Bioassay Procedure for Three Point and

Four Point Bioassay

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Contents
1. Introduction
2. Methods of Bioassay
3. Procedure
4. Three Point Bioassay
5. Four point bioassay
6. Conclusion
7. References

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Introduction
 Bioassays is also known as Biological assay or Biological standardization.

 Bioassays is defined as the estimation of potency of an active principle

in a unit quantity of test preparation using living tissue / microorganisms /
immune cells / whole animal by comparing with that of the standard drug
(International Units) by determining its pharmacological action.

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 End Point Method
Methods Direct
Methods Matching Method
of Bracketing Method
Bioassay Graphical /
Indirect Interpolation Method
Methods
Multiple Point
Method

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Procedure
1. Prepare the physiological salt solution
(Frog-Ringer Solution, Kreb’s Solution, Tyrode Solution, De Jalon Salt Solution)

2. Arrange the instrument and adjust the water bath

(Kymograph: Sherrington - starling kymograph, Student organ bath)

3. Adjustment of the lever and balance it

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 Source: Evaluation of the antispasmodic activity of Mahua in a Student organ bath [Image on the internet]. 2019 [cited 2021 Jan 30].
Available from: https://www.researchgate.net/figure/Evaluation-of-the-antispasmodic-activity-of-Mahua-in-a-Student-organ-
bath_fig5_335030367

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4. Tissue selection (fundus, ileum, colon, uterus etc.)

5. Surgical process and collection of appropriate tissue

6. Tissue attachment to the water bath

7. Relaxation time given to the tissue (30/45 min)

8. Prepare the standard drug (serial dilution)

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9. Select the lowest possible measurable concentration by trial and
error method

10. After relaxation test any concentration of drug for response

11. Then standardize the tissue response by with the same dose

12. Prepare DRC for the standard drug

13. Prepare test drug dilution (serial dilution)

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14. Prepare DRC for the test drug

15. Selection of assay method (i.e 3 point or 4 point assay)

16. After taking several responses measure height of each responses

17. Calculation

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Three Point Bioassay
Log dose response [LDR] curve plotted with varying concentration
of standard solutions and given test solution
Select two standard doses S1 & S2 from linear part of DRC
Choose a test dose response T between S1 & S2

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Then the three point bioassay is performed by following the Latin
square method.
Set-1 S1 S2 T
Set-2 S2 T S1
Set-3 T S1 S2

Then the heights of responses are measured and averages are

calculated.

The averages then substituted in the following formula to get the

concentration of the unknown solution.

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 Response (cm)
S1 S2 T
Set-1
Set-2
Set-3
Avg

𝑛1 𝑇−𝑆1 𝑛2
Conc. of Unknown = × 𝑎𝑛𝑡𝑖𝑙𝑜𝑔 ( × ) × 𝐶𝑠
𝑡 𝑆2−𝑆1 log 𝑛1
where
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

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Four Point Bioassay
Log dose response [LDR] curve plotted with varying concentration
of standard solutions and given test solution

Select two standard doses S1 & S2 from linear part of DRC

Choose two test doses response T1 &T2 between S1 & S2

Record 4 data sets [Latin square: Randomization reduces error]

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  Finally record the responses of S1, S2, T1 & T2 in the following sets.
Set-1 S1 S2 T1 T2
Set-2 S2 T1 T2 S1
Set-3 T1 T2 S1 S2
Set-4 T2 S1 S2 T1

Response (cm)
S1 S2 T1 T2
Set-1
Set-2
Set-3
Set-4
Avg

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 Then the heights of responses are measured and averages are calculated.

 The averages then substituted in the following formula to get the

concentration of the unknown solution.
𝑛1 𝑆1+𝑆2 − 𝑇1+𝑇2 𝑛2
Conc. of Unknown = × 𝑎𝑛𝑡𝑖𝑙𝑜𝑔{ }×
𝑡1 𝑆2+𝑇2 − 𝑆1+𝑇1 log 𝑛1

where
n1 = lower dose of standard
n2 = higher dose of standard
t1 = test dose of lower concentration
t2 = test dose of higher concentration
S1 = response of n1
S2 = response of n2
T1 = response of t1
T2 = response of t2

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Conclusion
 Bioassay is less elaborate, less accurate, more troublesome, more

laborious and more expensive.

 It is a successful tool in estimation and discovery of biologically

active substances and important application in sensitivity and
specificity of pharmacological applications.

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 REFERENCES

 Medhi B, Prakash A. Practical Manual of Experimental and Clinical

Pharmacology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2010.
Chapter 2, Bioassay; p. 45-69.

 Reddy M, Nagakannan P, Hunasagi R, et al. Isolated goat ileum preparation –

an alternative to isolated ileum preparation for the three point bioassay of
acetylcholine. IJRPC [Internet]. 2011 April. Available from:
http://www.ijrpc.com/files/018.pdf

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