Shariq bioassay

Bioassy

1. Bioassay Syed Shariq Naeem

2. Outline Types of assays Introduction Definition Indication and principles of bioassay Basic procedure Calculations Source of errors Summary

3. Types of Assays Biological assays Chemical assays: Spectrophotometer, Spectrofluorimetry, Chromatography, Radio Immunoassays Microbiological assays

4. Introduction Late 18 th centaury- standardization of Diphtheria antitoxin by Paul Ehrlich Bioassay literal meaning Bio – living tissue Assay- assessment / measurement Bioassay: Assessment of a biological substance

5. 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

6. 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

7. 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.)

8. Procedure Prepare the physiological salt solution Arrange the instrument and adjust the water bath. Balance the lever Tissue selection Surgical process and collection of required tissue. Tissue attachment to the water bath Relaxation time given to the tissue Prepare the standard drug( serial dilution) Select the lowest possible measurable concentration by trial and error method. Prepare DRC for the standard drug. Prepare DRC for the test drug.( serial dilution) Select a assay method (3 point or 4 point assay) Calculation

9. Step 1: Prepare the physiological salt solution

10. Various Physiological salt solutions Frog-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 MgCl ² . 6H ² O --- 1.1 g 1.0 g --- --- --- NaH 2 PO 4 . H ² O 0.1 g 1.4 g 0.5 g --- --- 1.4 g NaHCO ³ 2 g 21 g 10 g 1.5 g 5 g 21 g CaCl ² 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 O ² + 5%C O ² O ² or air Pure O ² O ² + 5% CO ² O ² + 5% CO ² For 10 litres pH- 7.3-7.4 Calcium chloride to be added last. Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.

11. 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

12. Electrolytes Ingredients Functions NaCl Maintain osmolarity K + Nerve conduction, muscle contraction, maintain heart rate & rhythm Ca + Contraction Mg+ Neurotransmission , decrease spontaneous activity NaHCO ³ & NaH 2 PO 4 Buffer Glucose Nutrient

13. 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

14. 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)

15. 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.

16. 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

17. Magnification : = Distance from the fulcrum to the writing point Distance form the fulcrum to the tied tissue For slow contracting muscles:- 10-15 times For fast contracting muscles:-5-10 times

18. Step:4-Tissue selection S.No Compound Tissue used 1. Acetylcholine Guinea-pig ileum Frog rectus abdominis muscle Leech dorsal muscle Rat uterus preparation Isolated guinea-pig auricles 2. Serotonin Isolated oestrous uterus of rat Isolated fundic strip of rat Guinea pig ileum Rabbit ear preparation Isolated heart of the mollusc Venus mercenaria

19. 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 colon Non pregnant rat uterus Rat fundus Rabbit aortic strip Rabbit jejunum Tracheal chain of guinea pig

20. 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.

21. 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

22. Aeration Pure oxygen ( O2 ) For heart Air For intestine Carbogen ( 95% O2 & 5% CO2 ) For uterus Mixing of the test drug Homogenisation of the solution Keeping the tissue lumen patent To maintain pH ( aeration by pure O2 causes losing of CO2 & solution becomes alkaline )

23. 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

24. 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 amount Giving a antagonist E.g. Acetylcholine for blocking spontaneous contraction of ileum.

25. Step 8: Prepare the standard drug ( serial dilution) Serial dilution : 10---10 -9

26. 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

27. Time cycle Contact time Time 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 Start the kymograph 2 Add the Acetylcholine 2.5 Stop the kymograph & wash the preparation 10 Wash the preparation 15 Start the kymograph

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

29. 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

30. [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

31. 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

32. Multiple Point Assays 3 point assay 4 point assay

33. 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

34. 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]

35. Step 11: Calculation Calculate the height of each response. Take mean of all S1, S2, T1 and T2 values. Plot a graph

36. S1 S2 T1 T2 D1 D2 M

37. S1 S2 T1 T2 D1 D2

38. 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

39. 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

40. 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

41. Strength of test solution = s1/t1 × antilog of M Dilution of the inner water bath has to be taken in to account

42. Calculation of the percentage error:- Percentage error = ACT-OCT × 100 ACT Where, ACT = Actual concentration of test OCT = Observed concentration of test The permissible limit of percentage error is <10%

43. Errors in bioassays Margin of error of bioassay should be < 10% Two types:- Biological variation: Methodological variation

44. Biological variation:- Variation in response to a drug. Down regulation of receptor (repeated washing of tissue) Loss of tissue sensitivity (change the tissue) Laboratory condition may be variable.

45. Methodological variation :- Human error: done by the experimenter Experimental error: faulty procedure selection or calibration error.(proper balancing the lever, and by maintaining the ph and temperature at a physiological level.)

46. Reasons for methodological error: Lack of standardization of procedure Over handling of tissue Preparation of physiological salt solution. Drug preparation or in dilution

47. Summary

48. Thank you

49. Summary Prepare the physiological salt solution Arrange the instrument and adjust the water bath. Balance the lever Tissue selection Surgical process and collection of required tissue. Tissue attachment to the water bath Relaxation time given to the tissue Prepare the standard drug( serial dilution) After relaxation test any concentration of the drug Then standardize the tissue response with same drug. ( take subsequent two response) Select the lowest possible measurable concentration by trial and error method. Prepare DRC for the standard drug. Prepare DRC for the test drug.( serial dilution) Select a assay method (3 point or 4 point assay) Measure the height of each response Calculation

50. Time cycle Time ( mins ) Event 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 time Time allowed for the drug (agonist) to remain in contact with the tissue Frog rectus abdominis muscle Guinea-pig ileum 90 sec 30 sec

51. 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

52. 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