2. Introduction
Assay :
The experimental study of drugs or chemical
substances for quality and purity, which is called assay
Assay is the estimation of the amount or the activity of
an active principle in a unit quantity of the preparation
An assay is an investigative (analytic) procedure in
laboratory medicine, pharmacology, environmental
biology, and molecular biology for qualitatively
assessing or quantitatively measuring the presence or
amount or the functional activity of a target entity (the
analyte) which can be a drug or biochemical substance
or organic sample.
3. Types of Assay
It can be divided into three types :
1) Physical assay
Where carried out of any drugs or chemical substance
physical quality and purity test e. g. colour, taste, smell, pH,
M. P, B. P etc.
2) Chemical assay
There is used of drugs or chemical substance chemical
quality and purity e. g. acidic, basic, chelating form etc.
It is the study of the separation, identification, and
quantification of the chemical components of natural and
artificial materials.
4. 3) Biological assay:-
Biological, means the scientific study of drugs in living
organisms (plant, animal and man) and observation of
pharmacological effect of living tissue, micro/macro or
immune cells or animal is known as biological assay or
bioassay.
Bioassay is defined as the estimation of the potency of
an active principle in a unit quantity of preparation.
Detection and measurement of the concentration of
the substance in a preparation using biological
methods.
5. Comparison Of Chemical & Bioassay
Bioassay Chemical Assay
Less Precise
More time consuming
More expensive
Active constituent &
structure not known.
More sensitive
More men power Required
Difficult to handle
More Precise
Less time consuming
Less expensive
Active constituent &
structure fully established.
Less sensitive
Less men power required
Easy to handle
6. The purpose of bioassay
The purpose of bioassay is to ascertain the potency of a
drug and hence it serves as the research.
Other purpose of bioassay is to standardize the
preparation so that each contains the uniform
specified pharmacological activity. It is also help in
diagnosis of various condition.
e. g
gonadotrophins for pregnancy.
7. Indications for bioassay:
(a) When the chemical composition is not known but
the substance has a specific biological action e.g. long
acting thyroid stimulator (LATS).
(b) When the chemical assay method is too complex or
insensitive e.g. adrenaline and histamine can be
bioassayed in microgram quantities.
(c) When drugs differ in composition but have the
same pharmacological action e.g. the digitalis
glycosides obtained from various sources.
(d) When the active principle is unknown, cannot be
isolated easily, e.g. peptide hormones.
8. Importance of Bio-assay
Bioassays, as compared to other methods of assay (e.g.
chemical or physical assay) is very important.
Because it 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.
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.
9. Principle of Bioassay
The basic principle of bioassay is to compare the test substance
with the International standard preparation (Standard
substance) of the same drug and calculate or find out how much
test substance is produce same biological effects as produced by
standard. The standard drug produced same effect in all animal
species and same dose given same degree of pharmacological
response in same animal or animal species under standard
condition.
e. g adrenaline will always show/elicit same effect or same
degree of rise blood pressure when given in the same dose in the
same animal and animal species.
In India, standard drugs are maintained in Central Drug
Research Institute (CDRI) Lucknow and Central Drug
Laboratory, Kolkata.
10. Agonist
It activates a receptor to produce an effect similar to
that of the physiological signal molecule.
Types/Methods:-
An agonist may produce graded response or quantal
response.
Graded response
Quantal response
11. : Graded response :
Graded response means that the response is proportional to
the dose and response may lie between no response and the
maximum response.
Drugs producing graded effect can be bioassayed by :
a) Matching or bracketing method
b) Graphical method
: Quantal response :
Quantal response means, that the response is in the form of
“all or none” that is either no response or maximum response.
Drugs producing quantal effect can be bioassayed by End-point
method.
12. 1) Quantal (Quantity or amount)
End point method :
In this method the experimental based on test and
standard series and their potency and average results
calculate of two groups of animals (one receiving standard
and other the test) are done.
Two series of such experiments –one using standard
digitalis and other using test preparation of digitalis is
done and then potency is calculated as follows :
13. e.g. bioassay of digitalis in cats.
The cat is anaesthetized with chloralose
and its blood pressure is recorded.
The drug is slowly infused into the
animal
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 .
potency is calculated
14. 2) Graded (Management order)
a) Matching or bracketing method :
In this method a constant dose of test is compared with
different dose of standard till the exact match is
obtained between test dose and standard dose.
Initially two responses of the standard are taken.
In end take the response of the double dose of the
standard and test, which matches.
These should give equal response.
Concentration of the test sample can be determined.
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 this method.
15. Concentration of the test sample can be determined as follows :
Structure of the matching or bracketing method
17. 2) Graded (Management order)
b) Graphical method :
This method is based on the assumption or intake of
dose and its response relationship.
The log dose response curve is plotted and the dose of
std. producing the same response as produced by the
test sample is directly read from the graph.
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.
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
mathematically.
18. Concentration of the test sample can be determined as follows :
Structure of the graphical method
19. Bioassay of Insulin
Standard preparation and unit:-
It is pure, dry and crystalline insulin. One unit
contains 0.04082 mg. this unit is specified by Ministry of
Health, Government of India and is equivalent to
international unit.
Preparation of standard solution:-
Accurately weigh 20 units of insulin and dissolve it in NaCl
solution.
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.
20. Preparation of test sample solution:-
The solution of the test sample is prepared in
the same way as the standard solution.
Method of bioassay:-
There are two methods
1. Rabbit method
2. Mouse method
21. 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 Dilution:-
These are freshly prepared by diluting with normal
NaCl solution so as to contain. 1 units/ml and 2
units/ml.
22. 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 standard preparation of insulin.
Any other suitable method can also be used.
Experimental procedure:-
Animals are divided into four groups of 3 rabbits each.
The rabbits are then put into an animal holder. They
should be handled with care to avoid excitement.
23. First phase/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 subcutaneous
injection of insulin as follows:
24. From each rabbit, a sample of blood is withdrawn upto 5 hrs. at the
interval of 1 hr. each Blood sugar is determined again. This is known as
‘Final Sugar Level’.
25. Second phase/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.
26. 2) Mouse method
Mice show characteristic convulsions after subcutaneous
injection of insulin at elevated temperatures.
The percentage convulsions produced by the test and standard
preparation are compared.
Selection of mice:-
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.
Standard and Sample Dilution:-
Dilutions are prepared with sterile saline solution, so as to
contain 0.064 units/ml (Standard dilution I) and 0.096 units/ml.
(Standard dilution II).
Similarly, test sample solutions are also prepared.
27. Mice are divided into 4 groups each containing 25 and insulin is
injected subcutaneous as follows
•Mice are put in an air incubator at 33°C and observed for one and 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.
Convulsive mice may be saved by an injection of 0.5 ml. of 5% dextrose
solution. Percentage convulsions produced by the test sample are compared
with those of the standard sample.
28. Those animals which survive may be used again for another expt. After an interval of
one week.
29. Some other mathods are :
3) Rat diaphragm method
4) Rat epididymal fat-pad method
5) Radioimmunoassay
30. Advantages of Bio-assay
Bioassays are procedures that can
determine the concentration of 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.
31. 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.
32. Disadvantages
Key problem is variability in response
Large number of animal to be used
Expertise in experimental design, execution of assay &
analysis of data required
Leads to expensive & time consuming.
Tachyphylactic responses of substance being assayed.
33. Errors in bioassays
Biological variation
Loss of tissue sensitivity.
Different species/sex/age/weight/health status.
Laboratory condition may be variable.
Housing and handling of animals.
Methodological error
Lack of standardization of procedure.
Set-up of apparatus.
Tissue isolation/preparation for experiment.
Drug preparation or dilution.
34. Some other examples of
Bioassayes are :
Bioassay of Digitalis
Bioassay of Adrenaline
Bioassay of d-Tubocurarine
Bioassay of Acetylcholine
35. REFERENCES
Subhash Chandra Kothiyal, Sarla Saklani, Shrivatsa
Kothiyal and Sarvesh Kumar, Basic Concepts in
Pharmacology, Book Publisher International, India, United
Kingdom, First Edition; 2019. 44-52
Satoskar RS, Bhandarkar SD, Rege NN, Pharmacology and
Pharmacotherapeutics, Popular Prakashan (p) Ltd,
Mumbai, (20) Twentieth Edition; 2007. 76-82 .
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