Biological tests and assays of Tetanus antiserum & Oxytocin.pptx

Oxytocin and tetanus antiserum biological tests and assays

1. Biological tests and assays of tetanus anti serum and oxytocin ADVANCED PHARMACEUTICAL ANALYSIS (MPA 102T) Presented on 1 Presented By Archana 1 st Sem M.Pharm Department of Pharmaceutical Analysis KLE College of Pharmacy, Bengaluru. Presented To Dr. Vanita Somasekhar Professor and Head Department of Pharmaceutical Analysis KLE College of Pharmacy, Bengaluru.

2. contents INTRODUCTION TO TETANUS ANTI-SERUM BIOLOGICAL ASSAY OF TETANUS ANTI-SERUM INTRODUCTION TO OXYTOCIN BIOLOGICAL ASSAY OF OXYTOCIN REFERENCES Presented on 2

3. TETANUS ANTI SERUM INTRODUCTION: Tetanus antiserum, also known as Tetanus I mmune G lobulin (TIG), is a liquid or freeze-dried preparation containing immunoglobulins, mainly immunoglobulin G. It contains antibodies against the tetanospasmin toxin produced by the bacterium Clostridium tetani. Tetanus is a serious disease characterized by muscle stiffness and spasms, often leading to severe complications or death. Presented on 3

4. The primary use of tetanus antiserum is for post-exposure prophylaxis in individuals who have sustained wounds at risk of tetanus infection, particularly if they have incomplete vaccination histories. It can neutralize the toxin and provide immediate immunity until the body can develop its own antibodies through vaccination. Tetanus antiserum is administered alongside a tetanus vaccine to ensure comprehensive protection. The preparation is intended for intramuscular administration. Category : Immunological product. Presented on 4

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6. BIOLOGICAL ASSAY OF TETANUS ANTITOXIN: Principle: The potency of tetanus antitoxin is determined by comparing the dose necessary to protect mice against the paralytic effects. Fixed dose of tetanus antitoxin is compared with the dose of the Standard Preparation of tetanus antitoxin necessary to give the same protection. Standard Preparation: It is the 2 nd International Standard consisting of freeze-dried hyper immune horse serum, the potency is determined in relation to the International Standard. Presented on 6

7. Test animals: Use healthy mice, weighing between 17g and 22g, from the same stock. Preparation of test toxin: Prepare tetanus toxin from a sterile filtrate of an 8 to 10 days culture of Cl. tetani . Test toxin may be prepared by adding 1 volume of the filtrate to 1 or 2 volumes of glycerin, stored below 0°. Test toxin prepared in stable form by saturating the filtrate with ammonium sulphate, collecting the resulting precipitate, drying it over phosphorus pentoxide and reducing it to a fine powder. The powder so obtained is preserved in the dry condition at a low temperature in Presented on 7

8. sealed ampoules. Determination of test dose of toxin ( Lp /10 dose): Limes paralyticum /10 ( Lp /10): This is the smallest quantity of the toxin mixed with 0.1 Unit of the Standard Preparation and injected subcutaneously into mice causes paralysis within 4 days. Procedure: Prepare a solution of the Standard Preparation in a suitable liquid such that 1 ml contains 0.5 Unit. Accurately weigh a quantity of the test toxin and dilute it with, a suitable liquid. Prepare mixtures contains 2.0 ml of the solution of the Standard Preparation Presented on 8

9. (1 Unit), add sufficient of a suitable liquid to give a final volume of 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Inject 0.5 ml of each mixture subcutaneously into mice, six mice being used for each mixture, observe the mice for 4 days. The test ( Lp /10) dose of the toxin(0.5 ml) mixture that contains the smallest amount of toxin sufficient to cause tetanic paralysis in all six mice injected within 4 days. Presented on 9

10. Determination of potency of the antitoxin: Sample preparation for test: Prepare a solution of the Standard Preparation in a suitable liquid such that it contains 0.5 Unit per ml. Accurately weigh a quantity of the test toxin and dilute with a suitable liquid, 1.0 ml contains five times the Lp /10 dose(0.5 Unit per ml). Procedure: Prepare mixtures contains 2.0 ml of the solution of the test toxin +one of a series of graded volumes of the preparation +sufficient suitable liquid to give a final volume of 5.0 ml. Presented on 10

11. Prepare similar mixtures with standard preparation. Allow the mixtures to stand at room temperature, protected from light for 60 minutes. Inoculate 0.5 ml of each mixture subcutaneously into each mouse, six mice being used for each mixture, observe the mice for 4 days. The mixture contains the largest volume of the preparation that fails to protect the mice from paralysis contains 1 Unit. The test is valid only if all the mice injected containing 2.0 ml or less of Standard Preparation show paralysis and all those injected with more do not. Calculate the potency of the preparation being examined in Unit per ml. Presented on 11

12. Potency: The potency is determined by comparing the antibody titre of the immunoglobulin under examination with that of a reference preparation calibrated in International Units, using an immunoassay of suitable of suitable sensitivity and specificity. The stated potency is not less than 100 Units of tetanus antitoxin per ml. The estimated potency is not less than the stated potency. The confidence limits (P=0.95) of the estimated potency are not less than 80% and not more than 125%. Storage: Protected from light, the liquid preparations in sealed, colourless glass containers and the freeze-dried preparation in an airtight colourless  glass container Presented on 12

13. OXYTOCIN INTRODUCTION: Oxy –Rapid Tocos -Labor Oxytocin synthesized in both sexes (male and female), well recognised physiological effects only in women. Pituitary gland consist posterior lobe which produce Oxytocin and diuretic hormone Presented on 13

14. ROLE OF OXYTOCIN : It stimulates the contraction of the uterine smooth muscle and memory gland. Oestrogen progesterone and prolactin- Responsible for production of milk by mammary gland but ejection require Oxytocin. It facilitates the contraction of uterus. Presented on 14

15. MECHANISM OF ACTION OF OXYTOCIN: Neuropeptide made in hypothalamus that stimulates contractions, that expel the infant from uterus. Responsible for milk letdown and triggered by the nipple stimulation of suckling. Oxytocin called love and binding hormone. It has a very special affect on mothering. Psychologically, Oxytocin promotes a feeling of well being and tranquility. It enables the growing sense of love and attachment to the infant. The more the infant suck the more Oxytocin is produced. Presented on 15

16. BIOLOGICAL ASSAY OF OXYTOCIN: PRINCIPLE: The potency of oxytocin is determined by comparing its activity with that of the standard preparation of oxytocin under the suitable method of assay. Potency is determined by comparing its activity Depression of BP. Contraction of uterus. Milk ejection Pressure. Vasopressor activity. Presented on 16

17. STANDARD PREPARATION OF OXYTOCIN: The standard preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 units), or another suitable preparation the potency of which has been determined in relation to the International Standard. Presented on 17

18. EXTRACT OF STANDARD PREPARATION: Presented on 18

19. Method - A By depression of the Blood pressure in the chicken Test animals: cockerel (young male chicken), Weight: 1.2-2.3 kg Method - B By c ontraction of the rat uterus Test animals: female rat, Weight: 120-200 g Method - C By measurement of milk ejection pressure in a lactating rat Test animals: lactating rat in the 3-21 days after parturition, Weight: 300 g Method - D Vasopressor activity Test animals: male rat, Weight : 300 g Presented on 19

20. Method–A DEPRESSION OF THE BLOOD PRESSURE IN CHICKEN Test Animals: Cockerel (young male chicken), 1.2 - 2.3 Kg , Anaesthetized cock-prolonged & constant high B.P Expose gluteus primus muscle(thigh) & remove popliteal artery & crural vein. Cannulate the popliteal artery & record B.P response Cannulate the crural or brachial vein . Prepare standard solution with saline. Inject 0.1 - 0.5ml Inject 2 doses of standard solution into cannulate vein is record B.P response Presented on 20

21. Dose should cause decrease in B.P (reqd. dose between 20- 100mUnits ) Interval between 2 injection, between 3-10mins depend on rate at which B.P return to normal Dil . test preparation with saline so as to get same response as standard The ratio between standard & test should be equal If animal rapidly becomes insensitive to repeated injection the solution another must used. Measure all responses and calculated result of the assay by standard statistical method. Presented on 21

22. Method-B BY CONTRACTION OF THE RAT UTERUS Test animals: Female rat 120-200g Inject 100 µg of oestradiol benzoate IM into female rat before the assay Immediately before assay confirm by vaginal smear that rate in oestrus or pre-oestrus. Kill rat & suspend one horn of uterus in organ bath containing a solution of following Sodium chloride ( NaCl ), P otassium chloride ( KCl ) , Calcium chloride (CaCl 2 ), Sodium bicarbonate (NaHCO 3 ), Disodium phosphate (Na 2 HPO 4 ), Monosodium phosphate (NaH 2 PO 4 ), Magnesium dichloride ( MgCl   2 ), Dextrose Presented on 22

23. Maintain the bath at temp at of 30°C Bath liquid required dose between 10-50 units/ml . Oxygenate solution with mix of 95% of O 2 , 5% of CO 2 record - contraction of muscle . Record contraction produces by addition of two dose of standard preparation (Required Dose 10 & 50munits/ml of bath liquid) W hen maximum contraction has been reached replace - bath liquid by fresh solution . Dose should be added at regular interval (3-5minutes) Similarly record the contraction of test preparation as standard. Presented on 23

24. Ratio between two dose of test & two dose of std should be equal. This ratio kept constant through out the assay . Measure all response & calculate result of assay by standard statistical method . Presented on 24

25. Method-C MILK EJECTION PRESSURE IN LACTATING RAT Test animals: lactating rat in the 3-21 days after parturition, Weight: 300 g Separate from litter & 30-60 minutes later anaesthetize (IP Pentobarbitone Na ). Tie rat to an operating table, at 37°, by its hind legs leaving front legs free. Cannulate trachea with a short PE tube of i.d. 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary . Cannulate an external jugular or femoral vein with a PE tube of i.d. 0.4 mm filled with saline & closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of Presented on 25

26. one teat , preferably the lower inguinal teat . Insert a PE tube of i.d. 0.3 mm & e.d. 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3-10 mm depth),into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature . Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial BP in rat) and fill with a 3.8% w/v of Na citrate /saline contain 50 Units of heparin Na/ ml to prevent clotting of milk . After cannulation, inject 0.05 -0.2 ml of this solution into teat duct through transducer to clear milk from tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula ) Presented on 26

27. Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of 5.3 kPa . Inject all solutions through the venous cannula using a 1ml syringe graduated in 0.01ml and wash them in with 0.2ml saline . Prepare a solution of Standard & Test preparation in saline solution so that the volume to be injected is between 0.1 - 0.4 ml. Choose two doses of Standard preparation such that the increase in milk-ejection pressure is about 1.35 kPa for Lr dose and about 2.7 kPa for Hr dose. Presented on 27

28. As an initial approximation, a lower dose of between 0.1 and 0.4 milli Unit and an upper dose of 1.5 to 2 times this amount may be tried . Choose two doses of the Test preparation with the same inter-dose ratio, matching effects of doses of the Standard preparation as closely as possible . Inject four doses (2 doses of Standard & 2 doses of Test) at intervals of 3- 5 minutes 2 doses of Standard and 2 doses of test should be given according to randomized block or a Latin square design & at least four responses to each - recorded. Measure all responses & calculate result of the assay by standard statistical methods . Potency: 90 % - 111 %. Fiducial limits of error are 80% - 125 % stated potency. Presented on 28

29. Method-D VASOPRESSOR ACTIVITY Test Animals: Male rat 300g NMT 0.5 Unit /20 Units of oxytocic activity by biological assay for vasopressor activity- comparing activity of Test & Standard Preparation of arginine vasopressin Freeze-dried syn. arginine vasopressin peptide acetate with human albumin & citric acid (supplied in ampoules containing 8.20 Units ) Inject slowly into tail vein of male albino rat weighing 300g -solution of a suitable a- adrenoreceptor blocking agent, (10 ml/kg body weight of solution prepared by dissolving 5 mg of phenoxy benzamine HCl in 0.1 ml of ethanol (95%), adding 0.05 ml of 1 M HCI & dil to 5ml with saline. Presented on 29

30. After 18 hours, anaesthetize rat - that will maintain -prolonged & uniform BP . After 45-60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate trachea with short PE of E.D. 2.5 mm & dissect carotid artery ready for cannulation . Then cannulate the femoral vein close to the inguinal ligament Retract the abdominal muscles to expose the inguinal ligament Retract superficial pudendal vein to one side & dissect femoral vein towards inguinal ligament from corresponding artery . When dissecting, a deep branch reaching femoral vein must be found & tied off to prevent bleeding during cannulation Presented on 30

31. Tie a short PE cannula of E.D. about 1 mm into femoral vein by two ligatures & join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline at about 37°C Firmly fix wet absorbent cotton swab to thigh so as to cover incision and cannula. At this stage inject through venous cannula 200 Units of heparin, dissolved in saline /100 g of body weight Then tie in a carotid cannula of E.D. about 1 mm & connect by a column of saline contain heparin with a pressure measuring device such as Hg manometer of I.D. about 2-3 mm. C entral & Peripheral nervous system including both vagus & associated sympathetic nerves is left intact. Presented on 31

32. No artificial respiration is necessary . No air is injected, inject all solutions through venous cannula by means of a 1 ml syringe & wash in with 0.2ml of saline from burette. Dil extract of Standard & Test preparation with saline so that volume to be injected is between 0.1 & 0.5 ml . Choose 2 doses of the Standard preparation such that the elevation of the BP is about 4 kPa for Lr dose & about 7 kPa but always submaximal for higher, ratio of low to high dose being determined by response & usually being 3-5. As an initial approximation doses of 3 and 5 M Units may be tried. Choose 2 doses of Test preparation with same inter-dose ratio, matching effects of dose of Standard preparation . Inject doses at intervals of 10 - 15 minutes. Presented on 32

33. 2 doses of Standard & 2 doses of Test preparation should given in randomized block / Latin square design & 4-5 responses to each recorded . Measure all responses & calculate result of the assay by Standard statistical methods. Presented on 33

34. reference Indian pharmacopeia 2007; volume: 3. European pharmacopeia 2010; volume : 3. https ://www.slideshare.net/slideshow/biological-assay-of-oxytocin-208932533/208932533 Presented on 34

35. Thank you Presented on 35