BIOASSAY PPT

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DETAILED DESCRIPTION OF BIOASSAY OF PITUTARY HORMONE

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1 BIOASSAY OF INSULIN AND POSTERIOR PITUTARY HORMONE by Vanitha

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BIOASSAYS DEFINITION: Estimation of the potency of an active principle in a unit quantity of preparation or detection and measurement of the concentration of the substance in a preparation using biological methods (observation of Pharmacological effects of living tissue micro/macro or immune cells or animal)is known as bioassay or biological assay. 2

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Importance: If active principle of drug is unknown e.g.: insulin, streptokinase, urokinase etc. If Chemical method is not available e.g.: acetylcholine, glucagon If chemical composition is not known e.g.: vaccines, antitoxins, toxins purification for chemical assay is not known e.g: vitaminD, oxytocin T o ascertain the potency hence served as quantitative part of screening methods I nvestigate function of endogenous mediators To measure drug toxicity and un wanted effects 3

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Principle: To compare the test substance with the international standard preparation of the same and to find out how much of test substance is required to produce same biological effect, as produced by the standard. Standard preparation: Representative of substance serves as basis for comparative measurement of activity. In India maintained and distributed by: Central Drug Laboratory, Kolkata . Central Drug Research Institute, Lucknow. 5

Factors affecting the Bioassay:

Factors affecting the Bioassay Biological Variation Animals Environment Temperature Diet Season Experience Planning of Experiment Solvent or vehicle for the drug substance Route of administration. Time of administration Number of animals Dose Design Selection of Method Selection of animals 6

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TYPES OF BIOASSAYS 1. Q uantal Assays {Direct-End point} Elicits “All or None” response in different animals E.g: Digitalis induced cardiac arrest in guinea pigs . Hypoglycemic convulsions in mice Digitalis induced head drop in rabbits Calculation of LD 5 0 in mice or rat 7

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2) Graded response Assays {mostly on tissues} Proportionate increase in the responses due to increase in dose or concentration E.g.: concentration of smooth muscle for histamine assay 8

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METHODS OF BIOASSAY End-Point method Matching method Graphical method Multiple point bioassay method 9

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END-POINT METHOD Threshold dose producing positive effect is measured on each animal and compare the average results E.g.: Bioassay of digitalis in cats 10

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Matching Method Firstly response of test is taken and it is matched with response of the standard drugs Done till a closed matching is observed 11

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Advantages: Simple method If the sensitivity of the preparation is not stable this method is useful Disadvantages: Time consuming Match is purely subjective so there are chances of errors Method does not give any idea of dose-response relationship 12

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Graphical Method This is based on dose response relation ship Log-dose-response curve is plotted and the dose of standard producing the same response as produced by the test is directly read from the graph 13

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MULTIPLE-POINT BIOASSAY METHOD Three point assay [2+1 dose assay]: Fast & convenient method Procedure: Log Dose Response curve[LDR] is plotted by taking two standard and one test concentration. n 1 = Lower Std. Dose n 2 = Higher Std. Dose t = test dose S 1 =Response of n 1 S 2 = Response of n 2 T = Response of test (t) C s = conc. of Standard 14

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FOUR POINT BIOASSAY METHOD (2+2 DOSE ASSAY) Procedure: LDR is plotted with varying concentrations of standard and test samples Select two std. doses (s₁ & s₂) and two test doses (t₁ &t₂) Record the data t 1 = Low Dose of Test t 2 = High Dose of Test T 1 = response of t 1 T 2 = response of t 2 15

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Six Point Bioassay Method{3 + 3 dose} Excellent reliable technique. Three concentration of standard & test are used. Recent applications: - Microbiological assay of vit B12 - Analgesic assay of sublingual buprenorphine & morphine - Diazyme’s cystatin -C assay –emerging marker–renal disease 16

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BIOASSAY OF INSULIN Standard preparation: 1 unit of insulin = 0.04082mg specified by Ministry Of Health, Govt. Of India Preparation of standard solution: weigh 20 units of insulin Dissolve in normal saline Acidify with HCl to pH 2.5 0.5% phenol-preservative & add 1.4% to 1.8% glycerin Preparation of test sample solution: same way as prepared by the standard sample solution 17

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METHODS OF INSULIN BIOASSAY Rabbit method Mouse method Rat Diaphragm Method Rat Epididymal Fat-pat method Radioimmunoassay Method 18

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Rabbit Method Selection of rabbits: weight-1800 to 3000 gm Fasted for 18 hrs Standard & sample dilutions: 1 unit/ml &2 unit/ml diluting with normal Nacl Doses: which can produce suitable fall in blood sugar level. Principle: The potency of a test sample is estimated by comparing the hypoglycemic effect of the standard preparation of insulin. 19

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12 RABBITS S.C. INJECTION OF INSULIN Standard Dilution (I) Standard Dilution (II) Test Sample Dilution (I) Test Sample Dilution (II) 20

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Procedure: First part- Blood collected from marginal ear vein Initial blood sugar level From each rabbit blood is withdrawn up to 5hrs at the interval of 1 hr Final blood sugar level 21

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Second part {cross over test}: After one week Again fasted Initial blood sugar level Grouping is reversed-”Twin cross over Test” Mean percentages decrease in blood sugar of the first and second part is calculated. 22

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Mouse method Principle: S.C. injection of insulin produces convulsions in mice. The percentage convulsions produced by the test and the standard are compared. Experimental procedure: 100 mice weighing 18-22gms Fasted for 18 hrs 23

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100 Mice 24 25 25 25 25 Test Sample Dilution Test sample Dilution Standard Dilution (0.096 units/ml) Standard Dilution (0.064 units /ml) Four groups are given with s.c. injection of insulin Mice are put in air incubator at 33°c and observed for 1 ½ hr The mice which convulse or die are taken out and observed Convulsive mice may be saved by an injection of 0.5ml of 5% dextrose solution Percentage convulsions produced by test sample are compared with the standard sample

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Rat diaphragm method Selection of animals : male albino rats weight-100 to 150gm Standard sample solution : 23.2 units/mg of Insulin. Standard dose – 2mu/ml 25

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Principle: Increase in glycogen content of the muscle or glucose uptake by muscle in response to insulin. Experimental Procedure : Buffer with 2.5mg/ml of glucose Glucose uptake is determined Human Blood Withdraw Buffer + Plasma mixture ( 2.5 mg of Glucose + 0.25 ml of plasma for each ml) centrifuge cups (Heparin + plasma) 26

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Hemi diaphragm placed in above solution Blotted and transfer to manometer flasks Incubate for 3 hrs at 37 0 C Shake rate --- 100 cycles /min After incubation 0.2 ml is removed and examined for glucose estimation 27

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Rat epididymal fat-pad method Ability of insulin to increase CO 2 production by the fat-pad is taken as the parameter for the measurement of potency of the insulin preparation. 28

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Radioimmunoassay Method Estimation of the concentration of the substance in a unit quantity of the preparation using radio labelled antigens RIA of insulin is based on ability of human insulin to displace beef’s insulin from the binding sites. 29

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Steps involved: Bovine insulin collected from sheep After 1 week AB’s are collected serum of AB’s is exposed to radio labelled insulin Bound Vs Free ratio is determined Standard curves are plotted Concentration of test insulin is determined using the standard curve 30

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Bioassay of posterior pitutary hormone Bio assay of oxytocin: Principle: Potency of oxytocin is determined by comparing its activity with that of the standard preparation of oxytocin. Standard preparation: 1 Unit of preparation = 0.5mg oxytocin Synthetic oxytocin peptide + human albumin + citric acid (12.5 units). Suggested methods: By depression of the blood pressure in chicken By contraction of the rat uterus By measurement of milk-ejection pressure in a lactating rat Pressor activity 31

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By depression of the blood pressure in chicken: Anaesthetize adult cockerel weighing 1.2-2.3kg. Expose gluteus primus muscle Popliteal artery Crural/brachial vein Cannulate Inject 0.1ml - 0.5ml saline solution Record Blood Pressure 32

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Inject standard solution 20-100 mu Dilute the preparation to be examined with saline solution Inject test solution Randomized readings are taken Test and standard are compared Calculate by statistical methods 33

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By contraction of the rat uterus: Female rat weighing 120-200gm Inject 100mcg of oestradiol benzoate Suspend one horn of uterus in a bath(32 0 c) Oxygenate the solution Record contractions Inject standard doses of 10-50mcg/ml Inject test sample Calculate the result by standard statistical methods 34

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By measurement of milk-ejection pressure in a lactating rat: Lactating rat (3 rd to 21 st day after parturition) weighing 300gm Anaesthetise (intraperitonial) Tie the rat by its hind legs, front legs free. Cannulate the trachea with PE(polyethylene) tube Cannulate external jugular or femoral vein Cannulate tip of a teat Connect this with strain gauge transducer 35

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Prepare 3.8%w/v solution of sodium citrate+ 50 units of heparin/ml Inject (0.05-0.25ml) of the above solution Clamp the strain gauge Potentiometric recorder Inject solutions through venous cannula Inject standard and test solutions(0.1-0.4ml) Measure responses 36

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Pressor activity: Spinal cat weighing 3.5 to 4.5Kg (Anaesthetise) Initial BP is recorded Cannulate trachea by tracheal tube(artificial respiration) Incision to vertebral column Brain is destroyed Constant temp. by warm Cu plate Cannulate femoral vein(venous cannula) Cannulate carotid artery (arterial cannula) 37

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Joined to manometer to record BP on kymograph Injections of pitutary extracts Dose - 0.05-1 unit for 30 min. Rise in Blood pressure Normal saline is passed Adjust test and std. doses to produce equal rise in BP Repeat it until both produce same rise in BP Activity expressed in units/ml 38

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Bioassay of vasopressin Principle : vasopressin activity of test is compared with the standard preparation of Arginine vasopressin. Standard preparation: (8.2 units) Freeze dried synthetic vasopressin peptide acetate + Human albumin + citric acid 39

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Procedure: Male Albino rat weighing 300gm Anaesthetise the rat After 45-60min tie the rat Cannulate trachea with PE tube Cannulate femoral vein by two ligatures Join to1ml burette (filled with saline solution) Inject 200 units of heparin/100g body weight 40

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Tie carotid cannula Taking care that no air is injected Inject all solutions (venous cannula) Injection volume of saline 0.1-0.5 ml Inject std. preparation 3-5mu (time interval 10-15 min) Test preparation is injected Examined by randomized method Calculate by statistical methods 41

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REFERENCES: K.Goyal. Elements of pharmacology, seventeenth edition cology4u.blogspot.com/2012/05/bioassay-of-insulin.html www.iasri.res.in/ebook/EBADAT/6.../4-BIOASSAYS.pdf http://www.nebiosolids.org pdf/Bioassay%20Page/McCarthy-PrinciplesOfBioassay-May11.pdf http://www.nebiosolids.org pdf/Bioassay%20Page/McCarthy-PrinciplesOfBioassay-May11.pdf http://nadjeeb.files.wordpress.com/2009/05/9058230511.pdf 42

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