL ASSAY OR BIOASSAY
 Estimation or determination of concentration or potency of a physical,
chemical or biological substance by mean of measuring and comparing
the magnitude of response of the test with that of a standard over a
suitable biological system under standard set of conditions

OR
 The estimation of the concentration or potency of a substance by
measuring the biological response that it produces
 Basic theme of biological assay is stimulus applied to the subject and
response of the subject to the stimulus.
PURPOSE OF BIOASSAY
Bioassay is carried out for a number of purposes. Some of which are;
 To ascertain the potency of drug and is the quantitative part of any
screening procedure
 To measure pharmacological activity of new or chemically undefined
substance
 Compare test sample with standard substance to determine quantity of
test sample required to produce an equivalent biological response to that
of the standard substance
 To standardize drugs, vaccines, toxins, poisons, disinfectants and
antiseptic so that each contains the uniform specified pharmacological
activity
 To determine the specificity of a drug e.g. Penicillin is effective
against Gram positive bacteria not on Gram negative
 Test method employed in measuring the response of living animals
to toxicity of chemical contaminant. Certain no. of individuals of
sensitive species are exposed to specific conc. of contaminant for
specific period to examine toxic effects
 For investigating function of endogenous mediators
 Improving and maintaining standards of basic environmental
conditions affecting well-being of people e.g. pollutants released
by particular source
COMPARISON OF CHEMICAL ASSAY AND
BIOASSAY

Bioassay Chemical Assay

Less precise More precise
More time consuming Less time consuming
More expensive Less expensive
More sensitive Less sensitive
More man power is required Less man power required
Difficult to handle easy to handle
Ethical issues are involved No ethical issues
INDICATIONS OF BIOASSAY
Bioassay is performed in the cases when;
 Chemical method is not available
 If available, too complex,
 Insensitive to low doses
 If active principle of drug is not known
 Unknown Chemical composition, e.g. long acting thyroid stimulator
 Biological activity of drug cannot be defined by a chemical assay
e.g. Cis and Trans form of methyl phenidate.
 Not possible to separate interfering substance e.g. Vitamin D.
 PRINCIPLES OF BIOASSAY
 All bioassays should be comparative against a standard drug.
Standards are internationally accepted samples of drugs
maintained under the conditions recommended by the Expert
Committee of the Biological Standardization of WHO. They
represent fixed units of activity (definite weight of preparation) for
the drug
 Standard and new drug should be as far as possible identical to
each other
 Activity assayed should be the activity of interest
 The degree of pharmacological response produced should be
reproducible under identical conditions. e.g. Adrenaline
 Method of comparison preferably (not essentially) tests therapeutic
property of drug
 Individual variations must be minimized
CLASSIFICATION OF BIOASSAY
There are three types of bioassay:
 Quantal: All or none response in all individuals, e.g.
 Digitalisinduced cardiac arrest in guinea pigs
 Hypoglycemic convulsions in mice by insulin
 Calculation of LD50 in mice or rats

 It is not a precise method and commonly applied for

 Comparison of LD50 and ED50
 Comparison of Threshold response

 Graded: Effect is produced gradually depending on dose

 e.g. Contraction of smooth muscle preparation
 Effect produced in confined period:
ADVANTAGES OF BIOASSAY
 Bioassays are procedures that can determine the concentration, purity or
biological activity of a substance such as vitamin, hormone and plant growth
factor. While measuring the effect on an organism, tissue cells, enzymes or
the receptor is preparing to be compared to a standard preparation.
 Bioassays may be qualitative or quantitative. Qualitative bioassays are used
for assessing the physical effects of a substance that may not be quantified,
such as abnormal development or deformity. Quantitative bioassays involve
estimation of the concentration or potency of a substance by measurement of
the biological response that it produces. Quantitative bioassays are typically
analyzed using the methods of biostatistics
 They not only help to determine the concentration but also the potency of
the sample.
 It is especially used to standardize drugs, vaccine, toxins or poisons
disinfectants, antiseptics etc. as these are all used over biological system
in some or other form.
 These also help determine the specificity of a compound to be used ex:
Penicillin's are effective against Gram positive but not on Gram negative.
 Certain complex compounds like Vitamin B-12 which can't be analyzed
by simple assay techniques can be effectively estimated by Bioassays.
 Sometimes the chemical composition of samples is different but has same
biological activity.
 For Drugs where no other methods of assays are available

 Biological products like toxin, anti-toxin, sera can be conveniently

assayed.
 Measure minute (Nano mole & Pico mole) quantities of active substances
can detect active substance without prior extraction or other treatment
LIMITATIONS OF BIOASSAY

 Key problem is variability in response

 Large number of animal to be used
 Expertise in experimental design, execution of assay & analysis
of data required
 Expensive and time consuming
 Time related changes in sensitivity of test organ
 Tachyphylactic responses of substance being assayed
ACCURACY LIMITS OF BIOASSAY

 Accuracy improves the efficiency of bioassay for pharmaceutical

biological products.”
 Accuracy within 20 % of true value is good
 Accuracy within 10 % of true value is excellent
TECHNIQUES OF BIOLOGICAL ASSAY
Classification of bioassay on the basis of techniques
 In vitro Techniques
 In Vivo Techniques
 Ex vivo Techniques
IN-VITRO TECHNIQUE
 These techniques employ a cell culture of recommended
biological system to study the effect of compound under
standard condition not similar to that of living environment.
Here the cell culture survives by utilization of the nutrition in
the media
 For Example; use of stem cells, cell culture, microbes
(bacteria) etc
IN-VIVO TECHNIQUE

 These techniques employ a living animal recommended for

the purpose of assay. The techniques aim to study the
biological effect or response of the compound under
screening in a living system directly.
 For Example; Use of rodents, rabbits etc
 EX VIVO TECHNIQUE
 These techniques employ a tissue or cells of recommended living
system to study the effect of compound under test in suitable
conditions within the stipulated time of organ survival outside the
body.
 For Example; Use of any isolated organ from animals in a glass
ware to study the effect of compound within the period of its
survival outside the living body with provision of only oxygen,
glucose and isotonic salts to maintain cell & cell organelles
integrity
TYPES OF BIOASSAY

Qualitative Bioassay
 It is used for assessing the physical effects of a substance that may
not be quantified, such as abnormal development or deformity.

Quantitative bioassays
 It involves estimation of concentration/potency of a substance by
measurement of the biological response it produces. These
bioassays are typically analyzed using the methods of biostatistics
METHODS OF BIOASSAY

Graded Response Assay:

 In these assays, as the dose increases, there is an equivalent rise in
response. The potency is estimated by comparing the Test sample
responses with the standard response curve. For Example; Acetyl-
choline producing contraction in the muscle of frog Rectus
abdominis
END POINT OR QUANTAL ASSAY:
 The threshold dose of the sample required to elicit a complete or a
particular pharmacological effect is determined and compared with
standard.
 For example Digitalis producing cardiac arrest

 Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED=

effective dose) is done by this method. Based on the method used
during the grade point assay procedure for determination of Type
of activity and Potency of the Sample, four methods of assays are
classified as:
 Matching point or bracketing method

 Interpolation assay

 Three point (2+1) assay

 Four- point (2+2) assay

MATCHING POINT OR BRACKETING
METHOD:

 Here a constant dose of the standard is bracketed by varying dose

of sample until an exact matching between the standard dose
responses and the particular dose response of the sample is
achieved. This technique is used;
 When test sample is too small
 Inaccurate & margin of error difficult to estimate Eg: histamine on
guinea pig ileum, Posterior pituitary on rat uterus
INTERPOLATION ASSAY:
 Bioassays are conducted by determining the amount of preparation
of unknown potency required to produce a definite effect on
suitable test animals/organs/Tissue under standard conditions. This
effect is compared with that of a standard. Thus the amount of the
test substance required to produce the same biological effect as a
given quantity the unit of a standard preparation is compared and
the potency of the unknown is expressed as a % of that of the
standard by employing a simple formula
MULTI POINT BIOASSAY:
 This method incorporates the principle of interpolation and
bracketing. 2+1 indicates- Two response of Standard and one
response of Test respectively. This procedure of 2+1 or 2+2 is
repeated 3 times or 4 times based on the method with crossing
over of all the samples. It can further divided as 3 point, 4 point
and 6 point bioassay
STANDARD PREPARATIONS
 A Standard preparation is a selected representative sample of
substance that is used as a basis of measurement. In other words
the result of a test drug is compared with that of a standard
preparation to get the result of the assay.
 The standards are internationally accepted samples of drugs
maintained and recommended by the Expert Committee on the
Biological Standardization of W.H.O. They represent the fixed
units of activity (definite weight of preparation) for drugs.
 It is essential that standard preparations shall be of uniform quality
and as stable as possible. From the economic point of view it is
recommended that local laboratory standards be prepared and
standardized against standard preparations.
TYPES OF STANDARD PREPARATIONS:
Standard preparations are of two kinds.

1. International standard and reference preparations: These are

established by the World Health Organization (W.H.O) i-e by the
WHO Expert Committee on Biological Standardization.

2. British standard and reference preparations: These are

established by the British Government according to B.P. The
standard preparations for Great Britain and Northern Ireland are kept
at The National Institute for Medical Research, London (NIMRL),
and may be obtained from there for the purpose of biological assays
and tests.
UNIT OF ACTIVITY
 “The Unit of activity refers to a specific quantity of a biologically
active substance, such as a hormone or vitamin etc, required to
produce a specific response/ particular biological effect”.
 Unit of activity is dependent on the potency of the substance, and
each substance would have a different IU to milligram
conversion. For example, 1000 IU of Vitamin C would have a
different weight than 1000 IU of Vitamin A.
 Greater the number of units per mg of a drug lesser will the
potency and vice versa.
UNITS OF ACTIVITY:
 The potency/ activity of certain drugs, antibiotics, endocrine
products and biological substances are expressed in units called as
unit of activity.
 For this purpose different units are used. Most commonly used
units of activity are IU (International Units), USP Units and B.P
Units.
 Unit of activity of a substance is based on a comparison of activity
of a sample of that substance on a milligram basis to the
corresponding reference standard.
 For example there are 1590 USP units of Penicillin G per mg of
the USP reference standards of the antibiotic.
 Similarly there are 22 IU of insulin per mg of the international
reference standards of the insulin. These units are based on the
biological activity or effect. These are used to quantify vitamins,
hormones, some medications, vaccines, blood products, and
similar biologically active substances.
 IU is Standard measure of the biological activity (biological effect)
of manufactured medicinal drugs and vitamins. For every substance
to which this unit is assigned, there is an internationally accepted
biological effect expected with a dose of 1 IU. Other quantities of
the standard preparation of the substance are expressed in multiples
of this dose and may be converted into mass units.
 For example, 1IU is equivalent to 45.5 microgram (0.0455
milligram) of insulin, 0.6 microgram (0.0006 milligram) of
penicillin, 0.3 microgram (0.0003 milligram) of vitamin-A, 50
micrograms (0.050 milligram) of vitamin-C, 25 nanograms
(0.000025 milligram) of vitamin-D.
BIOASSAY OF ANTIBIOTICS
 Antibiotics are the compounds used to kill or prevent the growth of
microorganisms. The bioassay of antibiotics is also known as
microbial assay.
 Microbial Assay: “It is the type of bioassay whereby
microorganisms are used to assay the presence of any compound
(drug) in a sample” Microbial assays are usually used for the assay
of antibiotics.
Purpose of Microbial Assay:
 Microbial assays are usually used to analyze the activity/ potency
of antimicrobial agents (antibiotics). A good microbial assay will
serve 2 basic purposes:
1. It verifies that the compound actually has the desired antimicrobial
activity.
2. It indicates the concentration of the antimicrobial agent needed to
inhibit the target microorganisms. In other words microbial assays are
used to determine MIC of an antimicrobial agent.
 MIC (Minimum Inhibitory Concentration): “It is the lowest
concentration of a compound (antibiotic) that still inhibits the
growth of certain bacterial strains” Thus MIC is a standard used to
gauge the effectiveness/ potency of antimicrobial agents. Higher
the MIC value of an antibiotic, lower is the potency.
 MLC (Minimum Lethal Concentration): “It is the minimum
concentration at which a compound (antibiotic) is lethal to a
microorganism”. Antimicrobials will normally kill a
microorganism at 2-4 times their inhibitory concentration.
PRINCIPLE OF THE MICROBIAL
ASSAY:
 The potency of a sample of an antibiotic is determined by
comparing the dose which inhibits the growth of a suitable
susceptible micro-organism with the dose of standard preparation
of that antibiotic which produces the same degree of inhibition.
 In other words the potency of an antibiotic is determined by
comparing its effects (extent of inhibition) on microorganisms with
those of reference standard of that antibiotic.
STANDARD PREPARATIONS AND UNIT:

 The standard preparations are supplied as dry powders in sealed

ampoules containing the approximate quantity of an antibiotic.
 The potency of an antibiotic is usually described as units contained
in 1 mg of the powder or in mg containing 1 unit; e.g., 641 units of
gentamycin sulphate is contained in 1 mg or 0.00156 mg contain 1
unit.
STANDARD PREPARATIONS AND UNITS OF VARIOUS ANTIBIOTICS.
SUGGESTED METHODS:

1. Diffusion Method:

Diffusion method consists of the following steps.

a) Preparation of the media & inoculam.
b) Preparation of the medium for drugs.
c) Preparation of the solutions of the sample and standard drug.
d) Addition of the solutions of the sample and standard drugs into
the media.
e) Incubation.
f) Calculation & result.
 A) PREPARATION OF THE MEDIA &
INOCULAM:
1). Suitable susceptible microorganism is selected for specific drug test. e.g Bacillus
subtilis is used for the test of streptomycin.

2). A suitable media is prepared for a particular type of microorganism. Usually nutrient
agar media is used.

3). Media is then inoculated with a 1% v/v suspension (inoculam) of the susceptible
microorganism.

4). Temperature of the media should not exceed from 48-50 ºC.

5). Then this media is put into previously sterilized Petri dishes, up to 3-4 mm depth.

6). Media with inoculated organisms is kept at a specific temperature (37 ºC) for a
given period of time.

7). The media is then removed & is dried at room temperature for 30 minutes or
solidified in refrigerator.
 B) PREPARATION OF THE MEDIUM FOR
DRUGS:

 The media is holed i-e holes of 5-8 mm diameter are made with the
help of sterile borer. These holes are used to add drugs to the
medium.
 C) PREPARATION OF THE SOLUTIONS OF
THE SAMPLE AND STANDARD DRUG:
 Solution of standard preparations of known concentration and
solution of substance being examined presumed to be of the same
order of concentration are prepared in a sterile solution of standard
PH of a suitable PH value for a particular antibiotic , e.g PH 8 for
streptomycin.
 When a monograph gives directions for the preparation of the
solution, the solution so prepared is diluted as necessary with the
appropriate sterile solution of standard PH.
D) ADDITION OF THE SOLUTIONS OF THE SAMPLE
AND STANDARD DRUGS INTO THE MEDIA:

 The solutions of the sample and standard drugs are added by

sterile pipette into separate holes previously made in the
inoculated medium.
E) INCUBATION:

 The medium with the drug and organisms is maintained at room

temperature for 2-4 hours, during which some diffusion of the
antibiotic into the medium occurs.
 The plates are then incubated at a specific temperature (37 ºC)
for 16-36 hours.
 In this period the drug diffuses well into the media & inhibits
the growth of microorganisms. Clear zone are formed in areas of
medium where the drug has inhibited the growth of
microorganisms. These zones are called zones of inhibition.
F) CALCULATION & RESULT:
 The diameters of the zones of inhibition produced by the various
concentrations of the standard preparation and of substance
being examined are measured with the greatest possible
accuracy.
 The diameters of the zones of inhibition of sample and standard
preparations are compared by standard statistical methods to
determine the potency or MIC of the sample.
2. TURBIDITY METHOD:
 In this method MIC of a given compound for a certain bacterial species is
determined by using a series of test tubes containing medium. In this
medium the microbes will normally grow. Each tube is inoculated with the
microorganism. Each test tube also contains progressively low concentration
of the test compound. After incubation period the tubes in which the growth
does not occur are noted.
 The tube containing the lowest concentration of the compound that still
prevents the growth defines the MIC of the test drug.
 The MIC of the standard preparation is also determined by the similar
procedure. The MIC of the test drug is then compared with that of the
standard preparation by standard statistical method.
BIOASSAY OF INSULIN INJECTIONS

 Insulin is a hypoglycemic agent i-e it lowers the blood glucose level.

Principle of the Bioassay: The potency of insulin injection (expressed
in International Units/ml) is estimated by comparing the hypoglycemic
effect of the test sample with that of the standard preparation of insulin.
Standard preparation and unit: It is pure, dry and crystalline insulin
supplied in ampoules containing approximately 110-125mg of insulin.
1mg contains 22 Units of insulin; 1 Unit is equivalent to 45.5
microgram (0.0455 milligram) of insulin. 1mg= 22 Units of Insulin. 1
Unit = 0.0455mg of Insulin.
PREPARATION OF STANDARD
SOLUTION:
 Take a specific quantity 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 (2-10 ºC) 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.
SUGGESTED METHODS:
 1. Mouse Convulsion Method:
 Principle:
 Mice show characteristic hypoglycemic convulsions after S.C. injection
of insulin at elevated temperatures. The percentage convulsions
produced by the test and standard preparations are compared.
 Experimental Conditions: Minimum 100 mice weighing between 18-
22 grams of the same strain are used. They should be maintained on a
constant and an adequate diet. They should be fasted 2 hrs prior to the
experiment.
 STANDARD AND SAMPLE DILUTIONS:
 Two dilutions of the standard solution are prepared with sterile
saline solution acidified with HCl to pH 2.5, so as to contain 0.030
units/ml (standard dilution-I) and 0.060 units/ml (Standard
dilution-II). Similarly, two dilutions of test sample solution are
also prepared.
 Dose: Suitable doses of insulin for mice weighing about 20g are
0.015 Units and 0.030 Units. Dilutions of standard and test
solutions are prepared in such a way that the respective doses are
contained in equal volumes not greater than 0.5ml.
PROCEDURE:
 100 Mice are divided into 4 groups each containing 25 mice and
insulin is injected S.C. to each group. A 4-point assay is performed
by injecting 2 doses (dilutions) of the standard solution (S1 & S2)
and 2 doses (dilutions) of the test solution (T1 & T2) into four
groups of mice.
 Mice are then kept at a constant temperature (29-35°C) in an air
incubator with a transparent font and observed for 1.5 hours
following insulin injection. The numbers of mice which convulse
or die are recorded. 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. Those
animals which survive may be used again for another experiment
after an interval of one week. The death or convulsion in the test
animals are due to the rapid onset of hypoglycemia.
RESULTS:
 Percentage convulsions produced by the test sample are
compared with those of the standard sample. The potency of the
test preparation is determined by a specific statistical method
based on the direct proportional relationship between % animals
showing convulsion and the potency of the preparation.
2. RABBIT BLOOD SUGAR METHOD:

 Principle:
 Insulin lowers blood sugar level, and the degree of blood sugar
lowering is proportional to the potency of the preparation. The
potency of a test sample is estimated by comparing the
hypoglycemic effect (average reduction in the blood glucose
level) of the sample with that of the std. preparation of insulin.
 Experimental Conditions: 12 healthy rabbits (1.5 to 2 kg) are used.
They should then be maintained on uniform diet but are fasted for 18 hrs
before assay.
 Standard and sample dilutions: Two dilutions of the standard solution
are prepared with sterile saline solution acidified with HCl to pH 2.5, so
as to contain 1 unit/ml (standard dilution-I) and 2 units/ml (Standard
dilution-II). Similarly, two dilutions of test sample solution are also
prepared.
 Dose: Suitable doses of insulin for rabbits is about 0.5 units/kg and 1
unit/kg body weight of the rabbit. Dilutions of standard and test solutions
are prepared in such a way that the respective doses are contained in
equal volumes not greater than 1ml.
 Procedure:
 12 Rabbits are divided into 4 groups each containing 3 rabbits.
Blood samples are withdrawn from the marginal ear vein of each
rabbit and the average concentration of blood glucose in mg/dl is
determined in each group (initial blood sugar). Then insulin is
injected S.C. to each group. A 4-point assay is performed by
injecting 2 doses (dilutions) of the standard solution (S1 & S2) and
2 doses (dilutions) of the test solution (T1 & T2) into four groups of
rabbits.
 Following insulin injection, blood samples are withdrawn from the
marginal ear vein every hour for 5 consecutive hours and the
average concentration of blood glucose in mg/dl is determined in
each group (final blood sugar).
 Results: For each dose of the test and standard insulin, the
reduction in blood sugar level is calculated and compared and the
potency of the test insulin is determined by a suitable statistical
method.
 BIOASSAY OF PREPARED DIGITALIS
 Digitalis is a drug that increases the force of myocardial contraction and is used
to treat cardiac failure. It is also known as cardio-tonic as it increases the
efficiency of failure heart. In higher doses it causes stoppage of the heart
(cardiac arrest) and death.
 Principle of the Bioassay: The Potency of prepared digitalis is estimated by
comparing the action of the test sample of prepared digitalis on the cardiac
muscles with that of the standard preparation of prepared digitalis.
 Standard preparation and unit: The standard preparation is a mixture of dried
and powdered digitalis purpurea leaves supplied in vials containing
approximately 2.5g of dried powdered leaves. 1 unit is equivalent to 76 mg;
1mg contains 0.01316 units of standard preparation of digitalis. 1mg= 0.01316
Units of Digitalis. 1 Unit = 76mg of Digitalis.
PREPARATION OF EXTRACTS:
Preparation of standard extract:
Standard extract of digitalis is prepared by the following procedure:
 The contents of a vial containing standard preparation of digitalis are
rapidly transferred to a stoppered weighing bottle and weighed.
 The contents are added to a stoppered glass container of at least 50ml
capacity.
 10ml of alcohol (80%) is added for each g of the powder.

 The container is stoppered and is shaken for 24 hrs at 20-30 ºC or for

48hrs at 10-20 ºC.
 After shaking the mixture is immediately centrifuged or filtered through a
sintered glass filter with precautions to avoid evaporation of the solvent.
 The final extract is transferred to a dry, hard glass bottle which is tightly
closed.
 The extract is stored at a temperature between 5 ºC and -5 ºC. The extract
should be used within one month.
Preparation of test sample extract:
 The extract of the test sample is prepared in the same way as the
standard extract described above.
SUGGESTED METHODS:

1. Guinea–pig Method
 Principle: At higher doses digitalis causes stoppage of the heart
(cardiac arrest) and death of guinea pigs, which indicates the
end point response of digitalis. The potency of the test sample is
estimated by comparing the lethal dose of the test sample to that
of the standard preparation of digitalis.
 Experimental Conditions: 12 guinea pigs (200-600 g) of the
same strain are used. The weight of the heaviest and the lightest
animals should not differ by more than 100 g. Later on when the
guinea pigs are divided into 2 groups each containing 6 guinea
pigs, the mean body weights of the two groups should not differ by
more than 10 %.
 Standard and sample dilutions: The standard and test sample
extracts are diluted with saline solution until the concentration of
alcohol are the same and do not exceed 10 % v/v.
 Procedure: 12 guinea pigs are divided in 2 groups each containing 6 guinea
pigs. One group is used for standard preparation and one for the test sample.
 The guinea pig is anaesthetized with urethane and the diluted standard
extract is injected at a slow and uniform rate into the vein of a guinea pig.
The injection is continued until the heart is arrested (this may conveniently
be determined from an electrical recording). The amount (volume) of
extract required to produce this effect is taken as the lethal dose of the
extract. Another set of 5 animals of the same species are used for this
experiment and the average lethal dose (ml per kg of the body weight) of 6
guinea pigs is determined for standard extract. The average lethal dose of
the test sample is determined in a similar way using 6 guinea pigs of the
same strain.
RESULTS:
 The potency of the test sample is calculated in relation to that of
the standard preparation by dividing the average lethal dose of
the sample to the test and expressed as units per gram.
2. Pigeon Method:
 Principle:
 At higher doses digitalis causes stoppage of the heart (cardiac
arrest). In pigeons, stoppage of heart is associated with a
characteristic vomiting response called „Emesis‟. This may be
taken as the end point response of digitalis. The potency of the test
sample is estimated by comparing the lethal dose of the test sample
to that of the standard preparation of digitalis.
 Experimental Conditions:
 12 pigeons are used. They should be free from gross evidence of disease,
obesity or emaciation (To become extremely thin, especially as a result of
starvation or 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. Later on when the pigeons are divided into 2
groups each containing 6 pigeons, the mean body weights of the two
groups should not differ by more than 30 %.
 Standard and sample dilutions:
 The standard and test sample extracts are diluted with saline solution so
that the estimated lethal dose per kg of the body weight is contained in
15ml.
 Procedure:

 12 pigeons are divided in 2 groups each containing 6 pigeons. One group is used for
standard preparation and one for the test sample.

 The pigeons are lightly anaesthetized with anesthetic ether and the diluted standard
extract is injected through cannula at a slow and uniform rate into the alar vein of a
pigeons. The dose is 1ml per kg of the body weight and is administered within few
seconds & repeated at 5 minutes

 interval until the heart is arrested which is associated with emetic response. The
amount (volume) of extract required to produce this effect is taken as the lethal dose of
the extract. Another set of 5 pigeons are used for this experiment and the average lethal
dose (ml per kg of the body weight) of 6 pigeons is determined for standard extract.
The average lethal dose of the test sample is determined in a similar way using 6
pigeons.
 Results: The potency of the test sample is calculated in relation
to that of the standard preparation by dividing the average lethal
dose of the sample to the test and expressed as units per gram.
BIOASSAY OF VITAMIN D
 Vitamin D is also known as anti-rachitic vitamin. The deficiency of
vitamin D leads to rickets. Rickets is a disorder caused by a lack of
vitamin D, calcium, or phosphate. It leads to softening and
weakening of the bones thus potentially leading to fractures and
deformity. Vitamin D helps the body to control calcium and
phosphate levels. If the blood levels of these minerals become too
low, the body may produce hormones that cause calcium and
phosphate to be released from the bones. This leads to weak and
soft bones. The deficiency of Vitamin D is the predominant cause
of rickets.
 Principle of the Bioassay: The potency of vitamin D is
determined by comparing the antirachitic activity of the test
sample of vitamin D with that of the standard preparation of
vitamin D. The antirachitic activity of vitamin D is measured in
rats.
STANDARD PREPARATION AND UNIT:
 The standard preparation of vitamin D consists of activated
crystalline 7- dehydrocholestrol supplied in bottles as solution in
vegetable oils containing 1000 units per gram of the solution. 1
unit is equivalent to 0.000025 mg; 1mg contains 40000 units of
pure vitamin D.
 1mg= 40000 Units of vitamin D.
 1 Unit = 0.000025mg of vitamin D.
EXPERIMENTAL CONDITIONS:
 Minimum of 40 young rats of either gender, shortly after being
weaned, are used for the test. About 10 litters (the offspring at one
birth of a multiparous mammal- those which produce more than
one at a birth) of 4 rats are selected. (Alternatively 5 litters of 8
rats can also be selected). 40 rats= 10 litters (groups) of 4 rats (10
×4 = 40)
 The weight of the heaviest rat in any litter should not exceed that
of the lightest by more than 10g. All of the 40 rats are fed for about
21 days (3 weeks) on a rachitogenic diet, which consists mostly of
carbohydrates, proteins and electrolytes and no fats. The
development of the necessary degree of rickets may be determined
in each rat under light anaesthesia by taking X-ray photographs of
the proximal ends of the tibia or the distal ends of the ulna and
radius.
PROCEDURE:
 40 rats are divided into 4 groups, each group containing 10 rats.
 The groups are made in such a way that one rat from each litter set of 4
or two rats from each litter set of 8, being assigned to each group. The
rats in two groups receive doses of x and nx units (where n is a suitable
value such as 1, 2, 3 or 4), respectively, of standard preparation, while
the rats of the other two groups receive, respectively, doses of test
sample preparation in the same ratio as the doses of the standard
preparation (i-e x & nx doses). Suitable doses of the standard preparation
may vary from 2 to 8 units (x=2 units; n=1, or 2, or 3, or 4). Each rat
may receive the whole of its dose at once or the dose may be divided into
8 daily doses.
 10-14 days after receiving the doses of standard preparation or of
the sample preparation if given in one amount (single dose), or 10-
14 days after receiving the first fraction of the dose (if given in
divided doses), the rats are killed and the extent to which rickets
has been cured is estimated by means of X-ray photographs, or by
examination of the bones after staining, in either instance by
reference to a standard scale.
 RESULTS:

 The extent to which rickets has been cured by the sample

preparation of vitamin D is compared with that of standard
preparation of vitamin D, and the potency of the sample
preparation is calculated by standard statistical methods.