Bioassay

Syed Shariq Naeem

Outline

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

Types of Assays

• Biological assays• Chemical assays: – Spectrophotometer, – Spectrofluorimetry, – Chromatography,

• Radio Immunoassays• Microbiological assays

Introduction

• Late 18th centaury- standardization of Diphtheria antitoxin by Paul Ehrlich

• Bioassay literal meaningo Bio – living tissueo Assay- assessment / measuremento Bioassay: Assessment of a biological substance

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

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

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.)

Procedure 1. Prepare the physiological salt solution2. Arrange the instrument and adjust the water bath.3. Balance the lever4. Tissue selection5. Surgical process and collection of required tissue.6. Tissue attachment to the water bath7. Relaxation time given to the tissue8. 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

Step 1: Prepare the physiological salt solution

Various Physiological salt solutionsFrog-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 litrespH- 7.3-7.4

•Calcium chloride to be added last.•Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.

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

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

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

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)

• 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.

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

• Magnification := Distance from the fulcrum to the writing point

Distance form the fulcrum to the tied tissue

o For slow contracting muscles:- 10-15 timeso For fast contracting muscles:-5-10 times

Step:4-Tissue selection

S.No Compound Tissue used

1. Acetylcholine Guinea-pig ileumFrog rectus abdominis muscleLeech dorsal muscleRat uterus preparationIsolated guinea-pig auricles

2. Serotonin Isolated oestrous uterus of ratIsolated fundic strip of ratGuinea pig ileum Rabbit ear preparationIsolated heart of the mollusc Venus mercenaria

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 colonNon pregnant rat uterusRat fundusRabbit aortic stripRabbit jejunumTracheal chain of guinea pig

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.

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

Aeration

Pure oxygen (O2 ) For heart

Air For intestine

Carbogen ( 95% O2 & 5% CO2 )

For uterus

Mixing of the test drugHomogenisation of the solutionKeeping the tissue lumen patentTo maintain pH ( aeration by pure O2 causes losing of CO2 & solution becomes alkaline )

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

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 amountGiving a antagonist

oE.g. Acetylcholine for blocking spontaneous contraction of ileum.

Step 8: Prepare the standard drug( serial dilution)

• Serial dilution: 10---10-9

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

Time cycle

Contact timeTime 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 preparatio

n

Wait for 11.5 min

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

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

[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

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

Multiple Point Assays• 3 point assay

• 4 point assay

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

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]

Step 11: Calculation

• Calculate the height of each response.• Take mean of all S1, S2, T1 and T2 values.

• Plot a graph

S1

S2

T1

T2

D1 D2

M

S1

S2

T1

T2

D1 D2

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

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

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

• Strength of test solution = s1/t1 × antilog of M

• Dilution of the inner water bath has to be taken in to account

Calculation of the percentage error:-• Percentage error = ACT-OCT × 100

ACTWhere,• ACT = Actual concentration of test• OCT = Observed concentration of test

• The permissible limit of percentage error is <10%

Errors in bioassays

• Margin of error of bioassay should be < 10%

• Two types:-1. Biological variation: 2. Methodological variation

• 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.

• Methodological variation:-1. Human error: done by the experimenter2. Experimental error: faulty procedure selection or

calibration error.(proper balancing the lever, and by maintaining the ph and temperature at a physiological level.)

• Reasons for methodological error:1. Lack of standardization of procedure2. Over handling of tissue 3. Preparation of physiological salt solution.4. Drug preparation or in dilution

Prepare Physiological sol.

Check instruments

Relaxation

Prepare DRC and Do a 4 point assay

Summary

Thank you

Summary 1. Prepare the physiological salt solution2. Arrange the instrument and adjust the water bath.3. Balance the lever4. Tissue selection5. Surgical process and collection of required tissue.6. Tissue attachment to the water bath7. Relaxation time given to the tissue8. Prepare the standard drug( serial dilution)9. After relaxation test any concentration of the drug10. 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 response16. Calculation

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 timeTime allowed for the drug (agonist) to remain in contact with the tissue

Frog rectus abdominis muscle Guinea-pig ileum

90 sec 30 sec

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

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