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    Bioassay Procedure For Three Point and Four Point

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    Lokesh Mahata
    This document describes procedures for three point and four point bioassays. The three point bioassay involves selecting two standard doses and one test dose between the standards. Responses are measured and the test concentration is calculated using the formula given. The four point bioassay similarly selects two standard and two test doses, and uses the four response measurements and a formula to determine the test concentration. Both methods use serial dilutions and a Latin square design to reduce errors. Bioassays provide a means to estimate potency of active substances and have applications in pharmacological research.

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    Bioassay Procedure For Three Point and Four Point

    Uploaded by

    Lokesh Mahata
    2K views17 pages
    This document describes procedures for three point and four point bioassays. The three point bioassay involves selecting two standard doses and one test dose between the standards. Responses are measured and the test concentration is calculated using the formula given. The four point bioassay similarly selects two standard and two test doses, and uses the four response measurements and a formula to determine the test concentration. Both methods use serial dilutions and a Latin square design to reduce errors. Bioassays provide a means to estimate potency of active substances and have applications in pharmacological research.

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    Bioassay Procedure for Three-Point and Four Point

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    Bioassay Procedure for Three Point and Four Point

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    This document describes procedures for three point and four point bioassays. The three point bioassay involves selecting two standard doses and one test dose between the standards. Responses are measured and the test concentration is calculated using the formula given. The four point bioassay similarly selects two standard and two test doses, and uses the four response measurements and a formula to determine the test concentration. Both methods use serial dilutions and a Latin square design to reduce errors. Bioassays provide a means to estimate potency of active substances and have applications in pharmacological research.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    2K views17 pages

    Bioassay Procedure For Three Point and Four Point

    Uploaded by

    Lokesh Mahata
    This document describes procedures for three point and four point bioassays. The three point bioassay involves selecting two standard doses and one test dose between the standards. Responses are measured and the test concentration is calculated using the formula given. The four point bioassay similarly selects two standard and two test doses, and uses the four response measurements and a formula to determine the test concentration. Both methods use serial dilutions and a Latin square design to reduce errors. Bioassays provide a means to estimate potency of active substances and have applications in pharmacological research.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 17

    Bioassay Procedure for Three Point and

    Four Point Bioassay

    01-02-2021 1
    Contents
    1. Introduction
    2. Methods of Bioassay
    3. Procedure
    4. Three Point Bioassay
    5. Four point bioassay
    6. Conclusion
    7. References

    01-02-2021 2
    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.

    01-02-2021 3
    End Point Method
    Methods Direct
    Methods Matching Method
    of Bracketing Method
    Bioassay Graphical /
    Indirect Interpolation Method
    Methods
    Multiple Point
    Method

    01-02-2021 4
    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

    01-02-2021 5
    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

    01-02-2021 6
    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)

    01-02-2021 7
    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)

    01-02-2021 8
    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

    01-02-2021 9
    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

    01-02-2021 10
    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.

    01-02-2021 11
    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

    01-02-2021 12
    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]

    01-02-2021 13
     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

    01-02-2021 14
     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

    01-02-2021 15
    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.

    01-02-2021 16
    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

    01-02-2021 17

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