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BIOASSAY 
Dr. RENJU.S.RAVI
OVERVIEW 
 BIOASSAY – Definition/Synonyms 
 PRINCIPLES OF BIOASSAY 
 INDICATIONS OF BIOASSAY 
 TYPES OF BIOASSAY 
 USES OF BIOASSAY 
 DRAWBACKS 
 BIOASSAY IN HUMANS
What is ASSAY 
 Amount or activity of an active principle in unit 
quantity of preparation 
 Types: 
3 
Physico-chemical 
assay 
Biological 
(Bioassay) 
Immunological assay 
Photometry 
Chromatography 
RIA 
ELISA
Bioassay 
Potency or concentration of an active 
principle in unit quantity of 
preparation 
by measuring its biological response 
on living tissues 
Introduced by Paul Ehrlich - biostandardization of 
Diphtheria antitoxin 
4
Synonyms 
5 
Biological 
assay 
Bio 
metrics 
Biological 
standadization 
Bio-standadizatio 
n
Principles of bioassay 
To compare the test substance with the 
International Standard preparation of the same 
To find out how much test substance is required to 
produce the same biological effect, as produced by 
the standard 
6
Contd.. 
Activity assayed should be the activity of interest 
Standard & test sample - similar pharmacological 
effects & mode of action 
Both should be compared for their established 
pharmacological effect using specified technique 
Ex: *Ach – contractile response on frog rectus 
*Histamine – contractile response on guinea 
pig ileum 
7
Contd.. 
Problem of biological variation must be minimized 
 Experimental conditions - kept constant 
 Animals - same species, sex and weight 
 Number of animals - large enough to minimize 
error (individual variation) 
 Isolated preparations - sensitive 
8
Indications of bioassay 
 No chemical method has been developed 
 Chemical assay is too complex /not sensitive enough to 
measure (ex: insulin, Ach) 
 To measure the pharmacological activity of new or 
chemically undefined substances 
 For biological standardization of drugs obtained from 
natural sources as these cannot be obtained in pure 
form. Eg: Oxytocin,Vasopressin,Insulin,Heparin.. 
9
Contd… 
 To compare the strength of a drug obtained 
from various sources due to different 
compositions (Eg:Cardiac glycosides) 
 Chemicals with similar structure, but different 
biological activity 
 Chemical structure of the active principle is 
unknown 
 Chemical structure known; cannot be actively 
purified. Eg: Peptide hormones 
10
Characteristics of a good assay 
method 
 Sensitivity 
 Specificity 
 Repeatability 
 Reproducibility 
 Precision 
 Accuracy 
 Stability – tissue has to stay “bioassay-fit 
11
Bioassay can be performed on 
12 
• Intact 
Invivo animals 
• Isolated 
tissues 
• Specific cells 
• Organisms 
Invitro
WHOLE ANIMALS 
 Nor Adrenaline – Spinal Cat 
 Cardiac Glycosides – Guinea Pig 
 Insulin – Mice 
 Estrogens – Ovariectamised Female Rat 
MICRO ORGANISMS 
Vit B12 – Euglena gracilis 
Tetracycline - Bacillus pumilus 
13
ISOLATED TISSUE 
Acetyl Choline – Frog Rectus Abdominus muscle 
 Histamine – Guinea Pig ileum 
 Adrenaline – Rat uterus 
Oxytocin – Rat uterus oestrogen primed 
DISPERSED CELLS 
• Plasma LH estimation by stimulation of 
testosterone synthesis - on isolated Leydig 
cells 
14
Types of Bioassay 
QUANTAL ASSAY 
GRADED ASSAY 
INDIRECT ASSAY 
DIRECT ASSAY
Quantal assay 
 Quantal response - the response is in the 
form of "all or none", i.e. either no 
response or maximum response 
 Drugs producing quantal effect can be 
bioassayed by end point method 
17
 The threshold dose producing a predetermined 
effect is measured 
 Comparison between the results of standard and 
the test 
 E.g: Bioassay of digitalis in cats,Insulin induced 
hypoglycemic convulsions in rat 
Threshold dose of the Std 
X Conc of Std 
Threshold dose of the Test 
Conc of Unknown =
Graded assay 
 Graded response - response is proportional to the dose 
and response may lie between no response and the 
maximum response. 
 Types: 
• Bracketing /direct matching 
• Interpolation 
• Multiple point assays 
Three point assay 
Four point assay 
Six point assay 
• Cumulative dose response 19
Graded DRC 
20 
standard Test/unknown
21 
DRC & Log DRC 
70% 
30% 
Sigmoid curve 
Wide range of doses 
can plot 
 Rectangular hyperbola 
• Potency 
• Efficacy 
• Slope of curve
22
Bracketing or Direct Matching 
 A constant dose of the standard is bracketed by varying 
dose of test sample 
 until an exact matching between the response of std & 
that of the sample is achieved 
 Strength of unknowm/test drug can be found by simple 
interpolation of bracketed response. 
23
24
Bracketing or Direct Matching 
25
ADVANTAGES 
 Simple & Faster 
 Amount of test drug available is small 
 Does not involve complicated 
calculations 
 Does not depend on DRC 
26
Disadvantages 
 less accurate,time consuming, troublesome 
 cannot get exact match of response 
 quantitative difference b/w test & std not 
obtained
Interpolation assay 
 A log dose-response curve is plotted with the 
standard on a simple graph paper or Semi-log 
paper 
 The concentration of the test is then read from the 
graph 
28
29 
standard Test/unknown
Interpolation 
100 
% standard 
R 
E 
S 
50 
P 
O 
N 
S 
E 
0 x x1 
LOG DOSE
Advantages 
 Sensitivity of tissue is 1st determined by prior 
plotting of a conc-response curve with known 
agonist 
 Dose can be plotted even if it varies over thousand 
fold range 
 Error is normally distributed
Disadvantages 
 Sensitivity of tissue changes with time 
 Timing of doses not taken into account 
 Variation in mode of application of drugs
Multiple point assays 
 Responses are repeated several times and the mean of 
each is taken 
 Chances of error are minimized 
 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 
bias 
33
34 
t s1 s2 
s1 s2 t 
s2 t s1 
3 - point assay 
3 cycles
3-POINT ASSAY 
% 
R 
E 
S 
P 
O 
N 
S 
E 
LOG DOSE 
35 
s1 
T 
t 
S2 
s2 
S1
Latin square design 
s1 s2 t 
t s1 s2 
s2 s1 t 
t s2 s1
CALCULATION 
• Mean responses of these 3 sets plotted 
• Log potency ratio (M) = 
(T-S1÷ S2-S1)× log d 
where, d – dose ratio = s2/s1 
• Strength of unknown = 
s1/t × antilog of M 
37
38 
4 - POINT ASSAY
4-POINT ASSAY 
% 
R 
E 
S 
P 
O 
N 
S 
E 
LOG DOSE 
39 
s1 
T1 
t1 
S2 
s2 
S1 
T2 
t2
Latin square design 
s1 s2 t1 t2 
s2 t1 t2 s1 
t1 t2 s1 s2 
t2 s1 s2 t1
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
Six point assay 
 3+3 dose assay 
 3 conc each of std & test drug are used 
 6 sets of experiments using 6 doses in each set 
 More time consuming,lesser in use 
 Reliability is excellent
Cumulative Dose Response Curve 
 Increase conc of drug in bath fluid step by step 
without washing out the preceeding doses 
 Continue till supramaximal effect is seen 
 Dose response curve is plotted
Cumulative dose response
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 
45
 Determination of potency, ED50/LD50 of 
drugs 
 New drug development 
 Measure clinical effectiveness 
46
Drawbacks 
• Biological variation 
• Troublesome 
• Time consuming 
• Expensive 
• Less accurate than physico-chemical 
methods 
47
HUMAN TISSUE BIOASSAY 
• Veins [surgery on varicose veins] 
• Tissues like larger blood vessels obtained during 
amputation 
• Organs removed during transplantation/ tumor 
surgeries/procedures requiring resection 
• Tissues collected Postmortem 
48
Conclusion 
 Successful tool in estimation & discovery of biologically 
active substances 
 Sensitivity & Specificity – important tool in pharmacology
Bioassay

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Bioassay

  • 2. OVERVIEW  BIOASSAY – Definition/Synonyms  PRINCIPLES OF BIOASSAY  INDICATIONS OF BIOASSAY  TYPES OF BIOASSAY  USES OF BIOASSAY  DRAWBACKS  BIOASSAY IN HUMANS
  • 3. What is ASSAY  Amount or activity of an active principle in unit quantity of preparation  Types: 3 Physico-chemical assay Biological (Bioassay) Immunological assay Photometry Chromatography RIA ELISA
  • 4. Bioassay Potency or concentration of an active principle in unit quantity of preparation by measuring its biological response on living tissues Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin 4
  • 5. Synonyms 5 Biological assay Bio metrics Biological standadization Bio-standadizatio n
  • 6. Principles of bioassay To compare the test substance with the International Standard preparation of the same To find out how much test substance is required to produce the same biological effect, as produced by the standard 6
  • 7. Contd.. Activity assayed should be the activity of interest Standard & test sample - similar pharmacological effects & mode of action Both should be compared for their established pharmacological effect using specified technique Ex: *Ach – contractile response on frog rectus *Histamine – contractile response on guinea pig ileum 7
  • 8. Contd.. Problem of biological variation must be minimized  Experimental conditions - kept constant  Animals - same species, sex and weight  Number of animals - large enough to minimize error (individual variation)  Isolated preparations - sensitive 8
  • 9. Indications of bioassay  No chemical method has been developed  Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach)  To measure the pharmacological activity of new or chemically undefined substances  For biological standardization of drugs obtained from natural sources as these cannot be obtained in pure form. Eg: Oxytocin,Vasopressin,Insulin,Heparin.. 9
  • 10. Contd…  To compare the strength of a drug obtained from various sources due to different compositions (Eg:Cardiac glycosides)  Chemicals with similar structure, but different biological activity  Chemical structure of the active principle is unknown  Chemical structure known; cannot be actively purified. Eg: Peptide hormones 10
  • 11. Characteristics of a good assay method  Sensitivity  Specificity  Repeatability  Reproducibility  Precision  Accuracy  Stability – tissue has to stay “bioassay-fit 11
  • 12. Bioassay can be performed on 12 • Intact Invivo animals • Isolated tissues • Specific cells • Organisms Invitro
  • 13. WHOLE ANIMALS  Nor Adrenaline – Spinal Cat  Cardiac Glycosides – Guinea Pig  Insulin – Mice  Estrogens – Ovariectamised Female Rat MICRO ORGANISMS Vit B12 – Euglena gracilis Tetracycline - Bacillus pumilus 13
  • 14. ISOLATED TISSUE Acetyl Choline – Frog Rectus Abdominus muscle  Histamine – Guinea Pig ileum  Adrenaline – Rat uterus Oxytocin – Rat uterus oestrogen primed DISPERSED CELLS • Plasma LH estimation by stimulation of testosterone synthesis - on isolated Leydig cells 14
  • 15. Types of Bioassay QUANTAL ASSAY GRADED ASSAY INDIRECT ASSAY DIRECT ASSAY
  • 16. Quantal assay  Quantal response - the response is in the form of "all or none", i.e. either no response or maximum response  Drugs producing quantal effect can be bioassayed by end point method 17
  • 17.  The threshold dose producing a predetermined effect is measured  Comparison between the results of standard and the test  E.g: Bioassay of digitalis in cats,Insulin induced hypoglycemic convulsions in rat Threshold dose of the Std X Conc of Std Threshold dose of the Test Conc of Unknown =
  • 18. Graded assay  Graded response - response is proportional to the dose and response may lie between no response and the maximum response.  Types: • Bracketing /direct matching • Interpolation • Multiple point assays Three point assay Four point assay Six point assay • Cumulative dose response 19
  • 19. Graded DRC 20 standard Test/unknown
  • 20. 21 DRC & Log DRC 70% 30% Sigmoid curve Wide range of doses can plot  Rectangular hyperbola • Potency • Efficacy • Slope of curve
  • 21. 22
  • 22. Bracketing or Direct Matching  A constant dose of the standard is bracketed by varying dose of test sample  until an exact matching between the response of std & that of the sample is achieved  Strength of unknowm/test drug can be found by simple interpolation of bracketed response. 23
  • 23. 24
  • 24. Bracketing or Direct Matching 25
  • 25. ADVANTAGES  Simple & Faster  Amount of test drug available is small  Does not involve complicated calculations  Does not depend on DRC 26
  • 26. Disadvantages  less accurate,time consuming, troublesome  cannot get exact match of response  quantitative difference b/w test & std not obtained
  • 27. Interpolation assay  A log dose-response curve is plotted with the standard on a simple graph paper or Semi-log paper  The concentration of the test is then read from the graph 28
  • 29. Interpolation 100 % standard R E S 50 P O N S E 0 x x1 LOG DOSE
  • 30. Advantages  Sensitivity of tissue is 1st determined by prior plotting of a conc-response curve with known agonist  Dose can be plotted even if it varies over thousand fold range  Error is normally distributed
  • 31. Disadvantages  Sensitivity of tissue changes with time  Timing of doses not taken into account  Variation in mode of application of drugs
  • 32. Multiple point assays  Responses are repeated several times and the mean of each is taken  Chances of error are minimized  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 bias 33
  • 33. 34 t s1 s2 s1 s2 t s2 t s1 3 - point assay 3 cycles
  • 34. 3-POINT ASSAY % R E S P O N S E LOG DOSE 35 s1 T t S2 s2 S1
  • 35. Latin square design s1 s2 t t s1 s2 s2 s1 t t s2 s1
  • 36. CALCULATION • Mean responses of these 3 sets plotted • Log potency ratio (M) = (T-S1÷ S2-S1)× log d where, d – dose ratio = s2/s1 • Strength of unknown = s1/t × antilog of M 37
  • 37. 38 4 - POINT ASSAY
  • 38. 4-POINT ASSAY % R E S P O N S E LOG DOSE 39 s1 T1 t1 S2 s2 S1 T2 t2
  • 39. Latin square design s1 s2 t1 t2 s2 t1 t2 s1 t1 t2 s1 s2 t2 s1 s2 t1
  • 40. 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
  • 41. Six point assay  3+3 dose assay  3 conc each of std & test drug are used  6 sets of experiments using 6 doses in each set  More time consuming,lesser in use  Reliability is excellent
  • 42. Cumulative Dose Response Curve  Increase conc of drug in bath fluid step by step without washing out the preceeding doses  Continue till supramaximal effect is seen  Dose response curve is plotted
  • 44. 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 45
  • 45.  Determination of potency, ED50/LD50 of drugs  New drug development  Measure clinical effectiveness 46
  • 46. Drawbacks • Biological variation • Troublesome • Time consuming • Expensive • Less accurate than physico-chemical methods 47
  • 47. HUMAN TISSUE BIOASSAY • Veins [surgery on varicose veins] • Tissues like larger blood vessels obtained during amputation • Organs removed during transplantation/ tumor surgeries/procedures requiring resection • Tissues collected Postmortem 48
  • 48. Conclusion  Successful tool in estimation & discovery of biologically active substances  Sensitivity & Specificity – important tool in pharmacology

Editor's Notes

  1. The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O. In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc
  2. Repeatability: Same observation using same instruments and operators, and over short time periods Reproducibility: Same observation using different instruments and operators, and over longer time periods
  3. Estrogens – Ovariectamised Female Rat / vaginal cornification
  4. . bioassay of digitalis in cats - Here the cat is anaesthetized with chloralose and its blood pressure is recorded. The drug is slowly infused into the animal and the moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done and then potency is calculated
  5. Initial portion of curve is so steep that it is impossible to guage the magnitude of response corresponding to small increase in dose When the response reaches max, large increase in dose required to produce small change in response In arithmetic scale, it is difficult to display wide dose range
  6. Biological standardization – comparison & adjustment of strength of the sample with that of std under controlled conditions
  7. Limitation of animal tissues: Species variable-cannot predict actual outcome in relation to the human