bioassays , methods used to test substances on isolated tissues preparations, acetycholine , rat ileum , DOSE response curves of drugs

1. BIOASSAY
RVS Chaitanya Koppala
SVCP, BHIMAVARAM

2. Outline
• Types of assays
• Introduction
• Definition
• Indication and principles of bioassay
• Basic procedure
• Calculations
• Source of errors
• Summary

3. Types of Assays
• Biological assays
• Chemical assays:
– Spectrophotometer,
– Spectrofluorimetry,
– Chromatography,
• Radio Immunoassays
• Microbiological assays

4. Introduction
• Late 18th centaury- standardization of
Diphtheria antitoxin by Paul Ehrlich
• Bioassay literal meaning
o Bio – living tissue
o Assay- assessment / measurement
o Bioassay: Assessment of a biological substance

5. Definition
 Comparative assessment of relative potency of
a test compound to a standard compound on a
living or biological tissue.
 Quantitative measurement of the amount of
active principle or substance in a
pharmaceutical preparation or biological
material using a suitable biological system

6. Indications for Bioassay
• Active principle of drug is unknown
• Active principle cannot be isolated, e.g. insulin, posterior
pituitary extract etc.
• Chemical method is either
– not available
– if available, too complex,
– insensitive to low doses e.g. Histamine can be assayed in microgram
conc.
• Chemical composition of drug is different but has same
pharmacological action e.g. cardiac glycosides isolated from
diff sources, catecholamines etc.
• To measure LD 50 and ED 50
• For biological standardization of drugs from natural sources
which cannot be obtained in a chemically pure form e.g.,
vasopressin, oxytocin, insulin, heparin

7. Principles of bioassay
• Bioassay involves the comparison of the main
pharmacological response of the unknown preparation
with that of the standard.
• The reference standard and test sample should have same
pharmacological effect and mode of action, so that their
DRC curve run parallel and their potency ratio can be
calculated.
• The test solution and standard should be compared for
their established pharmacological effect using a specified
pharmacological technique.
• The method selected should be reliable, sensitive,
reproducible and should minimize errors due to biological
variation and methodology. ( Animals should of same
species, sex and weight and number of animals should be
large enough to permit statistical analysis.)

8. Procedure
1. Prepare the physiological salt solution
2. Arrange the instrument and adjust the water bath.
3. Balance the lever
4. Tissue selection
5. Surgical process and collection of required tissue.
6. Tissue attachment to the water bath
7. Relaxation time given to the tissue
8. Prepare the standard drug( serial dilution)
9. Select the lowest possible measurable concentration
by trial and error method.
10. Prepare DRC for the standard drug.
11. Prepare DRC for the test drug.( serial dilution)
12. Select a assay method (3 point or 4 point assay)
13. Calculation

9. Step 1: Prepare the physiological salt
solution

10. Various Physiological salt solutions
Frog-
Ringer
Kreb’s Tyrode Ringer-
Locke
De
Jalon
Mc
Ewen
NaCl 65 g 69 g 80 g 91.5 g 90 g 76 g
KCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 g
MgCl². 6H²O --- 1.1 g 1.0 g --- --- ---
NaH2PO4. H²O 0.1 g 1.4 g 0.5 g --- --- 1.4 g
NaHCO³ 2 g 21 g 10 g 1.5 g 5 g 21 g
CaCl² 1.2 g 2.8 g 2 g 2.4 g 0.6 g 2.4 g
Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g
Aerating Gas air O² +
5%CO²
O² or air Pure O² O² +
5% CO²
O² + 5%
CO²
For 10 litres
pH- 7.3-7.4
•Calcium chloride to be added last.
•Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.

11. Uses: Physiological salt Solutions
Physiological salt
solutions
Uses
Frog-Ringer Amphibian tissue preparation
Kreb’s Mammalian/Avian skeletal muscle
preparation
Tyrode Intestine preparation
Ringer-Locke Heart muscle preparation
De Jalon Rat uterus preparation

12. Electrolytes
Ingredients Functions
NaCl Maintain osmolarity
K+ Nerve conduction, muscle
contraction, maintain heart rate &
rhythm
Ca + Contraction
Mg+ Neurotransmission , decrease
spontaneous activity
NaHCO³ & NaH2PO4 Buffer
Glucose Nutrient

13. Step 2: Arrange the instrument and adjust
the water bath.
 Kymograph: Sherrington- starling
kymograph
 To obtain a graphical amplified
measurable response of a muscle
or tissue
 Two important parts: motor box
and drum
 Speed lever: 1 revolution/ 96 min.
 Paper:
 glossy side outside – least
resistance
 Rough side inside – stick to the
drum.
 Fixing solution: shellac and
colophony saturated in alcohol

14. Student Organ bath
• Outer bath:-
 First designed by rudolph
magnus
 Perpex glass
 Store water outside the
inner bath to maintain the
temperature
• Inner bath:-
– Glass
– To observe the tissue
during experiment
– 5-50ml (usually 10ml)

16. • Tissue holder and oxygen supply:-
 Tissue is attached inside the inner water bath to a
tissue holder.
 Also supports the oxygen supply to the tissue.

17. Step:3 -Balance the lever
• Lever:
Three basic parts:
• Effort arm- where force in
applied
• Load arm- where effect of
force is observed
• Fulcrum
Classes of lever – 3
Types of lever

18. • Magnification :
= Distance from the fulcrum to the writing point
Distance form the fulcrum to the tied tissue
o For slow contracting muscles:- 10-15 times
o For fast contracting muscles:-5-10 times

19. Step:4-Tissue selection
S.No Compound Tissue used
1. Acetylcholine Guinea-pig ileum
Frog rectus abdominis muscle
Leech dorsal muscle
Rat uterus preparation
Isolated guinea-pig auricles
2. Serotonin Isolated oestrous uterus of rat
Isolated fundic strip of rat
Guinea pig ileum
 Rabbit ear preparation
Isolated heart of the mollusc Venus mercenaria

20. S. No Chemical Tissue used
3. Histamine  Guinea pig isolated ileum
 Guinea pig tracheal chain.
 Fall in BP of dog/cat
4. Adrenaline and
noradrenalin
Rat colon
Non pregnant rat uterus
Rat fundus
Rabbit aortic strip
Rabbit jejunum
Tracheal chain of guinea pig

21. Step 5: Surgical process and collection
of required tissue.
• Animal sacrificed by cervical dislocation.
• Tissue identified and isolated.
• Carefully dissect and separate unwanted tissue.
• Tissue kept in a physiological salt solution.
• Avoid excessive handling of tissue.

22. Step 6 : Tissue attachment to the
water bath
• Attach the ends of the tissue:-
– One end:- tissue holder
– Other end:- lever
• Method of attachment of tissue:
– Attach the thread at the end by a needle
– Intestine:- care should be taken not to block the lumen

23. Aeration
Pure oxygen (O2 ) For heart
Air For intestine
Carbogen ( 95% O2 &
5% CO2 )
For uterus
Mixing of the test drug
Homogenisation of the solution
Keeping the tissue lumen patent
To maintain pH
( aeration by pure O2 causes losing of CO2 & solution
becomes alkaline )

24. Temperature
Rabbit intestine Physiological temp.(37°C ) is needed for
mammalian tissues
Guinea-pig ileum Temp. should be decreased in some
experiment to decrease spontaneous
contractions
Frog rectus muscle Amphibian tissue can survive in room
temperature
Temperature should be constant through out the experiment

25. Step 7:Relaxation time given to the
tissue
1. Intestine 30-45 min
2. Frog rectus 45-60 min
Measures to decrease spontaneous contraction:-
Hanging a weight of appropriate amount
Giving a antagonist
oE.g. Acetylcholine for blocking spontaneous
contraction of ileum.

26. Step 8: Prepare the standard drug
( serial dilution)
• Serial dilution: 10---10-9

27. Step 9: Prepare DRC for the standard
and test drug
•Select two std doses s1& s2 from linear part of DRC [ Let the
corresponding response be S1, S2]
•Also s2/s1 = t2/t1 = 3/2

28. Time cycle
Contact time
Time allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec
Time (
min )
Event
0 Start the kymograph
2 Add the Acetylcholine
2.5 Stop the kymograph & wash
the preparation
10 Wash the preparation
15 Start the kymograph
Start
kymogarp
h
Add Ach
Stop
kymograp
h
Wash
preparati
on
Wait for
11.5
min

29. Step 10: Perform a assay (3 or 4 point
assay)

30. Types of Bioassays
• [1] Quantal Assays [ Direct endpoint ]
 Elicits an ‘All or None’ response in different
animals
 E.g.
 Digitalis induced cardiac arrest in guinea pigs
 Hypoglycaemic convulsions in mice.
 Digitalis induced head drop in rabbits
• [2] Graded Response Assays
 Graded responses to varying doses
 Unknown dose response measured on same
tissue

31. [2] Graded Response Assays [ Direct comparison
on same tissues]
Interpolation:
 Conc. of unknown is
read from a standard
plot of a log dose
response curve of at
least 4 sub maximal
concentrations

32. Matching & Bracketing:
Const dose bracketed with varying doses of standard till
exact match is obtained
• 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

33. Multiple Point Assays
• 3 point assay
• 4 point assay

34. 4 point assay [2 +2 dose assay]
• Procedure [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 response T1 &T2 between S1 & S2 ;
 Also s2/s1 = t2/t1 = 2/3
 Record 4 data sets [Latin square: Randomisation reduces error]
• s1 s2 t1 t2
• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1

35. 3 point assay [2+1 dose assay]
• Fast & convenient
• Procedure [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 [ in 2:3 dose ratio] from linear
part of LDR [ Let the corresponding response be S1, S2]
 Choose a test dose t with a response T between S1 & S2
 Record 4 sets data [Latin square: Randomisation reduces
error] as follows
 s1 s2 t
 t s1 s2
 s2 t s1
 s1 s2 t
 Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X log d
[d = dose ratio]

36. Step 11: Calculation
• Calculate the height of each response.
• Take mean of all S1, S2, T1 and T2 values.
• Plot a graph

37. S1
S2
T1
T2
D1 D2
M

38. S1
S2
T1
T2
D1 D2

39. Calculation of the strength of the solution from
graph :
• We know that D1=D2
• EG..
• 0.675 ml of 1 µg/ml= 0.425 of D2 conc.
• D2 = 0.675/ 0.425
= 1.59 of 1 µg/ml
• Strength of D2 = 1.59 µg/ml

40. Log potency ratio :
• The horizontal separation M of the two curves
represents the log potency ratio of the
concentration of test solution and of standard

41. Direct calculations
• M={(T1-S1) +(T2 –S2)}/{(S2-S1) +(T2-T1)}×log d
• Log d = log[s1/s2]
Where,
• M = Potency of the drug
• S1 & S2 = Length of the standard dose
response selected between 25-75 %
• T1 & T2 = Length of the test drug response
• s1 & s2 = Standard drug dose which came in
contact with tissue and had given the
response S1 & S2 respectively
• Dilution of the inner water bath has to be taken in to
account

42. • Strength of test solution = s1/t1 × antilog of M
• Dilution of the inner water bath has to be taken
in to account

43. Calculation of the percentage error:-
• Percentage error = ACT-OCT × 100
ACT
Where,
• ACT = Actual concentration of test
• OCT = Observed concentration of test
• The permissible limit of percentage error is <10%

44. Errors in bioassays
• Margin of error of bioassay should be < 10%
• Two types:-
1. Biological variation:
2. Methodological variation

45. • Biological variation:-
1. Variation in response to a drug.
2. Down regulation of receptor (repeated washing
of tissue)
3. Loss of tissue sensitivity (change the tissue)
4. Laboratory condition may be variable.

46. • Methodological variation:-
1. Human error: done by the experimenter
2. Experimental error: faulty procedure selection or
calibration error.(proper balancing the lever, and
by maintaining the ph and temperature at a
physiological level.)

47. • Reasons for methodological error:
1. Lack of standardization of procedure
2. Over handling of tissue
3. Preparation of physiological salt solution.
4. Drug preparation or in dilution

48. Prepare Physiological sol.
Check instruments
Tissue collection and mounting
Relaxation
Prepare DRC and Do a 4 point assay
Summary

49. Summary
1. Prepare the physiological salt solution
2. Arrange the instrument and adjust the water bath.
3. Balance the lever
4. Tissue selection
5. Surgical process and collection of required tissue.
6. Tissue attachment to the water bath
7. Relaxation time given to the tissue
8. Prepare the standard drug( serial dilution)
9. After relaxation test any concentration of the drug
10. Then standardize the tissue response with same drug. ( take subsequent
two response)
11. Select the lowest possible measurable concentration by trial and error
method.
12. Prepare DRC for the standard drug.
13. Prepare DRC for the test drug.( serial dilution)
14. Select a assay method (3 point or 4 point assay)
15. Measure the height of each response
16. Calculation

50. Time cycle
Time ( mins) Event
0 Raise the 1 gm weight & start the kymograph
2 Add acetylcholine
3.5 Stop the kymograph, wash rectus & lower the 1 gm weight
6 Raise the weight & start the kymograph
Contact time
Time allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec

52. Principles of Bioassay
• Active principle to be assayed should show the same
measured response in all animal species
• The degree of pharmacological response produced should
be reproducible under identical conditions [Eg Adrenaline
shows same rise in BP in the same species under identical
conditions: wt, age, sex, strain / breed etc]
• The reference standard must owe its activity to the
principle for which the sample is being bioassayed
• Activity assayed should be the activity of interest
• Individual variations must be minimised / accounted for
• Bioassay might measure a diff aspect of the same
substance compared to chemical assay [Eg testosterone &
metabolites

53. Biological objects
Whole
animal
Isolated organ Isolated
tissue
Isolated
cells
Assay of
insulin in
rabbits
Assay of
gonadotropins
on ovary
Assay of
oxytocin on
isolated
uterine
tissue
Assay of
antibiotics
on bacterial
cells

54. Thank you