"The determination of the relative strength of a substance (e.g., a drug or hormone or toxicant) by comparing its effect on a test organism with that of a standard preparation." is called bioassay.

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Concept of bioassay
Bioassay:
"The determination of the relative strength of a substance (e.g., a drug or
hormone or toxicant) by comparing its effect on a test organism with
that of a standard preparation." is called bioassay.
Or
Bioassays are a set of techniques relevant to the comparisons between the strength
of alternative but similar biological stimuli (a pesticide, fungicide, a drug etc.)
based on the response produced by them on the subjects typically, a bioassay
involves stimulus, subject and response. The change produced on the subject due
to application of stimulus (such as an analytical value like blood sugar content)
normally two preparations of the stimulus one known strength (standard
preparation) and another of unknown strength (test preparation) both with
quantitative doses are applied to a set of living organisms. the general objective of
a bioassay is to draw statistically valid conclusions on the relative potency of test
preparation with respect to standard one.
Purpose:
1. Measurement of the pharmacological activity of new or chemically undefined substances
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)

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5. Assessing the amount of pollutants being released by a particular source, such
as wastewater or urban runoff.
6. Determining the specificity of certain enzymes to certain substrates.
Types of bioassay :
1. Matching Bioassay
2. Interpolation Method
3. Bracketing Method
4. Multiple Point Bioassay (i.e.-Three-point, Four-point and Six Point Bioassay)
Matching Bioassay
It is the simplest type of the bioassay. In this type of bioassay, response of the test substance
taken first and the observed response is tried to match with the standard response. Several
responses of the standard drug are recorded till a close matching point to that of the test
substance is observed. A corresponding concentration is thus calculated. This assay is applied
when the sample size is too small. Since the assay does not involve the recording of
concentration response curve, the sensitivity of the preparation is not taken into consideration.
Therefore, precision and reliability is not very good.
Interpolation bioassay:
Bioassays are conducted by determining the amount of preparation of unknown potency
required to produce a definite effect on suitable test animals or organs or 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.
Many times, a reliable result cannot be obtained using this calculation. Therefore it may be
necessary to adopt more precise methods of calculating potency based upon observations of

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relative, but not necessarily equal effects, likewise, statistical methods may also be employed.
The data (obtained from either of assay techniques used) on which bioassay are based may be
classified as quantal or graded response. Both these depend ultimately on plotting or making
assumption concerning the form of DRC.
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:
Conc. of Unknown = Dose of the Standard X Conc. of Std.
Dose of 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 this method.

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Fig.1: Bioassay of histamine by the matching method
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.
Fig.2: Graphical method of bioassay
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.

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Fig.3: Bioassay of histamine by three point method.
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:
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
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:

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2. Alternatives to conventional bioassays includes
a) radio immune assays
b) enzyme linked immunosorbent assays
c) ligand binding assays
d) receptor binding assays
e) functional assays (using fluorescence techniques, chemiluminiscence
techniques).
1.radio immune assays.
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise
formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to
measure concentrations of substances, usually measuring antigen concentrations (for example,
hormone levels in blood) by use of antibodies.
Although the RIA technique is extremely sensitive and extremely specific, requiring specialized
equipment, it remains among the least expensive methods to perform such measurements. It
requires special precautions and licensing, since radioactive substances are used.
In contrast, an immunoradiometric assay (IRMA) is an immunoassay that uses radiolabeled
molecules but in an immediate rather than stepwise way.
A radioallergosorbent test (RAST) is an example of radioimmunoassay. It is used to detect the
causative allergen for an allergy.
Method.
Classically, to perform a radioimmunoassay, a known quantity of an antigen is made radioactive,
frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to
tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that
antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a
patient containing an unknown quantity of that same antigen is added. This causes the unlabeled
(or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody

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binding sites. As the concentration of "cold" antigen is increased, more of it binds to the
antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the
unbound ones, and the radioactivity of the free(unbound) antigen remaining in the supernatant is
measured using a gamma counter.
This method can be used for any biological molecule in principle and is not restricted to serum
antigens, nor is it required to use the indirect method of measuring the free antigen instead of
directly measuring the captured antigen. For example, if it is undesirable or not possible to
radiolabel the antigen or target molecule of interest, a RIA can be done if two different
antibodies that recognize the target are available and the target is large enough (e.g., a protein) to
present multiple epitopes to the antibodies. One antibody would be radiolabeled as above while
the other would remain unmodified. The RIA would begin with the "cold" unlabeled antibody
being allowed to interact and bind to the target molecule in solution. Preferably, this unlabeled
antibody is immobilized in some way, such as coupled to an agarose bead, coated to a surface,
etc. Next, the "hot" radiolabeled antibody is allowed to interact with the first antibody-target
molecule complex. After extensive washing, the direct amount of radioactive antibody bound is
measured and the amount of target molecule quantified by comparing it to a reference amount
assayed at the same time. This method is similar in principle to the non-radioactive sandwich
ELISA method.
2. enzyme linked immune sorbentassays (ELISA)
The enzyme-linked immunosorbent assay (ELISA) (/ɪˈlaɪzə/, /ˌiːˈlaɪzə/) is a commonly used
analytical biochemistry assay, first described by Engvall and Perlmann in 1972.[1] The assay uses
a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a
protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA
has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a
quality control check in various industries.[2]
In the most simple form of an ELISA, antigens from the sample are attached to a surface. Then, a
matching antibody is applied over the surface so it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step, a substance containing the enzyme's substrate is
added. The subsequent reaction produces a detectable signal, most commonly a color change.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The
sample with an unknown amount of antigen is immobilized on a solid support (usually a
polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically
(via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the
antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The
detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary
antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is
typically washed with a mild detergent solution to remove any proteins or antibodies that are
non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic
substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

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Principle
As an analytical biochemistry assay and a "wet lab" technique, ELISA involves detection of an
analyte (i.e., the specific substance whose presence is being quantitatively or qualitatively
analyzed) in a liquid sample by a method that continues to use liquid reagents during the analysis
(i.e., controlled sequence of biochemical reactions that will generate a signal which can be easily
quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid
and remains inside a reaction chamber or well needed to keep the reactants contained.[3][4] This is
in opposition to "dry lab" techniques that use dry strips. Even if the sample is liquid (e.g., a
measured small drop), the final detection step in "dry" analysis involves reading of a dried strip
by methods such as reflectometry and does not need a reaction containment chamber to prevent
spillover or mixing between samples.[5]
3. ligand binding assays.
Ligand binding assays (LBA) is an assay, or an analytic procedure, whose procedure or method
relies on the binding of ligand molecules to receptors, antibodies or other macromolecules.[1] A
detection method is used to determine the presence and extent of the ligand-receptor complexes
formed, and this is usually determined electrochemically or through a fluorescence detection
method.[2] This type of analytic test can be used to test for the presence of target molecules in a
sample that are known to bind to the receptor.[3]
There are numerous types of ligand binding assays, both radioactive and non-radioactive.[4][5][6]
As such, ligand binding assays are a superset of radiobinding assays, which are the conceptual
inverse of radioimmunoassays (RIA). Some newer types are called "mix-and-measure" assays
because they do not require separation of bound ligands.[5]
Ligand binding assays are used primarily in pharmacology for various demands. Specifically,
despite the human body’s endogenous receptors, hormones, and other neurotransmitters,
pharmacologists utilize assays in order to create drugs that are selective, or mimic, the
endogenously found cellular components. On the other hand, such techniques are also available
to create receptor antagonists in order to prevent further cascades.[7] Such advances provide
researchers with the ability not only to quantify hormones and hormone receptors, but also to
contribute important pharmacological information in drug development and treatment plans.[8]
Applications for Ligand Binding Assays
Ligand binding assays provide a measure of the interactions that occur between two molecules,
such as protein-bindings, as well as the degree of affinity (weak, strong, or no connection) for
which the reactants bind together.[10] Essential aspects of binding assays include, but are not
limited to, the concentration level of reactants or products (see radioactive section), maintaining
the equilibrium constant of reactants throughout the assay, and the reliability and validity of
linked reactions.[10] Although binding assays are simple, they fail to provide information on
whether or not the compound being tested affects the target's function

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4. receptor binding assays.
Receptor-binding assays are a critical component in lead indentification and later lead
characterization processes. They are used to characterize most known drug targets and typically
use
filter-based separations technology to obtain necessary “bound vs. free” fractions for assay
validation. For sensitivity and specificity, radiolabeled known drugs are used in competitive
binding assays. The assay is designed as a competitive inhibition assay using the radiolabeled
known drug/ligand receptor interaction to screen chemical or natural product libraries for more
effective NCEs (new chemical entities).
These quantitative binding parameter determinations indicate the minimal effective drug
concentrations. MultiScreenHTS plates for quantitative binding parameters provide a more
accurate and reliable alternative to homogeneous assays. They are widely used in high
throughput screening campaigns and provide a reliable platform that incorporates a range of
glass fiber filters to retain the receptor and “bound” ligand fraction. Operation by vacuum
manifold allows for more convenient characterizations since the bound fractions are easily
collected from the top of the plate.
The improved plate design of MultiScreenHTS automation-compatible
filter plates facilitates use with gripper arms and is compatible with microplate scintillation
counters.
MultiScreenHTS+ Hi Flow filter plates: improve washing efficiency and reduce data variation.
MultiScreen Harvest plates are also available to withstand the numerous washes and batch
pretreatment (PEI) required in cell harvest protocols.
Highly sensitive and specific radiometric assays
 Compatible with liquid scintillation cocktail
 Improved automation compatibility with new MultiScreenHTS filter plates

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5. functional assays
a) using chemiluminiscence technique.
Chemiluminescent immunoassay is a variation of the standard enzyme immunoassay (EIA),
which is a biochemical technique used in immunology. They can also be used as diagnosis tools
in medicine, as well as being in used in several other different industries for various applications.
How does it work?
During EIA the process uses enzyme labeled antibodies and antigens to detect the small
biological molecules required. The technique makes use of the basic immunology concept that an
antigen binds a specific antibody. Such antigen molecules, which can be identified in a fluid
sample, include molecules such as peptides, hormones and proteins. The enzymes used in
chemiluminescent immunoassay convert a substrate to a reaction product, which emits a photon
of light instead of developing a particular colour. Luminescence means that light is emitted by a
substance when it returns from an excited state to a ground state. There are different types of
luminescence and the various forms differ in the way that they achieve the excited state. For
chemiluminescence it is light produced by a chemical reaction. This chemiluminescent substance
can be excited by an oxidation reaction forming an intermediate. It is when this immediate return
to a stable ground state happens, that a photon is released and this is detected by the luminescent
signal instrument. The particular luminescence indicates the presence of the antigen. The amount
of the particular biological molecule, which is being looked for and is present in the sample, is
based on the luminescence observed. There are different types of substrate that are used for
chemiluminescence, with the most popular types being luminol or its derivatives.
The benefits of chemiluminescence immunoassay
There are benefits of using chemiluminescence rather other types of immunoassay, including
using fluorescence detection or a light absorption method, such as the fact that this technique is
ultra-sensitive and can detect small amounts of the biological molecule. In addition to this, it has
a wider dynamic range, as well as having a linear relationship between luminous intensity and
the concentration of the measured substance. These chemiluminescent reactions can also be
enhanced with an enhancer, which acts as an enzyme protector and allows the reaction to occur
for a longer period of time without a large reduction in the light output. The enhanced reaction
gives an intense light emission for a prolonged period of time, as well as the substrate only
needing to be added minutes before detection.

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b) using fluorescene technique.
Fluorescence immunoassay is a sensitive technique that can be used in the measurement of many
compounds, including drugs, hormones, and proteins; in the identification of antibodies; and in
the quantification of antigens such as viral particles and, potentially, bacteria. Homogeneous
fluorescence immunoassay, fluorescent excitation transfer immunoassay, fluorescence
polarization immunoassay, solid-phase "dipstick" immunoassay, solid-phase microbead
fluorescence immunoassay, substrate-labeled fluorescence immunoassay, and fluorescence
immunoassays using internal reflectance spectroscopy or phycobiliprotein conjugates are
reviewed.
imunoassays are preferred for the quantitation of many clinically relevant analytes.The high binding affinities and
the specificity of analyte recognition dis-played by antibodies greatly simplify the accurate determination of
analytes,despite the presence ofmany othersubstances in a sample ofinterest.Usefulimmunoassaystrategies require
appropriate levels ofanalyte sensitivity.The sensitivity ofthe earliest immunoassays was dramatically improved by the
introductionofradioisotopesas tracers.Radioimmunoassays (RIAs) became a benchmark against which subsequent
immunoassay technologies were measured. Moreover, RIAs provided guiding princi-ples that were extended for
immunoassay development with tracers otherthan radio-isotopes.The pursuit ofalternative tracers gained momentumdue
to the hazards and precautionsrequired in working with radioactive materials,theirrelative instability,and due to the
mounting problems and expense associated with their manufacture and disposal.Thus, reporters based on direct and
enzyme-generated fluorescence and absorbance became more widely used to fulfill the growing need for
nonradioisotopic tracers.As a result,RIA technology is declining in commercial use,and although the absorbance-based
enzyme immunoassay(EIA)is a rathermature technology,RIAs and EIAs are stillwidely used.Fluorescent labels
have long been used for immunological staining in fluores-cence microscopy, both prior to and since the
introduction of the RIA. Fluorescence technologyhas been extensively applied to instrumented immunoassay
development,not only since high detectionsensitivities are possible,but also since fluorophores display a variety of
measurable properties.The emission intensity,orientation,wave-form, lifetime, and the interrelationships between
these properties contribute to the continuing evolution of fluorescence immunoassays and to their commercial
develop-ment.The sophistication of fluorescence technologyforconducting clinicalimmu-noassays is evident in
instrumentation and immunoassay kits using advanced methods,and furtheradvances are indicatedby the rapid paceofnew
technologydevelopment.