Lecture includes definition of bioassay, Types of Assay and Bioassay , Indications, principles, advantages of bioassay. Example of a Bioassay with calculations. This lecture will be of help for postgraduate pharmacology students as well as undergraduates

1. Bioassay & DRC
Dr Naser Ashraf Tadvi
Associate Professor

2. Assay
• Analysis done to determine the presence of a
substance & amount of that substance
(Known or unknown) in biological fluids is
known as Assay.
• Determines Activity and potency of drug

3. Types of Assays
• Biological Method (Bioassay)
• Physico-chemical methods
– Chromatography, spectrophotometry
• Radioimmunoassay
• Microbiological methods

4. Bioassay
• Measurement of the concentration or potency
of a drug from magnitude of its main
biological effect
Main Pharmacological effect of drug
Compared with
Pharmacological effect of standard drug
Potency ratios are compared quantitatively

5. Indications(Uses) of Bioassay
• Measure pharmacological activity of new/
chemically undefined substance
• Measure concentration of known substance in
tissue extract or body fluid e.g Ach, histamine
• Measure ED50 / LD50 of a drug
• Biological standardization of natural drugs
which cannot be obtained in chemically pure
form e.g Vasopressin, oxytocin

6. Parameters recorded
• Blood pressure (Dogs and cats):
Sympathomimetics
• Ach: contraction of rabbit duodenum or frog
rectus abdominis
• Skeletal muscle relaxants: rabbit head drop
method
• Histamine: Guinea pig ileum
• Serotonin on rat colon

7. Principles of Bioassay
• Experimental conditions should be constant
• Standard should be in pure form and known
conc. Should be available
• Unknown and standard drug should have
same mode of action and pharmacological
effects
• Use a specified pharmacological technique like
rectus muscle preparation for Ach
• System should be insensitive to other drugs

8. Characteristics of a good bioassay
• Sensitivity - ability to detect smallest
concentration
• Specificity – the response which is being
measured should specific.
• Reproducibility - same observations using
different instrument and operator, over longer
time periods.
• Stability – sensitivity of preparation should be
constant.
• Availability – the particular tissue should be easily
available

9. BIOASSAY Can be performed
IN VIVO
-On intact animal
IN VITRO
-Isolated tissues
-Specific cells
-Organisms in culture

10. Advantages of isolated tissue experiments over
intact animal experiments
Several preparations can be obtained from a
single animal.
Small amount of the test material is required.
Cheap, less time consuming.
Interference due to pharmacokinetic factors
and compensatory reflexes is avoided.

11. Assembly for recording contraction of isolated
tissue
Organ Bath:
• Rudolph Magnus was first to design organ bath.
Outer organ bath: made up of perplex with electrical
thermostat and stirrer for uniform temperature.
Inner organ bath: made of glass , capacity of bath is
30ml.

12. Assembly for recording contraction of isolated tissue
• Lever:
Simple lever is used.
Stylus

13. Magnification and Adjustment
Magnification for Frog rectus is 5-10
Fast contracting- low
Slow contracting- high
BA
A
B

14. Role of Aeration / Aeration tube
1). Provides oxygen/ air to mounted tissue.
2). Allows uniform distribution of drug in organ bath
through bubbles.
3). Areation tube helps in mounting tissue in organ
bath.
Aeration – 30-40 bubbles / min (air)

15. Assembly of instruments for bioassay
Mariottes Bottle
Sherringtons starling drum
Simple writing lever
Aeration tube
Inner Organ bath
Outer Organ bath
Kymograph

16. 1 gm weight is added
Writing lever
Assembly of instruments for bioassay

17. Dissection and mounting the tissue
Procedure:
 A pithed frog is laid on its dorsal surface in a dissection tray.
 The skin of the abdomen cut to expose the whole abdomen.
 The rectus muscle is identified, and the border of the muscle
dissected out from adjacent abdominal wall.
 A thread is passed with the help of needle through the pubic
attachment of muscle and tied ,muscle is cut.
 The upper end of muscle i.e, xiphisternum part also tied and cut.
 The lower end of the muscle is tied to aeration tube at ‘s’ bent.

18. Pithing and Dissection of Frog

19. Dissection and mounting the tissue
 Before mounting the tissue make sure the blood vessels are
removed.
 Aeration tube with mounted tissue is placed in inner organ bath
filled with PSS.
 The upper end of muscle is tied to lever with thread.
 A load of 1gm is attached to the other side of lever at same
distance as that of tissue from the fulcrum. This helps the muscle
to relax in between contraction.
 Tissue is aerated with air i.e 30-40 bubbles /min. and allowed to
relax for 30- 45 min before recording responses.

20. Mounting the tissue

21. Acetyl choline Dilutions
 Stock solution- 1 mg/ml Acetylcholine(1000μg/ml)
The dilutions are made as follows-
1 ml stock soln. +9 ml distilled water= 1/10 dilution(100 μg/ml)
1 ml 1/10 dilution +9 ml distilled water= 1/100 dilution(10 μg/ml)
1 ml 1/100dilution +9 ml distilled water= 1/1000 dilution(1μg/ml)

22. Recording cycle
 Base line:30 seconds(s) (start drum & allow to run freely)
 Contractions: 90 s (contact period of Ach with tissue)
 Relaxation: 3 minutes (tissue washed 2-3 times after 90
sec contact period)
 Total cycle is 5 mins
 Soon after contraction of 90 secs the weight of 1gm is
lowered down to allow muscle to relax slowly, until lever
comes to base line.
 The contractions are recorded with each dose of solution
till ceiling effect is obtained i.e no further increase in
height of contraction with subsequent doses.

23. Recording cycle
 After the ceiling effect is obtained with standard Ach
solution, the procedure is repeated with the test
solution(unknown strength of Ach) in different dilutions.
 The further experiment is done by Multiple point assay
i.e. 2+1 assay
Sensitivity of Rectus muscle can
be increased by adding
Physostigmine(0.5μg/ml)/ Alcohol to perfusion
fluid

24. Types of Bioassay
• A. Quantal (Direct end point) Assay
• B. Graded Assay
-Bracketing assay
-Matching assay
-Interpolation assay
-Multiple point assay

25. Quantal response (All or none assay)
• In this assay, dose of standard and unknown
which provide predetermined ‘all or none’
response, then their potency ratios compared.
Ex: Digitalis induced cardiac arrest in cats, insulin
induced hypoglycemic convulsions in mice.

26. Quantal response (All or none assay)

27. Graded response assay

28. Multiple Point Assay
• Bracketing and Matching assay methods are
not ideal because lack of accuracy, sensitivity
• So , to minimize those errors multiple point
assays preferred (3,4 or 6 point assays)

29. 0.2 100
25.612.86.43.21.6 51.20.80.4 10
U
400
200 40
U
20
U
80
U
16
U
32
U
64
U
S2S1
S1
S1
S1S2
S2
S2
t
t
t
t
2+1 Bioassay of Ach on Isolated Frog
Rectus Muscle
S2S1
T

30. 6
Dose response curve

31. Test 1/10
10 U
80 U40 U20 U
Test SS
64 U32 U16 U
Dose response curve of test solution
in graded doses

32. Latin square
S1 S2 t
t S1 S2
S2 S1 t
X

33. Calculations
Response Height of
contraction
(mm)
Mean height
(mm)
Dose (ml)
1 2 3
S1 34 32 37 35.2 0.1
S2 67 65 80 72.7 0.2
T 42 42 43 40.7 0.32
Dose ratio(d) = = = 2
S1
S2 0.2
0.1

34. • From data obtained log potency ratio (M) is calculated from the
formula:
M = × log d
M = × log 2
M = × 0.301 = 0.047
Calculations
T – S1
S2 – S1
41 - 35
73 - 35
6
38

35. • From M ,the strength of the unknown can be calculated –
Strength of = × antilog of M
test solution
= × 1.114
= 0.348125 mg/ml
= 348 μg/ml
The given concentration was 300 μg/ml , so the error is 16%
Percentage error(%)= × 100
Calculations
S1
t
0.1
0.32
ACT
ACT - OCT

36. Calculation
DRUG DOSE (μg) RESPONSE (mm) LOG DOSE
2 0 0.30103
4 0 0.60206
8 0 0.90309
16 4 1.20412
32 8 1.50515
64 24 1.80618
128 30 2.10721
256 40 2.40824
512 42 2.70927
1024 45 3.0103
2048 45 3.31133

37. Dose Response Curve
-10
0
10
20
30
40
50
0 500 1000 1500 2000 2500
RESPONSE (mm)
DOSE (μg)
RESPONSE(mm)

38. Calculation of ED50

39. LDR CURVES OF 2 DRUGS WITH
DIFFERENT POTENCY

40. EFFICACY & POTENCY

41. FREQUENCY DISTRIBUTION CURVE

42. Therapeutic Index
ED50 LD50

43. Certain Safety Factor

44. Therapeutic window

45. Matching/ Bracketing Assay

46. Matching/ Bracketing Assay

47. Matching/Bracketing Assay
Advantage :
 Faster
 Can be completed when amount of test drug
available is small
 Does not involve complicated calculations
Disadvantage:
 Match is subjective
 Exact match may not always be possible

48. Interpolation method

49. Interpolation method

50. Thank You