pharmacology -bio assay

1. BIOASSAY
Dr Manish Mohan
JR Pharmacology

2. Contents
• Introduction
• Objectives of assay
• Characteristics of good assay
• Errors in bioassay
• Methodology
• Organ bath
• Physiological solution
• Different types of assay

3. ASSAY
“Estimation of the amount or activity of an active principle in a unit
quantity of preparation”

4. Types of assay
1.Physiochemical
- gas chromatography
- spectrophotometry
-fluorimetry
- mass spectrometry
2. Biologically mediated/bioassay
3.Immunological assay – RIA, ELISA
4.Microbiological assay

5. DEFINITION
“Bioassay is defined as comparative assessment of
relative potency of a test compound (T) to a standard
compound (S) on any living animal or biological tissue”

6. INTRODUCTION
Standardization of diphtheria antitoxin
-Paul Ehrlich
Basic step towards the drug discovery
 Determine the effect of any natural source of unknown substances
 Assessment of unknown substance on any living tissue

7. A bioassay experiment - Quantal
- Quantitative
Substances Used in Bioassay
-Derived from plants and animal sources

8. Objective or Purpose of Bioassay
Identification of various compounds
 Quantify the screening procedure
 Commercial production of drugs like antibiotics

9. Cont…….

10. Characteristics of good assay method
• High sensitivity
• High specificity
• Repeatability
• Reproducibility
• Accuracy
• Stability

11. PRINCIPLES OF BIOASSAY

12. ERROR IN BIOASSAY
Biological variation
Methodological error

13. Reason and its correction for biological variation
• Downregulation of receptors (repeated washing of tissue; avoid over washing)
• Loss of tissue sensitivity (change the tissue)
• Animal use of different species, sex, age, weight and health status (must use same
species)
• Laboratory condition may be variable (must be kept constant)
• Housing and handling of animals (must be handled by a qualified experienced
staff)

14. Reasons for Methodological error and its correction
 Lack of standardization of procedure
(standardize your procedure beforehand)
 Set-up of apparatus
(Check every instrument used in the experiment for their proper working)
Tissue isolation/extraction and preparation for the experiment
(minimize handling and excess cleaning of the tissue while mounting in the organ
bath)

15. Cont…..
Preparation of physiological salt solution (PSS) and maintenance of its
pH
(Follow the standard procedure to prepare and maintain the pH of PSS)
Drug preparation or it dilution
(while making serial dilution of the drug, mix it slowly; Avoid the vigorous mixing).

16. Bioassay may be performed on
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• Intact animals
Invivo
• Isolated tissues
• Specific cells
• OrganismsInvitro

17. WHOLE ANIMALS
• Nor Adrenaline – Spinal Cat
• Cardiac Glycosides – Guinea Pig
• Insulin – Mice
• Estrogens – Ovariectomized Female Rat
MICRO ORGANISMS
• Vit B12 – Euglena gracilis
17

18. ISOLATED TISSUE
• Acetyl Choline – Frog Rectus Abdominus muscle
• Histamine – Guinea Pig ileum
• Adrenaline – Rat uterus
• Oxytocin – Rat uterus estrogen primed
DISPERSED CELLS
• Plasma LH estimation by stimulation of testosterone
synthesis - on isolated Leydig cells
18

19. METHODOLOGY

20. ORGAN BATH
“ Traditional experimental set-up that is commonly used to
investigate the physiology and pharmacology of in vitro tissue
preparations”
Perfused tissues can be maintained for several hours in a temperature controlled
organ bath

23. Essential Parts of Organ Bath which is Required to
Record the Response of a Tissue/Muscle
Sherrington rotating drum
(standard speed of the drum is 1 revolution/96 min.)
Kymogram is the paper used to record the response
Outer bath/Outer water jacket
Inner organ bath

24. Cont.….
• Tissue holder and oxygen supply
• Fulcrum
• Heating iron coil and thermostat
• Fixing of the recording
(shellac and colophony saturated in the alcohol)

25. METHODOLOGY

27. Physiological Salt Solution
• Also called as PSS / Ringer solution
• Maintains tissue outside the animal body
• Select PSS in which tissue last longer
• Prepare the solution with the help of distilled or double distilled or
deionized water
• Prepare fresh solution

28. PHYSIOLOGICAL SALT SOLUTION (PSS)

29. Role of Each Ingredient
• Sodium (Na+): maintain the osmolarity
• Potassium (K+): maintaining rate and rhythm.
• Calcium chloride (CaCl2): excitation
• Magnesium chloride (MgCl2): reduce the spontaneous activity of
tissue

30. • Amphibian tissue - Frog-Ringer solution
• heart muscle preparation- Ringer-Locke
• Tyrode - mammalian smooth muscle
• Krebs, De Jalon and McEwen - mammalian isolated organ

31. PHYSIOLOGICAL SALT SOLUTION (PSS)
Tyrode may be used for experiment with non-innervated muscle while
Krebs is used for nerve muscle preparation
PH- 7.3-7.4

32. LEVER
“Load force × length of load arm = Effort force × length of effort arm.”
1) Effort arm
2) Load arm
3) Fulcrum

34. Different types of levers, commonly used in
experimental pharmacology
Simple lever Frontal writing lever

35. Gimbal lever Sprung lever
Auxotonic lever Starling heart lever

36. Magnification

37. TYPE OF TISSUE
1. According to tissue/muscle obtained:
– Smooth muscle preparation, e.g.: Uterus, tracheal smooth muscle,
vas deferens, aorta, ileum, ascending or descending colon, etc.
– Skeletal muscle, e.g.: Rectus abdominus muscle, etc.
– Cardiac muscle, e.g.: Frog heart etc.

38. 2. According to response obtained by tissue/ muscle
- Slow contracting tissue/muscle
e.g. Frog rectus abdominus muscle, stomach fundus, guinea pig
tracheal smooth muscle
– Fast contracting tissue/muscle
e.g. Ileum, uterus, ascending and descending colon

39. CLASSIFICATION OF BIOASSAY
• 1. Direct end point assay (DEPA)
• 2. Quantal assay (all or none assay)
• 3. Graded assay
a. Bracketing assay
b. Matching assay
c. Interpolation assay
d. Multiple point assay (3-point, 4-point, 6-point and 8-point)

40. 1. End Point Method
• Threshold dose producing a positive effect is measured
• e.g. bioassay of digitalis in cats
• The threshold dose of standard = Total period of infusion × Rate of
drug administration

41. • Concentration of test = TDS X CSD
TDT
• TDS = Threshold dose of standard
• TDT = Threshold dose of test
• CSD = Concentration of standard drug

42. Disadvantages
• Only toxicity study or high dose study is possible
• Dose ranging study cannot be done.

43. 2. Graded response assays
• Response is proportional to the dose
• Response may lie between no response and the maximum response

44. Bracketing assay
• Test sample volume is too small
• Single /few responses – test drug
• This response is bracketed b/w 2 responses of the standard drug
• Potency – conc. Standard drug / dose response curve

46. Matching assay
• Compare both test and standard – trial and error method
• Response is matched b/w test and standard drug
• Require very small volume of test drug
• It require most sensitive tissue

48. Disadvantage
• Purely subjective
• Experimental error cannot be determined
• Require Most sensitive tissue

49. Interpolation Assay
• Concentration of unknown is interpolated from the DRC graph

51. Limitations of above mentioned assays
• Lack of sensitivity
• Lack of accuracy
• Methodological errors such as tissue sensitivity error
• Variable temperature error
• Dilution error

52. Multiple point assays
 Responses are repeated several times and the mean of each is taken
 Chances of error areminimized
 3 point method - 2 doses of std+1 dose of test
 4 point method - 2 doses of std+2 doses of test
 6 point method - 3 doses of std+3 doses of test
 Latin square method of randomization to avoid any bias33

53. 3 POINT METHOD
- 2 doses of std+1 dose of test

55. Calculation
S1 and S2 = Length of standard dose response
T = Length of test dose response
s1 and s2 = Doses which produces responses S1 and S2

56. CONT……
• Percentage error (%) = ACT – OCT X 100
ACT
• where, ACT = Actual concentration of test
• OCT = Observed concentration of test

57. 4 point method - 2 doses of std+2 doses of test

58. 38
4 - POINT ASSAY

59. Calculation
• Mean responses of 4 sets plotted
• Log potency ratio (M)
(T2-S2)+(T1-S1) × Log d
(S2-S1)+(T2-T1)
where, d-dose ratio = s2/s1
• Strength of unknown =
s1/t1 × antilog of M
41

60. 6-point and 8-point Assay
• Time consuming lengthy procedure
• Reduced error
• Greater specificity

61. APPLICATIONS OF BIOASSAY
1. Estimate potency of natural drug.
2. Standardization of drugs of natural origin (plant and animal origin)
3. Screening of new compound for biological activity
4. Estimation of biologically active substance like histamine,
acetylcholine, adrenaline.

62. Thank you