Bioassay is a method used to determine the presence and amount of a substance in a biological system. There are several types of bioassays including those using isolated tissues, intact animals, and specific cells or organisms. When performing a bioassay on an isolated tissue like the frog rectus muscle, the tissue is dissected, mounted, and contractions are recorded in response to increasing doses of a standard drug like acetylcholine. The potency of an unknown substance can then be determined by comparing its effects to the standard drug using methods like multiple point bioassay.

1. Dr Agboola, Samuel S.
BIOASSAY

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

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

4. • 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
Bioassay

5. • 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 or LD50 of a drug
• Biological standardization of natural drugs
which cannot be obtained in chemically pure
form e.g Vasopressin, oxytocin
Indications(Uses) of Bioassay

6. • 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
Parameters recorded

7. • Experimental conditions should be constant
• Standard should be in pure form and known
concentration 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
Principles of Bioassay

8. • Sensitivity - ability to detect smallest
concentration
• Specificity – the response which is being
measured should be 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
Characteristics of a good bioassay

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

10. 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.
Advantages of isolated tissue experiments over
intact animal experiments

11. 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.
Assembly for recording contraction of isolated
tissue

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

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

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

15. Mariottes Bottle
Sherrington’s starling
DrumSimple writing lever
Aeration tube
Inner Organ bath
Outer Organ bath
Kymograph
Assembly of instruments for bioassay

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

17. 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.
Dissection and mounting the tissue

18. PithingandD
issectionofFrog

19.  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.
Dissection and mounting the tissue

20. Mounting the tissue

21.  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)
Acetyl choline Dilutions

22.  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.
Recording cycle

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) to perfusionfluid

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

25. • 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.
Quantal response (All or none assay)

26. Quantal response (All or none assay)

27. Graded response assay

28. • 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)
Multiple Point Assay

29. Matching/ Bracketing Assay

30. Matching/ Bracketing Assay

31. 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
Matching/Bracketing Assay

32. Interpolation method

33. Interpolation method