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ON THE PRODUCTION OF DIPHTHERIA AND TETANUS TOXOIDS FREE FROM MEDIUM SENSITIZING
AGENTS

Master Theses from / 2004-10-27 10:25:04
Oleh : A. Koesdarminta, S2 - Pharmacy
Dibuat : 1979-00-00, dengan 1 file

Keyword : the elimination of antigens originating
from the culture medium employed.
Nomor Panggil (DDC) : T 615.373 KOE
Sumber pengambilan dokumen : 91/472

Seperti diketahui pengebalan dapat memberikan reaksi sampingan yang merugikan dan salah satu diantaranya ialah reaksi kepekaan yang berlebihan. Menghilangkan sama sekali reaksi ini tentu saja tidak mungkin, sebab antigen sendiri yang diperlukan juga dapatmenyebabkannya. Karena itu cukup baiklah apabila berhasil untuk memperkecil reaksi kepekaan lebih semaksimum mungkin atau dengan lain perkataan mengurangi antigen yang tidak diperlukan sebanyak-banyaknya. Penelitian ini dimaksudkan untuk menghilangkan akibat dari antigen tambahan yang berasal dari medium. Hal ini dapat dicapai dengan memisahkan diphtheria dan tetanus toxoid dari lain-lain antigen yang terdapat di medium pertumbuhan, jadi suatu cara pemurnian. Atau dibuatnya suatu medium yang tidak mengandung zat-zat penyebab kepekaan. Akan tetapi caranya harus dipilih yang sesuai untuk produksi secara "batch". Pada pemurnian dipakai toxoid yang berasal dari medium yang dipergunakan untuk produksi dan ini mengandung daging sapi yang telah dicerna oleh papain untuk diphtheria, atau ekstrak jantung sapi untuk tetanus. Ternyata medium ini memang mengandung zat penyebab kepekaan yang berasal dari sapi, jadi zat ini harus dihilangkan. Karena hasil penguraian papain dapat bervariasi dari molekul yang kecil hingga yang besar, maka pemisahannya amat sukar. Oleh sebab itu pada tiap-tiap tingkat proses pengerjaan, harus diteliti kemurniannya. Analisa yang dipakai untuk kemurnian ialah immunodifusi, immunoforesa, elektroforesa disk, analisa ultrasentrifuga dan uji antigenik (Lf) per mg protein yang tidak terdialisa. Kepekaan ditunjukkan dengan reaksi anafilaktik sistemik, reaksi aktip anafilaktik kutan dan reaksi pasip anafilaktik kutan. Adapun cara-cara pemurnian yang dilakukan ialah sebagai berikut :
Cara pemurnian yang pertama dimulai dengan pembuatan toxoid dari filtrat perbenihan bakteri. Kemudian ini di ultrafiltrasi lewat membrana kolodium dan diteruskan dengan fraksionasi ammonium sulfat. Fraksi yang aktip ialah antara 20%- 35% (b/v) untuk diphtheria sedangkan untuk tetanus ialah antara 13%-23% (b/v). Ini didialisa sehingga diperoleh bahan baku (bulk) untuk pembuatan vaksin. Ternyata larutan ini masih mengandung zatzat kepekaan yang berasal dari sapi. Maka usaha pemurnian diteruskan dengan kromatografi kolom dengan DEAE sellulosa dan kromatografi kolom dengan sephadex G 200. Fraksi toxoid yang terpekat dan paling sedikit atau sama sekali tidak mengandung zat-zat kepekaan diambil. Untuk mempertajam urutan 2 kolom sepharose 4B. Cara pemurnian yang kedua ialah alhydrogel dicampur dengan toxoid yang diendapkan dengan ammonium sulfat, dengan maksud agar supaya toxoid terpisah dari zat-zat kepekaan sapi, berdasarkan perbedaan dalam konstanta adsorpsi. Cara pemurnian yang ketiga ialah dengan jalan menyaring bakteri dari mediumnya, sehingga toxin yang masih terdapat dalam sel-sel bakteri dengan sendirinya dapat terpisah dari zat-zat kepekaan medium. Kemudian toxin diekstrak dari bakteri dengan larutan hypertonik, kemudian dijadikan toxoid dan dimurnikan menurut cara pemurnian yang pertama. Mengenai medium digunakan medium yang tidak mengandung zat-zat penyebab kepekaan, karena itu dipilih hasil hidrolisa protein yang mengandung peptida-peptida yang tidak dapat menyebabkan terjadinya zat anti. Dapat pula dipakai medium yang terdiri dari asam-asam amino saja, misalnya "Bacto casamino acids". Dengan sendirinya pada medium semi sintetik ini hendaknya dipergunakan bakteri yang telah teradaptasi terhadapnya.

Deskripsi Alternatif :

It is known that active immunization occasionally may induce harmful side-effects, one of which may be a hypersensitive reaction.In such par ticular cases it would be impossible to eliminate this reaction completely, since it may be caused by the used antigen itself in the vaccine. One of the possible measures to minimize hypersensitive reaction is to try to reduce the excess of unnecessary antigens as much as possible. The following experiments describe the elimination of antigens originating from the culture medium employed. This is achieved by separating the sensitizing agents already present in the medium from the diphtheria and tetanus toxoids or by preparing a medium which is free from sensitizing agents. It is essential that the selected method is suitable for batch method production.
In the purification procedure a toxoid is used originating from a medium usually used for routine production, containing papain digested beef meator extracted beef heart. It is shown that this kind of medium contains sensitizing agents originating from beef. Therefore these agents should be eliminated. Since digestion by papain may result in the formation of small as well as large molecules, the separation of sensitizing agents is very difficult and complicated; the products of each step of the purification procedure should have been examined for their purity and hypersensitivity. Analysis for purity is done by performing the immunodiffusion test, immunophoresis, disc electrophoresis, ultra centrifugal analysis and an antigenic test (Lf) per mg non-dialyzable protein. Hypersensitivity is demonstrated by systemic anaphylactic reaction, active cutaneous anaphylactic reaction and passive cutaneous anaphylactic reaction. The first purification procedure has been performed as follows:
The first step is the conversion of the toxin in the bacterial culture filtrate into toxoid followed by ultra-filtration through a collodion membrane and finally by ammonium sulfate fractionation. After dialyzing, the solution is used as a bulk solution for vaccine production. However, according to recent investigation, especially with diphtheria toxoid and most probably also with tetanus toxoid, this bulk solution may still contain beef sensitizing agents. Therefore the purification procedure should be continued by applying column chromatography using DEAE cellulose and Sephadex G 200 gels. Then a fraction is collected which contains the highest concentration of toxoid and which is free from or contains the minimum amount of beef sensitizing agents. If sensitizing agents are still present or if it is necessary to increase the purity of the final product, then column chromatography should be repeated by using another kind of gel, such as Sepharose 4B and by using two column in series. The second purification procedure is the adsorption of the ammonium sulfate precipitated toxoid into alhydrogel in order to separate the toxoid from sensitizing agents based in the differences of their adsorption properties. The third purification procedure is by filtering the bacteria from the culture medium. In this case the toxin still present in the bacteria cells, will be automatically separated from the medium sensitizing agents. The toxin is then extracted from the bacteria with a hypertonic solution and converted into toxoid which is then purified according to the first purification procedure. As far as the medium is concerned, this experiment is carried out on a medium free from sensitizing agents. For this purpose a production is satisfactory obtained from protein hydrolysis is selected. This medium consists of peptides which are unable to produce antibody response. Or an alternatively employed medium is the semisynthetic medium containing only amino acids such as Bacto Casamino acids. To increase the titre of the produced toxin by using a suitable semisynthetic culture medium, it would be better to employ bacteria which has been adapted to the particular medium. If we examine the results, we come to the conclusion, that the following procedure is the best. The active ammonium sulfate fraction in tetanus toxoid, contains hardly any sensitizing agents from the culture medium. These sensitizing agents could be eliminated by further purification with column chromatography using Sephadex G 200. An advanced method of diphtheria toxoid purification,could eliminate about 75% of the sensitizing agents. For this reason,perhaps a culture medium which does not contain sensitizing agents, for instance the so called Norlin's medium, is the desired solution. The used adapted strain is the DV 5-4
In submerged culture, with a little change in the medium, the titre of the diphtheria toxin obtained is 100 Lf ml and the flocculation time is about 11 minutes. This means that this procedure of production is satisfactory.

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