Absorbed Tatanus Vaccine

Introduction to Absorbed Tatanus Vaccine
Principle, Assay methods of ATV, Preparation, Symptoms,
Causes, Risk factor, Complications
Presnted by
SHAIK GOUSE UL AZAM
Department of Pharmaceuticals Analysis

1. ABSORBED TATANUS VACCINE A Seminar as a part of curricular for Master of Pharmacy, I Year - I semester Presented by SHAIK GOUSE UL AZAM (20L81S0708) PHARMACEUTICAL ANALYSIS Under the guidance of SHAIK SHAKIR BASHA M.Pharm, Assistant Professor Department of Pharmaceutical Analysis

2. Contents Introduction Principle Assay methods of ATV Preparation Symptoms Causes Risk factor Complications References 2

3. Itroduction Vaccine: A preparation of killed micro organisms that are attenuated /living fully virulent organisms that is administration to produce immunity to a particular disease 3

4. Absorded Tatanus vaccine Tatanus is a serious disease caused by a bacterial toxin that affects nervous system, leading to painful muscle contractions, particularly of jaw and neck muscles. Tatanus can interfere with ability to breathe and can threaten life. Tatanus is commonly known as “lockjaw” 4

5. Bio l o g ic a l Ass a y Of T etanus V acc i ne Principle : The potency of tetanus vaccine is determined by administration of the vaccine to animals i.e. guinea-pigs or mice followed administration of either challenge with tetanus toxin or by determining of the titre of antibodies against tetanus Toxoid in the serum of the guinea-pigs. In both cases the potency of the vaccine is calculated by comparison with a reference vaccine, calibrated in International Units. Tetanus vaccine (adsorbed) BRP is calibrated in International Units with reference to the International Standard 5

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7. A. CHALLENGE TOXIN IN GUINEA PIG S E L E C T ION A N D DIS T RI B U T ION OF T E ST A N IMA L S: Healthy guinea pigs from the same stock should be selected weight 250-350 gm. Animals are distributed in not less than 6 equal groups containing number of animals sufficient to obtain results If the validity of the challenge toxin is to be determined 3 further groups of the 5 animals each should be taken which are used as unvaccinated control. Animals should be of same sex if both sex are included then the should be equally distributed among groups. 7

8. SELECTION OF CHALLENGE TOXIN: Preparation of tetanus toxin containing not less than 50 times the 50 percent paralytic dose per millilitre is selected. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the paralytic dose for every assay. PREPARATION OF CHALLENGE TOXIN SOLUTION: The challenge toxin is immediately diluted to 50 times the 50 percent paralytic dose per millilitre with the, peptone buffered saline solution pH 7.4 etc to obtain stable challenge toxin. 8

9. DILU T ION OF THE T E ST AND RE F ER E NCE SOLUTION: The vaccine to be examined and the reference preparation are diluted with the 9g/l solution of NaCl. The dilution forms series which do not differ the by 2.5 folds from the alternating previous and next dilutions. When injected subcutaneously with dose of 1.0 ml per guinea pig should protect approximately 50% of animals from the paralytic effects of tetanus toxin prescribed for test. IMMUNIZATION CHALLENGE AND CHALLENGE: Allocate the dilutions to the groups. Then Inject subcutaneously 1 ml allocated dilution into the guinea pig of respective groups. 9

10. D E T E RMI N A TION OF A C TI V ITY OF THE CH A L L E N GE TOXIN 3 dilutions made from the challenge toxin and each dilution were allocated to 1 group of 5 guinea pigs i.e. 3 groups if necessary. Subcutaneously inject 1mL of allocated solution into each guinea pig of the group. The activity and stability of the challenge toxin are determined by carrying out a suitable number of determinations of the 50 percent paralytic dose. 10

11. REQUIREMENTS FOR A VALID ASSAY The test is not valid unless, for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses of the preparations given to the guinea-pigs, If applicable, the number of paralyzed animals in the 3 groups of 5 injected with the dilutions of the challenge toxin solution indicates that the challenge was approximately 50 times the 50 percent paralytic dose. The confidence limits ( P = 0.95) are not less than 50 per cent and not more than 200 percent of the estimated potency, The statistical analysis shows significant slope and no deviation from linearity and parallelism of the dose. 11

12. READING AND INTERPRETATION OF RESULTS: The guinea-pigs are examined twice daily. Remove and humanely kill all animals showing definite signs of tetanus paralysis. The number of guinea-pigs without paralysis 5 days after injection of the challenge toxin are counted. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the proportion of challenged animals without paralysis in each of the groups of vaccinated guinea-pigs, using the usual statistical methods 12

13. B.CHALLENGE TOXIN IN MICE: S E L E C T ION A N D DIS T RI B U T ION OF T E ST A N IMALS: Use in the test healthy mice from the same stock, about 5 weeks old and from a strain shown to be suitable. Rest same as guinea pig. SELECTION OF CHALLENGE TOXIN: Preparation of tetanus toxin containing not less than 100 times the 50 per cent paralytic dose per millilitre is selected. PREPARATION OF CHALLENGE TOXIN SOLUTION: DILU T I O N OF THE T E ST A N D R E FE R E N CE SOLUTION: IMMUNIZATION CHALLENGE AND CHALLENGE: D E T E RMIN A TION OF A C TI V ITY OF THE CHALLENGE: 13

14. READING AND INTERPRETATION OF RESULTS: The mice are examined twice daily. Remove and humanely kill all animals showing definite signs of tetanus paralysis. The number of mice without paralysis 4 days after injection of the challenge toxin are counted. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the proportion of challenged animals without paralysis in each of the groups of vaccinated mices, using the usual statistical methods. 14

15. C. DETERMINATION OF ANTBODIES IN GUINEA PIG S E L E C T ION A N D DIS T RI B U T ION OF T E ST A N IMALS: Healthy guinea pigs selected of weight 250-350 gm. Animals are distributed in not less than 6 equal groups containing number of animals sufficient to obtain results. Animals should be of same sex if both sex are included then the should be equally distributed among groups. Use a further group of non-vaccinated guinea-pigs of the same origin to provide a negative serum control. If test consistency has been demonstrated, a reference negative serum control may be used. 15

16. REFERENCE PREPARATION: A suitable reference preparation such as tetanus vaccine(adsorbed) BRP or a batch of vaccine shown to be effective in clinical studies, or a batch representative thereof, and which has been calibrated in International Units with reference to tetanus vaccine (adsorbed) BRP or the International Standard for tetanus toxoid (adsorbed) are used. DI L U T ION OF THE T E ST A N D R E FE R E N CE SOL U TIO N : The vaccine to be examined and the reference preparation are diluted with the 9g/l solution of NaCl. The dilution forms series which do not differ the by 2.5 to 5 folds from the alternating previous and next dilutions. Use not fewer than 3 dilutions within the range for example 0.5-16 IU/ml for each series. Use dilutions for immunization preferably wit hin 1 h of preparation. Alloc a te 1 di l ut i on to e a ch group of guinea-pigs. 16

17. IMMUNISATION: Inject subcutaneously in the nape of each guinea-pig 1.0 ml of the dilution allocated to its group. BLOOD SAMPLING: 35-42 days after immunization, take a blood sample from each vaccinated and control guinea-pig using a suitable method. PREPARATION OF SERUM SAMPLES: Avoid frequent freezing and thawing of serum samples. To avoid microbial contamination, it is preferable to carry out manipulations in a laminar-flow cabinet. 17

18. DE T ERMIN A TION OF AN T IBODY TITR E Determine the relative antibody titre or score of each serum sample by a suitable immunochemical method . The methods shown below (enzyme-linked immunosorbent assay (ELISA) and toxin-binding inhibition (ToBI)) have been found suitable. CALCULATION OF POTENCY: Calculate the potency of the vaccine to be examined in International Units relative to the reference preparation, using the usual statistical methods. 18

19. Tatanus vaccine preparation A process for the preparation of tatanus toxoid, which process comprises incubating purified tatanus toxin with 0.2 to 1%(v/v) formaldehyde in the presence of 0.005 to 0.25M lysine for 24 to 32 days at a PH of 6.0 to 8.0 and temperature of 30 to 45  c 19

20. Symptoms Common signs and symptoms of tatanus include: Spasms and stiffness in jaw muscles (trismus) Stiffness of neck muscles Difficulty swallowing Stiffness of abdominal muscles Fever Sweating Elevated blood pressure Rapid heart rate 20

21. Causes Tatanus is caused by a toxin made by spores of bacteria, Clostridium tetani, found in soil, dust and animal feces . When the spores enter a deep flesh wound, they grow into bacteria that can produce a powerful toxin, tetanospasmin. Nearly all cases of tatanus occur in people who have been vaccinated or in adult or in adults who have not kept up with their 10-years booster shots. 21

22. Risk factor Tatanus cases have developed from the following Puncture wounds Gunshot wounds Burns Surgical wounds Injection foot ulcers Dental infections Infected umbilical stumps in newborns born of inadequately vaccinated mothers 22

23. Complications Complications of tatanus infection may include Broken bones: The severity of spasms may causes the spine and other bones to break. Blockage of lung artery (pulmonary embolism): A Blood clot that travel from body can block the main artery of the lung or one of it branches. Death: severe tatanus –induced muscle spasms can interfere with or stop your breathing .lack of oxygen may also induce cardiac arrest and death . Pneumonia is another cause of death. 23

24. Prevention One can easily prevent tatanus by being vaccinated. The primary vaccine series The tatanus vaccine usually is given to childrens as part of the diphtheria and tatanus toxoids and a cellular pertussis vaccine . This vaccination provides protection against three diseases throat and respiratory infection(diphtheria),whooping cough etc. The Dtap vaccine is a series of five shots, typically given in the arms or thigh to childrens at ages:2months,4months,6months,15 to 18months,4 to 6years 24

25. Reference J Hamborsky, A Kroger, S Wolfe, Epidemiology and prevention of vaccine-preventable diseases 2015. V Baldo, P Bonanni, M Castro “combined hexavalent diphtheria –tatanus –acellular pertussis 2014. JH Brown Precision of potency assay of alum-precipitated tetanus toxoid in mice: an interinstitutional study. Journal of Immunology, 1953. J Lyng , G Nyerges The second international standard for tetanus toxoid (adsorbed). Journal of Biological Standardization, 1984. 25

26. Thank you 26