vaccines: vaccines By E.chandra sekhar M.pharm 1 st year Ph.analysis Slide 2: ABSORBED DIPTHERIA ANTI-TOXIN: DEFINITION: Dephtheria anti-toxin is preparation containing antitoxic globulins that they have the power of specifically neutralizing the toxin formed by Cornebacterium diphtheria . It is obtained by fractionation from the serum of horses or other mammals that have been immunized against diphtheria toxin. Slide 3: BIOLOGICAL ASSAY OF ADSORBED DIPHTHERIA ANTI-TOXIN: PRINCIPLE: The potency of diphtheria anti-toxin is determined by comparing the dose necessary to protect guinea-pig or rabbits against the erythrogenic effect of a fixed dose of the standard preparation of diphtheria anti toxin necessary to give the same protection. Slide 4: SELECTION OF TOXIN: It is based on the LR/100. If the LR/100 shows constant in 200 animals. The toxin should be selected for assay. Store the toxin at 0-5⁰c . add the toulene and other anti microbial preservatives which does not cause rapid decline in specific toxicity. DETERMINATION OF TEST DOSE TOXIN: Saline solution prepared having the 1ml contains 0.1unit toxin.(standard preparation) Prepare mixture of saline solution (a series of graded volumes make up to 2ml) 1ml std preparation + 0.1ml test toxin 1ml std preparation + 0.2ml test toxin 1ml std preparation + 0.3ml test toxin Slide 5: Stay 15-60min protected from the light. 0.2ml sample each mixtures inject intracutaneously into the animals. Observe the animals after 48hrs Mixture containing larger amount of toxin gives the larger reaction Mixture containing less amount of toxin gives the smaller reaction Slide 6: DETERMINATION OF POTENCY OF THE ANTITOXIN: Dilute the test toxin with saline solution so that 1 ml contains 10 times the test dose (LR/100) i.e 1ml contains 1unit toxin Preparation of mixture soln (2ml): 1ml dilution + 0.4ml antitoxin 1ml dilution + 0.5ml anti toxin 1ml dilution +0.6 ml anti toxin Each series of Inject 0.2ml and determine LR/100 Slide 7: DETERMINATION OF LR/100: Test dose and std dose contains 0.01unit of anti toxin in 0.2ml solution. They should show the same LR/100. the test dose should between the 90% - 111% storage: stored at room temperature. Protected from the light. Slide 8: Labelling : The no of units per ml Either it is from horse of other mammals Recommended dose preservatives Slide 9: RABIES VACCINE DEFINITION: Rabies vaccine is a suspension of a suitable strain of fixed rabies grown in suitable approved cell culture and inactivated by a suitable method. The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a suitable sterile liquid. Dried vaccine + sterile liquid suspension Slide 10: BIOLOGICAL ASSAY OF RABIES VACCINE: PRINCIPLE: The potency of rabies vaccine is determined by comparing a lethal intracerebral dose of a rabies virus with the dose of the standard preparation of rabies vaccines necessary to give for same protection . STANDARD PREPARATION: Freeze – dried preparation. Potency is determined by international reference Slide 11: TEST ANIMALS : White mice weight 11g-15g. 6 groups of 16 animals , 4groups of 10 animals. These two groups are used for titration of LD50 of challenge suspension. Injected 0.03ml/mice intracerebrally. Slide 12: STANDARD CHALLENGE VIRUS SUSPENSION: Standard challenge virus suspension is prepared by injecting ↓ Intracerebrally 0.03ml of 10 fold dilution standard strain horse serum to the test animal. ↓ Showing characteristic symptom of encephalitis are sacrified ↓ Harvest the brain aseptically. ↓ Slide 13: Wash it saline solution to remove blood clot ↓ Homogenized the brain with 10%digested casein hydro lysate [PH7.2]. ↓ Centrifuge. ↓ Separated liquid is distributed into sterile ampoules. ↓ Stored at 2-8 0 . Slide 14: DET.OF CHALLENGE VIRUS [DETETERMINATION OF VIRUS TITRE OF THE CHALLENGE VIRUS] :- Prepare 3-10 fold dilution of std.challenge virus suspension. ↓ 0.03ml inject intracerebrally to a groups of 10 mice. ↓ Observe mice for 14 days. ↓ Count the number of mice surviving in each group. ↓ Calculate the virus titre of std challenge vurus suspension by statistical method. Slide 15: DETERMINATION OF POTENCY OF THE VACCINE: Prepare 3-5 fold serial dilution of standard and test soln of vaccines ↓ Separate mice in 6 groups of 16each ↓ Inject 0.03ml intraperitoneally and after 7days prepare same solution and inject ↓ Slide 16: Both standard and test should prepared in such a way ↓ After 7days inject 0.03ml standard virus suspension to vaccinated mice ↓ Observe it for 14 days and calculate its potency by statistical method. Lowest dilution should protect the 50% of the animal Highest dilution protect less than 50% of the animal. Slide 17: RABIES ANTI SERUM Rabies anti serum is a preparation containing the specific globulin [or] its derivatives obtaines by purification of hyper immune serum[or] plasma of healthy horses [or] other animals having the specific activity of neutralizing the rabies virus. BIOLOGICAL ASSAY OF RABIES ANTISERUM: The potency of rabies antiserum is determined by comparing a lethal intracerebral dose of rabies virus with the dose of standard preparation of rabies antiserum necessary to give same protection. Slide 18: PROCEDURE: STANDARD PREPARATION: Standard preparation is dried serum (or) Other preparation , the potency of which has been determined in relation to international standard. TEST ANIMALS: Mice, 10g-14g -animal same sex. TEST VIRUS: Any suitable strain rabies virus of known potency, such as the CVS strain may be used. TEST DOSE OF VIRUS: 20-1000 LD50 intracerebral injection to each mouse. Slide 19: DET. OF TEST DOSE OF VIRUS: Virus dilution equal quantity of 2%v/v solution of heat inactivated horse serum in water . ↓ Maintain at 37⁰c at one hour. 2) Prepare is 10 fold dilution in a 2%v/v solution of heat –inactivated normal horse serum Inject into mouse. ↓ The test is not valid unless the quantity of virus used lies between 20-1000 LD50 . Slide 20: DETERMINATION POTENCY OF RABIES ANTISERUM: Prepare 2 fold dilution of std preparation and test preparation with 2% v/v heat inactivated normal horse serum in water. ↓ To each dilution add a quantity of suspension of test virus ↓ Keep the mix at 37⁰c for 1hr ↓ Inject 0.03ml intracerebrally to mice ↓ Slide 21: Observe mice for 14days ↓ Mice dying before 5th day after inoculation of virus are eliminate from test, all the mice dying between 5th -14th day after showing signs of rabies ↓ Count the no of mice surviving ↓ Calculate potency of the test preparation by std statisticalmethod The preparation pass the test it found to have 80units /ml The preparation for the test also same as standard Slide 22: TETANUS ANTI –TOXIN DEFINATION : Tetanus anti-toxin is a preparation containing antitoxic globulins that have the power of specifically neutralizing the toxin formed by clostridium tetani. It is obtained by fractionation from horse serum or other mammals that have been immunized against tetanus toxin. POTENCY LIMIT: NLT 1000 units/ml for prophylactic use, NLT 3000 units/ml for therapeutic use. Slide 23: BIOLOGICAL ASSAY OF TETANUS ANTI-TOXIN: PRINCIPLE: The potency of tetanus anti-toxin is determined by comparing the dose necessary to protect the mice against the paralytic effects. PROCEDURE: STANDARD PREPARATION: Standard preparation containing freeze dried hyper immune horse serum. TEST ANIMALS: mice- 17-22 gm. Slide 24: PREPARATION OF TEST TOXIN: Extract the c.tetani toxin from the 8-10days culture ↓ (filtrate) 1vol toxin + 2 vol glycerin Saturate the filtrate with the ammonium sulphate ↓ Collecting the ppt and dried over phosphorous pentaoxide ↓ Obtained powder is preserved in ampoules at temperature below 0c Slide 25: DETERMINATION OF TEST DOSE OF TOXIN:[LP/10DOSE]. Standard: Prepare standard preparation with saline solution final concentration-0.5units/ml . Prepare mixture of solution: 2ml standard preparation+ one of the serial dilution of toxin ↓ Final volume 5ml with saline soln. ↓ Inject 0.5ml of mix subcutaneously to the mice. ↓ Observe for 4 days. Slide 26: The test [LP/10] dose of the toxin is the amount present in 0.5ml of the toxin efficient to cause titanic paralysis in all six mice injected with it within 4 days. LP/10dose: LP means limes paralyticum This is the smallest quantity of the toxin when mixed with 0.1unit of the std preparation and injected subcutaneously into mice cause paralysis with in 4 days DETERMINATION OF POTENCY OF THE TETANUS ANTI-TOXINS STANDARD PREPARATION 0.5 UNITS/ML TEST PREPARATION 2ml of test toxin + one of the serial dilution of standard preparation 2ml of test toxin+ one of the serial dilution of test preparation Final volume 5ml dilution with saline solution Same Stand 60 minutes Slide 27: Inject 0.5ml subcutaneously into 6 mice Observe 4 days Observe 4 days Slide 28: THE TEST FAIL :- if largest volume of test preparation fails to protect the mice from paralysis. if lowest volume of standard preparation fails to protect the mice from paralysis. Then calculate the potency in unit/ml. Slide 29: MICRO BIOLOGICAL ASSAY OF NEOMYCIN SULPHATE: Neomycin sulphate is a mixture of compounds obtained by the growth certain strains of streptomyces fradie. CATEGORY:- anti-microbial. MICROBIAL ASSAY: TEST ORGANISM:- Staphylococcus Epidermsis. PREPARATAION OF MEDIUM: Peptone --- 6gm Pancreatic digest of casein --- 4gm Yeast extract --- 3gm Beaf extract --- 1.5gm Dextrose --- 1gm Agar --- 15gm Final pH after sterlisation --- 7.8—8.00 Slide 30: PREPARATION OF INOCLUM: Suggested inoculums composition amount - 0.4ml/100ml. Suggested dilution factor : 1:40. Incubation condition : temp – 32-35. Time—24 hours. STOCK AND TEST SOLUTION OF STANDARD PREPARATION: Final stock conc. per ml : 1mg/ml. Standard solution conc. : 1mg/ml. Test dilution : use before14days Intial and final diluents : B2. Media dose ug or units/ml : 1.0ug/ml. Incubation temperature : 36-37oc. Slide 31: METHOD OF ASSAY:-[A] Plate diffusion [or] cylindrical plate [or]cup plate. cylindrical plate method: This method depends on the diffusion of an neomycin sulphate from a vertical cylinder or a cavity through the solidified agar layer of a petri-dish or plate, to prevent the growth of added microorganism entirely in a circular area or “ZONE” around the cylinder or cavity containing a solution of neomycin SO 4 . Slide 32: A previously liquefied medium appropriate to the assay, is inoculated with the requisite quantity of suspension of the micro organism, the suspension is added to the medium at a temperature t/t 40-500 and the inoculated medium is poured immediately into petri-dish or large rectangular plates to occupy a depth of 3to 4mm. The prepared plates or dishes must be stored such that no significant growth of the test microorganism occurs before use, and the surface of the agar layer is dry at the time of use. Slide 33: Solution of known concentration of the std. preparation and test neomycin sulphate are prepared in appropriate buffer solutions. The volume of solution added to each cylinder or cavity must be uniform and sufficient to fill the holes. Then they are incubated or about 24 hours at about 370 and the diameter or areas of the circular inhibition zones are measured. POTENCY:- potency of neomycin sulphate NLT - 95% NMT – 108% CALCULATION OF POTENCY: conc.in unit/mg vs diameter of zones. Slide 34: OXYTOCIN Oxytocin is a polypeptide hormone obtain from the posterior pituitary gland. It is a neurosecretary product mainly synthesize in the cell bodies of paraventracular nuclear of the hypothalamus. pituitary gland consist posterior labe which produce oxytocin and diuretic hormone. ROLE OF OXYTOCIN: Oxytocin stimulate the contraction of the uterine smooth muscle and the memory gland. Oestrogen progesterone and prolactin – responsible for production of milk by memory gland but milk ejection require oxytocin. Oxytocin facilitates the contraction of uterus. Slide 35: Oxytocin may be presented as a solid or as a solution in a solvent containing an appropriate antimicrobial preservative such as 0.2% w/v of chlorobutanol. If it is derived from animal species. Oxytocin contains NLT 90% NMT 110% the stated number of units of oxytocin activity If it is synthetic product presented as a solid it contains NLT 560 units/mg Calculated with reference to the peptide content and when presented as a liquid NLT 150 units/ml Bacterial endotoxins:- NMT 100 endotoxis units per 200 units of oxytocin Slide 36: BIOLOGICAL ASSAY OF OXYTOCIN: PRINCIPLE: The potency of oxytocin is determined by comparing its activity with that of the standard preparation of oxytocin of under the conditions of a suitable method of assay. PROCEDURE: STANDARD PREPARATION: Consisting free dried synthetic oxytocin peptide with human albumin citric acid Slide 37: METHOD-A By depression of the blood pressure in chicken Healthy adult cock[1.2—2.3kg] is anaesthesised with anaesthetic this maintain the prolonged and constant high B.P Exposure the gluteus primus muscle(thigh) and remove politeal artery and crural vein. Cannulate the popliteal artery is record the B.P response Cannulate the crural or brachial vein. Prepare standard solution with saline solution. Inject 0.1—0.5ml Inject two doses of std solution into cannulate vein is record the B.P response Dose should cause decrease in B.P Slide 38: Internal between two injection between 3-10 minutes depending on the rate at which B.P return to normal Dilute the preparation with saline solution so as to get same response as standard The ratio between standard and test should be equal If the animal rapidly becomes insensitive to the repeated injection the solution another must be used. Measure all the responses are calculated the result of the assay by std statistical method. Slide 39: METHOD—B: By contraction of the rat uterus: Inject 100mcg of oestradiol benzoate intramuscularly into female rat before the assay Immediately before the assay confirm by vaginal…….that the rate in oestrus or pre oestrus. Kill the rat and suspend uterus in organ bath containing a solution of following composition Nacl,Kcl,Cacl2, NaHco3 Disodium hydrogen phosphate[Na2Hpo4] Sodium dihydrogen phosphate Mgcl2 Dextrose Slide 40: Maintain the bath at temp at of 320c Bath liquid required dose between 10-50 units/ml. Oxygenate the solution with mix of 95% of O2 , 5% of co2 record the contraction of muscle. Record the contraction produces by the addition of two dose of std. preparation when maximum contraction has been reached replace the bath liquid by fresh solution. Dose should be added at regular interval[3-5minutes] Slide 41: Similarly record the contraction of test preparation as standard. Ratio between two dose of test and two dose of standard should be equal This ratio kept constant through out the assay. Measure all the response and calculate the result of the assay by standard statistical method. Slide 42: MICROBIOLOGICAL ASSAY OF CYANOCOBALAMINE STANDARD CYANOCOBALAMINE STOCK SOLUTION : Weight std. cyanocobalamine reference standard dissolved in 25% alcohol [0.1µg/ml] Standard cyanocobalamine solution: Standard cyancoblamine stock solution diluted with water[0.01 to 0.04µg/ml] TEST SOLUTION: Weight material dilution H2o + dil. HCl [to PH 6.0] Final solution contains the B12 activity approximately same strength of standard. Slide 43: CULTURE MEDIUM: dried yeast extract -- 0.75 gm peptone -- 0.75gm anhydrous dextrose -- 1gm potassium dihydrogen po4 -- 0.2gm Dissolve in 50ml of H2o + 10ml tomato juice filtrate +1ml sorbitan mono oleate derivative solution ↓Adjust PH 6.8 with NaoH solution Make upto 100ml with H2o [sterile in auto clave] Slide 44: BASAL MEDIUM STOCK SOLUTION :[BMS]: Dissolve in following order L-cystine DL-trptophan dissolved in 1N – HCL and then Adenine-guanine uracil solution Xanthene solution Riboflavin – thiamine-biotin – nicotine acid solution P-amino benzoic acid – pyridoxine-pyridoxal- pyridoxamine solution. Slide 45: Calcium panthothenate. Folic acid solution. Salt solution – A. Salt solution-B. Asparagines solution. Acid digested solution. Acid digested casein solution. Dextrose , sodium acetate ascorbic acid dissolved in 100ml H2o ↓ AdjustPH 6.0 with NaoH [1N]. Add sorbition now oleate derivative solution ↓ And make up volume to 250 ml with H2O. Slide 46: SUSPENSION MEDIUM: Dilute 25ml of basal medium stock solution with equal volume of H2O sterile in autoclave. INOCULUM: Transfer few cells of lacto bacillus leichmanii from a subculture onto 2 sterile tubes with 10ml culture medium. Incubation at 370c for 16-24 hrs. Centrifuge decant supernatant liquid. Suspended cells in 10ml of sterile suspension medium . Centrifuge decant of supernatant liquid [repeat 3 times]. Suspend the cells in 10ml of sterile suspension medium 1ml of the suspension of cells 10ml sterile suspension mix the inoculums Slide 47: Standard preparation Test preparation To triplicate test tubes 0ml 0.5ml, 1ml,1.5ml upto 5mlof std. cyano cobalamine solution To other triplicate test tube add 1,2,3,4ml of test solution 5ml basal medium to each tube Basal medium solution of each tube Make up to 10ml with water Make upto 10ml with water Sterile the tube by heating for 5 mts at `121 o c Aseptically inoculate each tube with 1 drop of inoculums Incubate at 30-37 o – 64 to 72 hrs. Slide 48: UN INOCULATED BLANK: 2 tubes as the “ uninoculated blank” to which neither std. cyanocobalamine solution, nor test solution. Nor inoculums is added incubate at 64-72hcs. Transfer the contents to photo electric colorimeter tube is adjust the transmittance at 640 nm to 100% record the reading. CALCULATION: On a graph paper, plot the transmittance value against the corresponding levels of std. cyanocobalamine added, draw a smooth curve. Determine The interpolation of a vitamin B12 activity /ml of test solution for each level used. Discard any values falling beyond the range. 90-100% of provisional average, at least 8 to 12 assay values should remain. Calculate the potency from the average of these remaining values.