The document discusses bioassay, which is the estimation of the concentration or potency of a substance by measuring its biological response over a biological system under standard conditions. It describes the purposes of bioassay such as determining drug potency and specificity. The principles and types of bioassay techniques like graded response, quantal, and multi-point assays are explained. Examples of bioassays for important drugs like insulin are provided. The document highlights the applications, uses, advantages, and disadvantages of bioassay.

1. BIOASSAY
Submitted to
Dr. Monika Dwivedi
Asst. Prof.
Dept. of Pharmaceutical science
Submitted by
Govind yadav

2. 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.
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3. The purpose of bioassay.
• To ascertain the potency of a drug
• To standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics etc., so
that each contains the uniform specified pharmacological activity.
• 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. 3

4. 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.
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5. In India
• standard drugs are maintained
in Government institutions like
1. Central Drug Research
Institute, Lucknow
2. Central Drug Laboratory,
Calcutta.
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6. Bioassay can be performed on
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• Intact animalsInvivo
• Isolated tissues
• Specific cells
• Organisms
Invitro

7. 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.
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8. 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 mice, rabbits, dog etc.
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9. Partial List Of Official Quantitative Biological Tests
Drug and Dosage form Test Animal (s)
Antibiotics Suitable microorganism
Insulin Rabbit & Mice
Digitalis & other glycosides Pigeon & Guinea Pig
Vitamin A & D Rat
Parathyroid drugs Dog
Posterior pituitary Rat
Tubocurarine Chloride Rabbit
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10. Characteristics of a good assay method
• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Precision
• Accuracy
• Stability – tissue has to stay “bioassay-fit
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11. Types of Bioassays
[1] Quantal Assays [ Direct endpoint ]
• Elicits an ‘All or None’ response in different animals
• Eg.
• Digitalis induced cardiac arrest in guinea pigs
• hypoglycemic convulsions in mice.
• Digitalis induced head drop in rabbits
• Calculation of LD50 in mice or rats
[2] Graded Response Assays [mostly on tissues]
• Graded responses to varying doses
• Unknown dose response measured on same tissue
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12. 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.
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13. 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 13

14. 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. 14

15. 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.
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16. 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.
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17. 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)
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18. 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 18

19. Bioassays of some important drugs
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 normal saline. 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.
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20. Preparation of test sample solution: The solution of the test sample is
prepared in the same way as the standard solution described above.
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 Dilutions:These are freshly prepared by diluting with
normal NaCl solution so as to contain 1 unit/ml. and 2 units/ml.
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 std. preparation of insulin.
Any other suitable method can also be used. 20

21. Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each.
The rabbits are then put into an animal holder.They should be handled with
care to avoid excitement.
First part of theTest: 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 sc. injections of insulin as follows:
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22. From each rabbit, a sample of blood is withdrawn up to 5 hrs. at the interval
of 1 hr. each. Blood sugar is determined again.This is known as ‘Final Blood
Sugar Level’.
Second 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 OverTest’.
• Mean percentage decrease in blood sugar of the first and second part is
calculated.
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23. Mouse Method: Mice show characteristic convulsions after s.c. inj. of
insulin at elevated temperatures.The percentage convulsions produced
by the test and standard preparations are compared.
Experimental procedure: 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 dilutions: Dilutions are prepared with sterile
saline solution, so as to contain 0.064 units/ml. (std dilution I) and 0.096
untis/ml. (std. dilution II). Similarly, test sample solutions are also
prepared.
Mice are divided into 4 groups each containing 25 mice and insulin is
injected s.c. as follows:
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24. • The mice which convulse or die are taken out of the incubator and
observed.
• Percentage convulsions produced by the test sample are
compared with those of the standard sample.
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25. Other bioassay’s.
• Immunological assay (ELISA).
• Micro-bioassay.
• Radioimmunoassay.
• Biotechnology.
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26. 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.
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27. Uses of Bioassay
• To measure the pharmacological activity of new/ chemically
undefined substances
• To investigate the function of endogenous mediators
• To measure drug toxicity and unwanted effects
• To measure the conc of drugs and other active substances in
the blood or other body fluids
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28. •Determination of potency, ED50/LD50 of drugs
•New drug development
•Measure clinical effectiveness
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29. 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
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30. Conclusion
• Successful tool in estimation & discovery of biologically active
substances
• Sensitivity & Specificity – important tool in pharmacology
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