Bioassay techniques

BIOASSAY TECHNIQUES
Submitted to
Dr. Sujarani S.
Asst. Prof.
Dept. ofVPT, Pookode.
Submitted by
Dr. Sindhu K.
MVSc scholar,
Dept. ofVPT, Pookode.

Biological assessment.
• 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.
• The estimation of the concentration or potency of a substance by measurement of
the biological response that it produces.
• The structure of bioassay: STIMULUS-applied to subject.
RESPONSE-of the subject to the stimulus.

The purpose of bioassay.
• To ascertain the potency of a drug and hence it serves as the quantitative part of any
screening procedure (Research).
• To standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics etc., so
that each contains the uniform specified pharmacological activity. (standardization
required as these are all used over biological system in some or other form.)
• Helps to determine the specificity of a compound to be used e.g. Penicillin's are
effective against Gram +ve. but not on Gram –ve.
• From the clinical point of view, bioassay may help in the diagnosis of various
conditions. e.g. gonadotrophins for pregnancy.
• Sometimes the chemical composition of samples are different but have same
biological activity.
• Certain complex compounds likeVitamin B-12 which can't be analysed by simple
assay techniques can be effectively estimated by Bioassays.
• For samples where no other methods of assays are available.

Principle of Bioassay.
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
1. Central Drug Research
Institute, Lucknow
2. Central Drug Laboratory,
Calcutta.

Classification of
bioassay.
•Invitro.
•Invivo.
•Exvivo.

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.

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.

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.
• 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.

Types of bioassay.
• Qualitative bioassay is used for assessing the physical effects of a
substance that may not be quantified, such as abnormal development or
deformity.
Eg: 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
available.
• Quantitative bioassays  involve 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.

Bioassay Methods.
1. Graded Response Assay: : In these assays, as
the dose increases there is an equivalent rise in
response.The potency is estimated by comparing
theTest sample responses with the standard
response curve.
• 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.

2. 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.
• 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 ofType 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 ofTest 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.

Three point assay [2+1 dose assay]
• Fast & convenient:
• Log dose response [LDR] curve plotted with varying conc of std drug solutions and given
test solution
• Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part of LDR [ Let the
corresponding response be S1, S2]
• Choose a test dose t with a responseT between S1 & S2
• Record 4 sets data as follows
• s1 s2 t
• t s1 s2
• s2 t s1
• s1 s2 t
• Log Potency ratio [M] = [(T –S1) / (S2-S1)] X log (dose ratio)

4 point assay [2 +2 dose assay]
• [E.g. Ach bioassay]
• Log dose response [LDR] curve plotted with varying conc of std Ach solutions and
given test solution
• Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response
be S1, S2]
• Choose two test doses t1 & t2 with responseT1 &T2 between S1 & S2 ;
• Also s2/s1 = t2/t1 = 2/3
Record 4 data sets
• s1 s2 t1 t2
• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1

Other bioassay’s.
• Immunological assay (ELISA).
• Micro-bioassay.
• Radioimmunoassay.
• Biotechnology.

ELISA (immunological assay)
• ELISA is a popular format of a "wet-lab"
type analytic biochemistry assay that uses
a solid-phase enzyme immunoassay (EIA)
to detect the presence of a substance,
usually an antigen, in a liquid sample or
wet sample.
• The ELISA has been used as a diagnostic
tool in medicine and plant pathology, as
well as a quality-control check in various
industries.
• The substances detected by ELISA tests
include hormones, bacterial antigens and
antibodies.

Types of ELISA.
• Direct ELISA: involve attachment of the antigen to the solid phase, followed
by an enzyme-labeled antibody. This type of assay generally makes
measurement of crude samples difficult, since contaminating proteins
compete for plastic binding sites.
• Indirect ELISA: involve attachment of the antigen to a solid phase, but in this
case, the primary antibody is not labeled. An enzyme-conjugated secondary
antibody, directed at the first antibody, is then added. This format is used most
often to detect specific antibodies in sera.
• Competitive ELISA: involves the simultaneous addition of 'competing'
antibodies or proteins. The decrease in signal of samples where the second
antibody or protein is added gives a highly specific result.
• Sandwich ELISA: involve attachment of a capture antibody to a solid phase
support. Samples containing known or unknown antigen are then added in a
matrix or buffer that will minimize attachment to the solid phase. An enzyme-
labeled antibody is then added for detection.

Applications of ELISA
• ELISATest Applications in Antibody Concentration Determination
• ELISATest Applications in Monoclonal Antibody Screening
• ELISATest Applications inVirus test (HIV, West NileVirus, NDV)
• ELISATest Applications in Home PregnancyTest
• ELISATest Applications in Food industry (detecting potential food allergens
such as milk, peanuts, walnuts, almonds and eggs)
• ELISATest can be used to diagnostic diseases

Micro-bioassay (antibiotics)
• The potency (activity) of an antibiotic product is expressed as the ratio of the
dose that inhibits the growth of a suitable susceptible microorganism to the
dose of an International Biological Standard, an International Biological
Reference Preparation, or an International Chemical Reference Substance of
that antibiotic that produces similar inhibition.
• To carry out the assay a comparison is made between the inhibition of the
growth of microorganisms produced by known concentrations of the reference
material and that produced by measured dilutions of the test substance.
• This response can be measured by the diffusion method or in a liquid medium
by the turbidimetric method.

Radioimmunoassay:
• RIA is a very sensitive in vitro assay technique used to measure
concentrations of antigens (E.g. hormone levels in the blood) by use of
antibodies.
• It is the estimation of the concentration of the substance in a unit quantity of
preparation using radiolabelled antigens.
• It requires special precautions and licensing, since radioactive substances are
used.
• A number of drugs are estimated now days by radioimmunoassay methods
because these methods are highly specific and highly sensitive.
• Eg: the radioimmunoassay of insulin is based on the ability of human insulin
(unlabelled) to displace beef’s insulin (which may be labelled) from the
binding sites (i.e. antibodies).

Principle of radioimmunoassay.
It uses an immune reaction [antigen-
antibody reaction] to estimate a ligand.
Ag+Ag*+Ab → [Ag -Ab+ Ag*Ab + Ag + Ab*]
• - Unbound Ag* and Ag washed out
• - Radio activity of bound residue
measured.
• - Ligand concentration is inversely related
to the radio activity.
• - [Ag: ligand to be measured;Ag*:
radiolabelled ligand].

Method of RIA.
• Requirements:
• 1. Preparation and characterization of
an antigen
• 2. Radio labeling of the antigen
• 3. Preparation of the specific antibody
• 4. Development of assay system.
• Components of RIA Assay Kit:
• Drug
• Antibody
• Labeled Drug

Applications of RIA.
Endocrinology
• Insulin, HCG,Vasopressin
• Detects Endocrine Disorders
• Physiology of Endocrine Function
Pharmacology
• Morphine
• Detect DrugAbuse or Drug Poisoning
• Study Drug Kinetics
Epidemiology
• Hepatitis B
Clinical Immunology
• Antibodies for Inhalant Allergens
• Allergy Diagnosis
Oncology
• Carcino embryonic Antigen
• Early Cancer Detection and Diagnosis.

Biotechnology.
• “Any technological application that uses biological systems, living organisms
or derivatives thereof, to make or modify products or processes for specific
use" (UN Convention on Biological Diversity, Art. 2)
• The American Chemical Society defines biotechnology as the application of
biological organisms, systems, or processes by various industries to learning
about the science of life and the improvement of the value of materials and
organisms such as pharmaceuticals, crops, and livestock.
• The European Federation of Biotechnology defines Biotechnology is the
integration of natural science and organisms, cells, parts thereof, and
molecular analogues for products and services.
• Biotechnology also writes on the pure biological sciences (animal cell
culture, biochemistry, cell biology, embryology, genetics, microbiology, and
molecular biology).

Branches of biotechnology.
•Bioinformatics
•Blue biotechnology
•Green biotechnology
•Red biotechnology
•White biotechnology

Bioinformatics
• Is an interdisciplinary field which
addresses biological problems using
computational techniques, and makes the
rapid organization as well as analysis of
biological data possible.
• It plays a key role in various areas, such
as functional genomics, structural
genomics, and proteomics, and forms a
key component in the biotechnology and
pharmaceutical sector.

Blue biotechnology
• is a term that has been
used to describe the
marine and aquatic
applications of
biotechnology.

Green biotechnology
• is biotechnology applied to
agricultural processes.
• Eg:
1. The selection and domestication of
plants via micropropagation.
2. The designing of transgenic plants
to grow under specific environments in
the presence (or absence) of chemicals

Red biotechnology
• is applied to medical processes.
• Eg:
1.The designing of organisms to
produce antibiotics,
2.The engineering of genetic cures
through genetic manipulation.

White biotechnology
• Also known as industrial biotechnology,
is biotechnology applied
to industrial processes.
• Eg:
1. The designing of an organism to produce a
useful chemical.
2. The using of enzymes as industrial catalysts
to either produce valuable chemicals or destroy
hazardous/polluting chemicals.
White biotechnology tends to consume less in
resources than traditional processes used to
produce industrial goods.

Applications
• Health care (medical),
• Crop production and agriculture,
• Non food (industrial) uses of crops
and other products
(e.g. biodegradable plastics, vegetable
oil, biofuels),
• Environmental uses.

To Sum - up
• Bioassay & its principles,
structure.
• Types & methods of bioassay.
• Immunological assay (ELISA).
• Micro-bioassay.
• Radioimmunoassay.
• Biotechnology.

Bioassay techniques

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Bioassay techniques