It highlights the biological assays majorly requested for.

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