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Bioassay or Biological Assay

A bioassay or biological assay is a biological testing procedure for estimating the

concentration of a pharmaceutical active substance in a formulated product or bulk
material. In contrast to the common physical or chemical methods detailed information
on the biological activity of a substance is achieved. Over the last decade biological
assays (bioassays) have become more important to an effective quality control program in
biopharmaceutical development and manufacturing.
The general approach of most bioassays (biological assays) is to perform a dilution assay,
which measures the biological responses at several doses. A key assumption of a dilution
assay is that the active component follows the same principle of activity in standard and
sample preparation. In such case the unknown preparation may in theory be derived by
diluting with inert components or by concentrating the bulk solution. This concept of
similarity may be checked with the help of the parallel-line model (parallel-line assay) or
with the help of parallel-logistics models.
Both methods are suitable for the analysis of the results obtained by several biological
assays. An exemplary list of use is given below:
Several immunochemical methods
Microbiological assay of several antibiotics (diffusion method and turbidimetric
Assay of human coagulation factor VIII
Assay of diphteria vaccine
Assay of heparin
Assay of pertussis vaccine
Assay of tetanus vaccine
Assay of human anti-D immunoglobulin
Assay for erythropoietin

Bioassay (commonly used shorthand for biological assay), or biological
standardisation is a type of scientific experiment. Bioassays are typically conducted to
measure the effects of some substance on a living organism and are essential in the
development of new drugs, and in monitoring environmental pollutants. Both are
procedures by which the potency or the nature of a substance is estimated by studying its
effects on living matter.

Definition: "It is the comparable estimation of the nature, constitution or

potency of the active principles with that of the standard drug, by means of the
reaction on a living matter such as whole animal, isolated tissue or organism."

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 precipitation.
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. An example of a
qualitative bioassay includes Arnold Adolph Berthold's famous experiment on castrated
chickens. This analysis found that by removing the testes of a chicken, it would not
develop into a rooster because the endocrine signals necessary for this process were not
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.

1. measurement of the pharmacological activity of new or chemically undefined
2. investigation of the function of endogenous mediators
3. determination of the side-effect profile, including the degree of drug toxicity
4. measurement of the concentration of known substances (alternatives to the use of
whole animals have made this use obsolete)
5. assessing the amount of pollutants being released by a particular source, such as
wastewater or urban runoff.

Bioassays are of two types:
Quantal: A quantal assay involves an "all or none response". For example: Insulin
induced hypoglycemic convulsive reaction or the cardiac arrest caused by
digitalis. In both the cases, the end point is an all or none response e.g.either
convuslion occurs or doesn't occur; similarly is with cardiac arrest.
Graded: Graded assays are based on the observation that there is a proportionate
increase in the observed response following an increase in the concentration or
dose. The parameters employed in such bioassays are based on the nature of the
effect the substance is expected to produce. For example: contraction of smooth

muscle preparation for assaying histamine or the study of blood pressure

response in case of adrenaline.
A graded bioassay can be performed by employing any of the below-mentioned
techniques. The choice of procedure depends on:
1. the precision of the assay required
2. the quantity of the sample substance available
3. the availability of the experimental animals.


Matching Bioassay
Interpolation Method
Bracketing Method
Multiple Point Bioassay
Six Point Assay

Environmental bioassays
Environmental bioassays are generally a broad-range survey of toxicity. A toxicity
identification evaluation is conducted to determine what the relevant toxicants are.
Although bioassays are beneficial in determining the biological activity within an
organism, they can often be time-consuming and laborious. Organism-specific factors
may result in data that is not applicable to others in that species. For these reasons, other
biological techniques are often employed, including radioimmunoassays. See
Water pollution control requirements in the United States require some industrial
dischargers and municipal sewage treatment plants to conduct bioassays. These
procedures, called whole effluent toxicity tests, include acute toxicity tests as well as
chronic test methods. The methods involve exposing living aquatic organisms to samples
of wastewater.[1][2]
Biological assay
Once a pharmaceutical protein is isolated from the cells in which it was grown,
researchers perform tests to measure the protein's biological activity.
It must maintain a certain minimal level of biological activity to be used for animal or
clinical testing or, later, for market. Researchers also test to confirm that the isolated
protein is identical to the desired protein.
Bioassay :determination of the active power of a drug sample by comparing its effects on a live
animal or an isolated organ preparation with those of a reference standard.

1. Determination of the strength or biological activity of a substance, such as a drug or hormone,

by comparing its effects with those of a standard preparation on a test organism.
2. A test used to determine such strength or activity.
To undergo a bioassay: is to try the laboratory determination of the concentration of a drug or
other substance in a specimen by comparing its effect on an organism, an animal, or an isolated
tissue with that of a standard preparation. Also called biologic assay.

Bioassay : determination of the active power of a drug sample by comparing its effects on a
live animal or an isolated organ preparation with those of a reference standard.

What is Bioassay: Definition, Methods &

advantages of Bio assays
What is Bioassay? Definition & principle of bioassay
Bioassay is an assay designed to analyse any compound by use of a suitable biological
system like animals, tissues, microbes etc.
Bioassay definition: It is defined as estimation or determination of concentration or potency
of a physical, chemical or biological substance (agent) by means of measuring and
comparing the magnitude of the response of the test with that of standard over a suitable
biological system under standard set of conditions.
In bionalysis the response produced by the test compound is compared with that of standard
sample the way similar to other analytical methods but here the biological system is involved
in the determination.
Principle of bioassay: The bioassay compares the test sample with a same Internationally
applicable standard substance. It determines the quantity of test sample required to produce
an equivalent biological response to that of standard substance.
Standard samples are accepted by expert committee at international level and they
represent fixed units of activity.

Why Bioassay? Advantages & Uses of bioassay

Bioassays have some different role and purpose over other assay techniques.
1. They not only help to determine the concentration but also the potency of the sample.
(Potency is a term which denotes activity of the compound per molecule basis. i.e. if a
compound shows better activity at minute concentration, greater is the potency, and if its
activity is low at lower concentrations, lesser is the potency).

2.It is especially used to standardize drugs, vaccines, toxins or poisons, disinfectants,

antiseptics etc. as these are all used over biological system in some or other form.
3. These also help determine the specificity of a compound to be used ex: Penicillin's are
effective against Gram+ve but not on Gram-ve. Testing of infected patients sputum helps
determine which anti-biotic be given for quick recovery.
4. Certain complex compounds like Vitamin B-12 which can't be analysed by simple assay
techniques can be effectively estimated by Bioassays.
5. Sometimes the chemical composition of samples are different but have same
biological activity.

Bioassay Methods / types of bioassays

Basically there are two types of bioassays as per the technique used in determination of the
Sample under test.
1. Graded Response Assay
2. End Point or Quantal Assay
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.
In the graded dose response relationship, relates the size of the response to the drug in a
single biologic unit as the dose administered increased the pharmacological response also
increases and eventually reaches a steady level called the ceiling effect there will be on
further increase in response even with an increase in dose.
The graded dose response curve is obtained by plotting a graph with dose on the X-axis and
response on the Y-axis. It is usually sigmoid in shape however the log dose response curve
is almost a straight line and particularly useful in bio assay.
Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.
E.g. Acetyl-choline producing contraction in the muscle of frog Rectus abdominis.
End Point or Quantal Assay: As the name indicates, the threshold dose of the sample
required to elicit a complete or a particular pharmacological effect is determined and
compared with standard.

E.g., Digitalis producing cardiac arrest. Here the sample effect is identified by the response it
produces on the biological system. Digitalis produces cardiac stimulation on further doses it
produces cardiac arrest.
(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to
the neck muscle of the Rabbit, the neck starts to droop. On further doses there is complete
hanging of the neck and rabbit has no ability to lift the neck
Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is done by
this method.

Matching point method

Interpolation 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
(The Rectus abdominus muscle is a skeletal muscle of frog commonly used to see the
effect of cholinergic related drugs. The muscle is contractile and each contraction
response is recorded as spike or peak as you can see in the videos. The videos are
intended for your understanding of the concept of methods followed in bio-assay.)

a) Matching point or bracketing method

b) Interpolation assay
c) Three point (2+1) assay
d) 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.
To determine potency of test a log dose response curve is plotted. Check the video for more.
Interpolation assay Here a log dose-response curve is plotted with the standard. The
concentration of the test is then read from the graph.
Three point (2+1) & Four- point (2+2) assay: This method incorporates the principle of
interpolation and bracketing. 2+1 indicates- Tow 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.
Here cross over method is used during the assay as.S1 S2 T, T S1 S2, S2 T S1 doses.

Bioassay systems and techniques

The bioassay systems vary based on the biological system used like animals (mouse, rat,
guinea pig, rabbits etc), plant bioassay (using plant constituents to evaluate a sample
like(haemolytic activity) microbiological or cell based assay (using microbes like bacteria,
fungi or cultured cells for anti biotic compound screening etc).
Based on techniques they can be differentiated into two broad types like
a) In vivo techniques:These techniques employ a living animal recommended for the
purpose of assay. The techniques aims to study the biological effect or response of the
compound under screening in a living system directly. Ex: By use of rodents, rabbits etc.
b) In vitro techniques: 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. Ex: use of stem cells, cell culture, microbes (bacteria) etc
c) Ex vivo techniques: 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. The methods described in the
videos employ a living tissue of an animal in an apparatus to study the contractile effect of
Ex: 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.

Biological assay of glucagon in rabbits

DiabetologiaVolume 5, Number 3 / June,


F. Tarding1, 2, P. Nielsen1, B. Keiser-Nielsen1 and Aa. V. Nielsen1

Summary Bioassay of glucagon based on the hyper-glycaemic activity was
investigated in normal rabbits. It was shown that the index of precision ( )
derived from the dose response curve is about 1.2, which means that several
hundred animals are required per assay in order to obtain a reasonably narrow
confidence interval. Triple cross-over design improved the index of precision to
0.37, about 50 rabbits then being required to obtain a 95% confidence interval of
35%. The weight and sex of animals, as well as the fasting time and the route of
administration of glucagon, were shown not to influence the quality of the assay.
Pretreatment of rabbits with cortisone improves the blood sugar response to
glucagon. With a single dose of 25 mg of cortisone per rabbit, a considerable
increase in the response to glucagon was observed with only a moderate elevation
of the fasting blood sugar level. It was shown that the cortisone potentiation of the
glucagon effect is at a maximum around 24 days after the cortisone
adminstration, and that a cross-over assay can be performed within this period.

Further, it was shown that the cortisone treatment of the animals can be repeated
several times without causing any changes in the glucagon response or in the
animals as such. Variables in an assay, such as sampling time, dose ratio, and
response calculation, have been evaluated. Based on these results, an assay
procedure for the determination of the biological potency of glucagon employing
cortisonepretreated rabbits has been designed. With this procedure, a 95%
confidence interval of 20% is ordinarily obtained in assays employing 32 rabbits.

Key-words Glucagon - bio-assay - cortisone-treated rabbits - biological

standardization - cross-over assay - glucagon patency estimation

Biological Assay Detection Technologies.

Detection technologies comprise a broad range of techniques designed to quantitate
analytes resulting from chemical or biological processes, including biological assays.
Typically, the aim of a biological assay is to measure the effect of a substance on a
biological system which has been configured in vitro in either a cell-based or biochemical
format. While cell-based assays are sometimes erroneously referred to as in vivo, in
reality the more accurate term is ex vivo, due to the fact that the cells have been cultured
outside of a living organism. Biological assays are configured as such to gain insights
into physiological events that are typically associated with disease states and are
commonly employed at all stages of preclinical drug discovery. The substances
modulating biological processes can be either stimulatory or inhibitory and the detection
technology chosen is intended to provide an accurate read out of the respective effect.
Most commonly, biological assays are run in microtiter assay plates with densities of 96,
384, 1536 or 3456 wells/plate. The plates are used in high-throughput screening (HTS)
which is a process that utilizes a combination of robotics, liquid handlers, plate readers,
incubators, washers, shakers, barcode readers and other devices for assembling assay
components and compounds in high throughput and collecting the resulting data. [1][2]
Many widely utilized detection technologies are a form of optical information gathering.
These can be divided into four main classes: fluorescence, luminescence, radiometric and
absorbance. The links at the end of this article provide more detail on specific
technologies. In all cases, examples of assay formats utilizing particular detection
technologies are provided and are described in more detail in the literature.[3] Other
lesser-used detection formats that have seen more specialized application are automated
patch clamping, label free and rubidium flux.
Monitoring of assays is usually accomplished with the use of reagents whose optical
properties change following a chemical or biological event. Care needs to be taken to
ensure that interferences from compounds (either autofluorescent or quenching) and inner
filter effects are minimized whenever possible.