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bioassay.pdf
- 1. BIOASSAY By :Dr. Sumit Kumar Mahato Junior Resident( Academic) Guide: Dr. Uma Shanker Pd. Keshri Associate Professor Department of Pharmacology RIMS, Ranchi
- 2. An assay is an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology, and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity (the analyte) which can be a drug or biochemical substance or organic sample
- 3. Chemical Assay Immunoassay Bioassay
- 4. Chemical Assay: It is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Immunoassay: A technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample.
- 5. 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 Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin 5
- 6. Bioassay Bioassay Chemical Assay Less Precise More time consuming More expensive Active constituent & structure not known. More sensitive More men power Required Difficult to handle More Precise Less time consuming Less expensive Active constituent & structure fully established. Less sensitive Less men power required Easy to handle
- 8. To compare the test substance with the International Standard preparation of the same To find out how much test substance is required to produce the same biological effect, as produced by the standard Activity assayed should be the activity of interest 8
- 9. Standard & test sample - similar pharmacological effects & mode of action Both should be compared for their established pharmacological effect using specified technique Ex: *Ach – contractile response on frog rectus abdominis muscle *Histamine – contractile response on guinea pig ileum 9
- 10. Problem of biological variation must be minimized Experimental conditions - kept constant Animals - same species, sex and weight Number of animals - large enough to minimize error (individual variation) Isolated preparations - sensitive 10
- 11. No chemical method has been developed Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach) To measure the pharmacological activity of new or chemically undefined substances For biological standardization of drugs obtained from natural sources as these cannot be obtained in pure form. Eg: Oxytocin,Vasopressin,Insulin,Heparin.. 11
- 12. To compare the strength of a drug obtained from various sources due to different compositions Chemicals with similar structure, but different biological activity Chemical structure of the active principle is unknown Chemical structure known; cannot be actively purified. Eg: Peptide hormones 12
- 13. Sensitivity Specificity Repeatability Reproducibility Precision Accuracy Stability – tissue has to stay “bioassay- fit 13
- 15. WHOLE ANIMALS Nor Adrenaline – Cat Cardiac Glycosides – Guinea Pig Insulin – Mice Estrogens – Ovariectamised Female Rat ISOLATED TISSUE Acetyl Choline – Frog Rectus Abdominus muscle Histamine – Guinea Pig ileum Adrenaline – Rat uterus Oxytocin – Rat uterus oestrogen primed 15
- 16. Qualitative bioassay Is used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. Eg: Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available. Quantitative bioassays involve estimation of concentration/potency of a substance by measurement of the biological response it produces. These bioassays are typically analyzed using the methods of biostatistics
- 17. 1.Direct end point assay (DEPA) 2.Quantal assay (all or none assay) 3.Graded assay : a) Bracketting assay b) Matching assay c) Interpolation assay d) Multiple point assay ( 3-point, 4point, 6- point, 8-point).
- 18. Principle: is to measure direct response of dose of standard and test preparation. The threshold dose required for response is determined for each experimental unit. Ratio between these doses estimates the potency of the test preparation relative to the standard. Concentration of test= TDS/TDT×CSD Where, TDS= Threshold dose of standard TDT= Threshold dose of test CSD= Concentration of standard drug Threshold dose of standard = Total period of infusion × rate of drug administration
- 19. Advantage: Drug effects appear rapidly and are easily recognised Drug effect is directly proportional to drug dose Rapid end-point detection. Disadvantages: Only toxicity study or high dose study is possible Dose ranging study cannot be done
- 20. Quantal response – the unknown is compared with the standard with respect to potency which produces the quantal affect, i.e change is easily recognised sign or often death. In a quantal assay there is use of dose response relationship. quantal response to a drug is obtained and percentage of positive response at each dose is calculated. The response in quantal assay is varying, i.e some responses are irreversible and hence animal used is once and some responses have no permanent effect and animal can be used in next experiment e.g – Determination of LD50 Hypoglycemic convulsion in mice in the assay of insulin 20
- 21. Graded response - In these assays, as the dose increases there is an equivalent rise in response. The potency of a test agonist is determined by comparing its mean response to standard mean response. This process is also known as “analytical dilution assay” GRA is the simplest way of determining potency of a test drug because it does not require statistical analysis 21
- 23. 23 DRC & Log DRC 30% 70% Sigmoid curve Wide range of doses can plot Rectangular hyperbola • Potency • Efficacy • Slope of curve
- 24. 24
- 25. Used when test sample is too small. Comparison of potency between unknown and standard drug is done by trial and error method. Response is matched at only one dose Does not need dose respone curve of test compound It require most sensitive tissue Concentration of test= (Dose of standard/ Dose of test)× conc. Of standard Disadvantage: Experimental error is not excluded out There is no sign of parallelism as it lack dose response relationship.
- 26. Used when test sample is too small. Single or few responses is taken by using any test drug concentration. This response is bracketed between two responses one higher and one lower of the standard drug. The strength of the unknown can be found by simple interpolation of this bracketted response on dose axis
- 28. 28
- 29. less accurate,time consuming, troublesome cannot get exact match of response quantitative difference b/w test & standard not obtained
- 31. A log dose-response curve is plotted with the standard, Single or few responses of test drug are plotted. The dose of test drug which comes at the linear log dose- response is interpolated from the dose respone plot 31
- 33. Sensitivity of tissue is 1st determined by prior plotting of a conc-response curve with known agonist Dose can be plotted even if it varies over thousand fold range Error is normally distributed
- 34. Sensitivity of tissue changes with time Timing of doses not taken into account Variation in mode of application of drugs
- 35. Responses are repeated several times and the mean of each is taken Chances of error are minimized 3 point method - 2 doses of std+1 dose of test 4 point method - 2 doses of std+2 doses of test 6 point method - 3 doses of std+3 doses of test Latin square method of randomization to avoid any bias 35
- 36. 36 t s1 s2 s1 s2 t s2 t s1 3 cycles
- 38. s1 s2 t t s1 s2 s2 s1 t t s2 s1
- 39. • Mean responses of these 3 sets plotted • Log potency ratio (M) = (T-S1÷ S2-S1)× log d where, d – dose ratio = s2/s1 • Strength of unknown = s1/t × antilog of M S1, S2- length of standard dose response selected between 25-75% T-length of test dose response selected in between of two standard responses s2/s1 -standard drug dose which came in contact with tissueand given the response S1, S2. respectively t = Test drug dose which came in contact with tissu and given the response T 39
- 41. 41 4 - POINT ASSAY
- 43. s2 t1 t2 s1 t1 t2 s1 s2 t2 s1 s2 t1 s1 s2 t1 t2
- 44. • Mean responses of 4 sets plotted • Log potency ratio (M) (T2-S2)+(T1-S1) × Log d (S2-S1)+(T2-T1) where, d-dose ratio = s2/s1 • Strength of unknown = s1/t1 × antilog of M 44
- 46. 3+3 dose assay 3 conc each of std & test drug are used 6 sets of experiments using 6 doses in each set More time consuming,lesser in use Reliability is excellent
- 47. to measure the pharmacological activity of new/ chemically undefined substances to investigate the function of endogenous mediators to measure drug toxicity and unwanted effects to measure the conc of drugs and other active substances in the blood or other body fluids 47
- 48. Determination of potency, ED50/LD50 of drugs New drug development Measure clinical effectiveness 48
- 49. • Biological variation • Troublesome • Time consuming • Expensive • Less accurate than physico-chemical methods 49
- 50. Successful tool in estimation & discovery of biologically active substances Sensitivity & Specificity – important tool in pharmacology