BIOASSAY
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). Hence micro bioassay, radioimmunoassay are also
regarded as `bioassay'. Recently `biotechnology' has also been considered for
bioassay. Bioassay of the products like erythropoietin, hepatitis- B vaccine
etc. is being done through biotechnology.
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.
(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.
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.
The purpose of bioassay is to ascertain the potency of a drug and hence it
serves as the quantitative part of any screening procedure (Research). Other
purpose of bioassay is to standardize the preparation so that each contains the
uniform specified pharmacological activity. In this way, it serves as a pointer
in the Commercial Production of drugs when chemical assays are not available or
do not suffice. From the clinical point of view, bioassay may help in the
diagnosis of various conditions, e.g. gonadotrophins for pregnancy.
PRINCIPLE OF BIOASSAY
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.
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 and 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. The drugs producing quantal effect can be bioassayed by end
point method. The drugs producing graded responses can be bioassayed by (1)
Matching or bracketing method
(2) Graphical method.
1. 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 experimentsone using standard digitalis and the
other using test preparation of digitalis is done and then potency is
calculated as follows:
In case, if it is not possible to measure individual effective dose or if
animals are not available, fixed doses are injected into groups of animals and
the percentage of mortality at each dose level is determined. The percentage of
mortality is taken as the response and then the comparison is done in the same
way as done for graded response.
2. Matching 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 dose s 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. Concentration of the test sample
can be determined as follows:
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 this method.
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.
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. Other methods
which are based on the dose-response relationship include 3 point, 4 point, 5
point and 6 point methods. In these methods, the responses are repeated several
times and the mean of each is taken. Thus, chances of error are minimized in
these methods. In 3 point assay method 2 doses of the standard and one dose of
the test are used. In 4 point method 2 doses of standard and 2 doses of the
test are used. In 6 point method 3 doses of standard and 3 doses of the test
are used. Similarly one can design 8 point method also. The sequence of
responses is followed as per the Latin square method of randomization in order
to avoid any bias.
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:
Similarly, in 4 point method, amount of
standard producing the same response as produced by the test can be determined
by graphical method. It is determined mathematically as follows:
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.
