Principles & types of bioassay
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The document discusses principles and types of bioassay. It begins by defining a bioassay as a comparative assessment of the potency of a test compound to a standard compound on living tissue. It then outlines the principles of bioassays, including using a standard and test sample with the same mechanism of action and comparing them using a reproducible pharmacological technique. The document categorizes bioassays as either graded, involving measuring a biological response on a scale, or quantal, involving measuring an endpoint response. It provides details on specific graded bioassay methods and discusses quantal bioassays, LD50 determination, and bioassays of antagonists.
![Four Point Assay
19
M = [T1 – S1 + T2 - S2] x log s2
S2 – S1 + T2 - T1 s1
T (concentration) = s1 x antilog M
t1
Six Point & Eight Point Assay](https://image.slidesharecdn.com/principlestypesofbioassay-180309134847/85/Principles-types-of-bioassay-19-320.jpg)
Principles & types of bioassay
- 1. Principles and Types of Bioassay DR. SAHIL KUMAR
- 2. Outline Introduction Indications of Bioassay Principles of Bioassay Classification of Bioassay: Graded & Quantal Bioassay of antagonist Advantages and Disadvantages of Bioassay Error in Bioassay Human Tissue Bioassay Conclusion
- 3. INTRODUCTION What is a Bioassay? Comparative assessment of relative potency of a test compound to a standard compound on a living tissue. Qualitative identification & Quantitative measurement of the amount of active principle in pharmaceutical preparation or biological material. Measurement of conc. of a drug from magnitude of its biological effect.
- 4. Is Bio-standardization same as Bioassay? Historical aspect: Paul Ehrlich – Bio-standardization of Diphtheria antitoxin. INTRODUCTION
- 5. INDICATIONS OF BIOASSAY Active principle unknown. Active principle cannot be isolated. To study biological response of new drug. To ensure purity & potency. If chemical assay not available/ complex/ insensitive to low doses. To estimate concentration of endogenous mediators.
- 6. PRINCIPLES OF BIOASSAY Compare potency of unknown substance with standard (including assessment of errors). Standard & test sample should have same pharmacological effect & mode of action.
- 7. The test and standard should be compared using a specified pharmacological technique. Method selected should be sensitive, reproducible & should minimize errors d/t biological variations & methodology. PRINCIPLES OF BIOASSAY
- 8. TYPES OF BIOASSAY Quantal Direct end point assay (DEPA) LD50 determination Graded Matching Bracketing Interpolation Multiple point 8
- 10. METHODOLOGY: Graded Bioassay 1) Checking of apparatus for proper functioning. 2) Prepare Physiological Salt Solution 3) Arrange the instrument and adjust the water bath. 4) Balance the lever 5) Tissue selection 6) Surgical process and collection of required tissue. 7) Tissue attachment to the water bath 8) Relaxation time given to the tissue 9) Prepare the standard drug (serial dilution) 10) Select lowest possible measurable conc. 11) Prepare DRC for the standard drug. 12) Prepare DRC for the test drug. (serial dilution) 13) Select an assay method (3 point or 4 point assay) 14) Calculation
- 11. Graded Bioassay Intermittent dose method Cumulative dose method 0.2ml 0.4ml 0.8ml 1.6ml 3.2ml 11
- 12. Matching Assay 12 •Adv: Test DRC not reqd., small vol., fast. •Disadv: Trial & Error method, poor precision.
- 14. Interpolation Assay 14 Conc. of unknown is read from a standard plot of a log dose response curve.
- 15. Three Point Assay 15 S1 S2 T S2 T S1 T S1 S2
- 16. Three Point Assay •Mean responses of three sets taken. •Potency ratio calculated. M = T – S1 x log s1 S2 – S1 s2 •Strength of test solution = s1 x antilog M t 16
- 17. Four Point Assay 17 S1 S2 T1 T2 S2 T1 T2 S1 T1 T2 S1 S2 T2 S1 S2 T1
- 18. 18
- 19. Four Point Assay 19 M = [T1 – S1 + T2 - S2] x log s2 S2 – S1 + T2 - T1 s1 T (concentration) = s1 x antilog M t1 Six Point & Eight Point Assay
- 20. Multiple point assays: •Adv: Reduced error, reduced variability. •Disadv: Lengthy, Large amount of test sample required. 20
- 21. QUANTAL BIOASSAY
- 22. 22 •Threshold dose producing a required response is measured on each animal. •Eg. Bioassay of Digitalis in Cats, Hypoglycemic convulsions in mice. •Threshold dose = Period of infusion X Rate. Direct End-Point Assay
- 23. LD50 Determination: Graphical & Arithmetic methods
- 24. Bioassay of Antagonist Determination of the type of drug antagonism: Parallel shift of the log DRC. Double reciprocal (Lineweaver & Burk) plot Schild Plot and pA2 value.
- 26. Advantages Chemical assay too complex. If difference b/w results; bioassay given more importance. Toxicity of new drug. Time consuming. Requires much skill. Biological variations exist. Disadvantage s
- 27. Errors in bioassays Biological variation • Loss of tissue sensitivity. • Different species/sex/age/weight/health status. • Laboratory condition may be variable. • Housing and handling of animals. Methodological error • Lack of standardization of procedure. • Set-up of apparatus. • Tissue isolation/preparation for experiment. • Drug preparation or dilution.
- 28. Human Tissue Bioassay Animal tissues can’t predict accurate outcomes. Limitations: Ethical, costly, take time, cooperation of various specialties required, storage. Vascular tissue: Veins, cardiac blood vessels, large blood vessels after amputation. Cardiac tissue: Used fresh, stored at 4◦C, functional for 2 weeks. Brain tissue. Lung tissue.
- 29. Thank You
Editor's Notes
- #6: Chemical composition of drug is different but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc.
- #7: Principle means a fundamental concept.
- #11: Point # 10) Doses added in geometric progression Different dilutions of drug Doses added till plateau achieved Point # 11) Response is measured with the help of a ruler and percentage of maximal response is found out. Point #13) fixing with Shellac and colophony saturated with methanol.
- #12: What we usually do in our lab is Intermittent method
- #27: Advantages & Disadvantages of Bioassay
- #30: Conclusion