BioassaySyed Shariq Naeem
Outline•   Types of assays•   Introduction•   Definition•   Indication and principles of bioassay•   Basic procedure•   Ca...
Types of Assays• Biological assays• Chemical assays:  – Spectrophotometer,  – Spectrofluorimetry,  – Chromatography,• Radi...
Introduction• Late 18th centaury- standardization of  Diphtheria antitoxin by Paul Ehrlich• Bioassay literal meaning  o Bi...
Definition Comparative assessment of relative potency of  a test compound to a standard compound on a  living or biologic...
Indications for Bioassay• Active principle of drug is unknown• Active principle cannot be isolated, e.g. insulin, posterio...
Principles of bioassay• Bioassay involves the comparison of the main  pharmacological response of the unknown preparation ...
Procedure1.  Prepare the physiological salt solution2.  Arrange the instrument and adjust the water bath.3.  Balance the l...
Step 1: Prepare the physiological salt              solution
Various Physiological salt solutionsFor 10 litres     Frog-      Kreb’s      Tyrode      Ringer-     De         McpH- 7.3-...
Uses: Physiological salt SolutionsPhysiological salt   UsessolutionsFrog-Ringer          Amphibian tissue preparationKreb’...
ElectrolytesIngredients          FunctionsNaCl                 Maintain osmolarityK+                   Nerve conduction, m...
Step 2: Arrange the instrument and adjust              the water bath. Kymograph: Sherrington- starling  kymograph    To...
Student Organ bath• Outer bath:-    First designed by rudolph     magnus    Perpex glass    Store water outside the   ...
• Tissue holder and oxygen supply:-   Tissue is attached inside the inner water bath to a    tissue holder.   Also suppo...
Step:3 -Balance the lever• Lever:  Three basic parts:     • Effort arm- where force in       applied     • Load arm- wher...
• Magnification :  = Distance from the fulcrum to the writing point    Distance form the fulcrum to the tied tissue  o For...
Step:4-Tissue selectionS.No   Compound        Tissue used1.     Acetylcholine   Guinea-pig ileum                       F...
S. No   Chemical         Tissue used3.      Histamine           Guinea pig isolated ileum                            Gui...
Step 5: Surgical process and collection              of required tissue.•    Animal sacrificed by cervical dislocation.•  ...
Step 6 : Tissue attachment to the                water bath• Attach the ends of the tissue:-   – One end:- tissue holder  ...
AerationPure oxygen (O2 )            For heartAir                          For intestineCarbogen ( 95% O2 &          For u...
Temperature Rabbit intestine             Physiological temp.(37°C ) is needed for                              mammalian t...
Step 7:Relaxation time given to the                    tissue1.        Intestine       30-45 min2.        Frog rectus     ...
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 th...
Time cycle                          StartTime (     Event                                            kymogarp             ...
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  ...
[2] Graded Response Assays [ Direct comparison               on same tissues] Interpolation:  Conc. of unknown is   read ...
Matching & Bracketing: Const dose bracketed with varying doses of standard till  exact match is obtained   • Used when t...
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...
3 point assay [2+1 dose assay]• Fast & convenient• Procedure [E.g. Ach bioassay]    Log dose response [LDR] curve plotted...
Step 11: Calculation• Calculate the height of each response.• Take mean of all S1, S2, T1 and T2 values.• Plot a graph
T2                             S2               MT1                   S1     D1                 D2
T2                         S2T1               S1     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...
Log potency ratio :• The horizontal separation M of the two curves  represents the log potency ratio of the  concentration...
Direct calculations• M={(T1-S1) +(T2 –S2)}/{(S2-S1) +(T2-T1)}×log d• Log d = log[s1/s2]Where,• M        = Potency of the d...
• 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                        ACT  Where,  • ACT = Actual...
Errors in bioassays• Margin of error of bioassay should be < 10%• Two types:-  1. Biological variation:  2. Methodological...
• Biological variation:-  1. Variation in response to a drug.  2. Down regulation of receptor (repeated washing     of tis...
• Methodological variation:-  1. Human error: done by the experimenter  2. Experimental error: faulty procedure selection ...
• Reasons for methodological error:  1.   Lack of standardization of procedure  2.   Over handling of tissue  3.   Prepara...
SummaryPrepare Physiological sol.   Check instruments       Tissue collection and mounting          Relaxation            ...
Thank you
Summary1.    Prepare the physiological salt solution2.    Arrange the instrument and adjust the water bath.3.    Balance t...
Time cycleTime ( mins)   Event      0        Raise the 1 gm weight & start the kymograph      2        Add acetylcholine  ...
Principles of Bioassay• Active principle to be assayed should show the same  measured response in all animal species• The ...
Biological objectsWhole     Isolated organ Isolated        Isolatedanimal                   tissue          cellsAssay of ...
Shariq bioassay
Shariq bioassay
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  1. 1. BioassaySyed Shariq Naeem
  2. 2. Outline• Types of assays• Introduction• Definition• Indication and principles of bioassay• Basic procedure• Calculations• Source of errors• Summary
  3. 3. Types of Assays• Biological assays• Chemical assays: – Spectrophotometer, – Spectrofluorimetry, – Chromatography,• Radio Immunoassays• Microbiological assays
  4. 4. Introduction• Late 18th centaury- standardization of Diphtheria antitoxin by Paul Ehrlich• Bioassay literal meaning o Bio – living tissue o Assay- assessment / measurement o Bioassay: Assessment of a biological substance
  5. 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. 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. 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. 8. Procedure1. 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
  9. 9. Step 1: Prepare the physiological salt solution
  10. 10. Various Physiological salt solutionsFor 10 litres Frog- Kreb’s Tyrode Ringer- De McpH- 7.3-7.4 Ringer Locke Jalon EwenNaCl 65 g 69 g 80 g 91.5 g 90 g 76 gKCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 gMgCl². 6H²O --- 1.1 g 1.0 g --- --- ---NaH2PO4. H²O 0.1 g 1.4 g 0.5 g --- --- 1.4 gNaHCO³ 2g 21 g 10 g 1.5 g 5g 21 gCaCl² 1.2 g 2.8 g 2g 2.4 g 0.6 g 2.4 gGlucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 gAerating Gas air O² + O² or air Pure O² O² + O² + 5% 5%CO² 5% CO² CO²•Calcium chloride to be added last.•Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.
  11. 11. Uses: Physiological salt SolutionsPhysiological salt UsessolutionsFrog-Ringer Amphibian tissue preparationKreb’s Mammalian/Avian skeletal muscle preparationTyrode Intestine preparationRinger-Locke Heart muscle preparationDe Jalon Rat uterus preparation
  12. 12. ElectrolytesIngredients FunctionsNaCl Maintain osmolarityK+ Nerve conduction, muscle contraction, maintain heart rate & rhythmCa + ContractionMg+ Neurotransmission , decrease spontaneous activityNaHCO³ & NaH2PO4 BufferGlucose Nutrient
  13. 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. 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. 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. 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. 17. • Magnification : = Distance from the fulcrum to the writing point Distance form the fulcrum to the tied tissue o For slow contracting muscles:- 10-15 times o For fast contracting muscles:-5-10 times
  18. 18. Step:4-Tissue selectionS.No Compound Tissue used1. Acetylcholine Guinea-pig ileum Frog rectus abdominis muscle Leech dorsal muscle Rat uterus preparation Isolated guinea-pig auricles2. 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. 19. S. No Chemical Tissue used3. Histamine  Guinea pig isolated ileum  Guinea pig tracheal chain.  Fall in BP of dog/cat4. Adrenaline and Rat colon noradrenalin Non pregnant rat uterus Rat fundus Rabbit aortic strip Rabbit jejunum Tracheal chain of guinea pig
  20. 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. 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. 22. AerationPure oxygen (O2 ) For heartAir For intestineCarbogen ( 95% O2 & For uterus5% CO2 ) 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. 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 temperatureTemperature should be constant through out the experiment
  24. 24. Step 7:Relaxation time given to the tissue1. Intestine 30-45 min2. Frog rectus 45-60 min Measures to decrease spontaneous contraction:- Hanging a weight of appropriate amount Giving a antagonist oE.g. Acetylcholine for blocking spontaneous contraction of ileum.
  25. 25. Step 8: Prepare the standard drug ( serial dilution) • Serial dilution: 10---10-9
  26. 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. 27. Time cycle StartTime ( Event kymogarp hmin ) 0 Start the kymograph Wait for 2 Add the Acetylcholine 11.5 Add Ach min 2.5 Stop the kymograph & wash the preparation 10 Wash the preparation Wash Stop 15 Start the kymograph preparati kymograp on h Contact time Time allowed for the drug (agonist) to remain in contact with the tissueFrog rectus abdominis muscle Guinea-pig ileum90 sec 30 sec
  28. 28. Step 10: Perform a assay (3 or 4 point assay)
  29. 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. 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. 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. 32. Multiple Point Assays • 3 point assay • 4 point assay
  33. 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. 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. 35. Step 11: Calculation• Calculate the height of each response.• Take mean of all S1, S2, T1 and T2 values.• Plot a graph
  36. 36. T2 S2 MT1 S1 D1 D2
  37. 37. T2 S2T1 S1 D1 D2
  38. 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. 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. 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. 41. • Strength of test solution = s1/t1 × antilog of M• Dilution of the inner water bath has to be taken in to account
  42. 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. 43. Errors in bioassays• Margin of error of bioassay should be < 10%• Two types:- 1. Biological variation: 2. Methodological variation
  44. 44. • 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.
  45. 45. • Methodological variation:- 1. Human error: done by the experimenter 2. Experimental error: faulty procedure selection or calibration error.(proper balancing the lever, and by maintaining the ph and temperature at a physiological level.)
  46. 46. • Reasons for methodological error: 1. Lack of standardization of procedure 2. Over handling of tissue 3. Preparation of physiological salt solution. 4. Drug preparation or in dilution
  47. 47. SummaryPrepare Physiological sol. Check instruments Tissue collection and mounting Relaxation Prepare DRC and Do a 4 point assay
  48. 48. Thank you
  49. 49. Summary1. 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
  50. 50. Time cycleTime ( 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 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. 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. 52. Biological objectsWhole Isolated organ Isolated Isolatedanimal tissue cellsAssay of Assay of Assay of Assay ofinsulin in gonadotropins oxytocin on antibioticsrabbits on ovary isolated on bacterial uterine cells tissue
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