NO772704L - ATOXIC, IMMUNOGENIC PRODUCT OF TETANUS TOXIN - Google Patents
ATOXIC, IMMUNOGENIC PRODUCT OF TETANUS TOXINInfo
- Publication number
- NO772704L NO772704L NO772704A NO772704A NO772704L NO 772704 L NO772704 L NO 772704L NO 772704 A NO772704 A NO 772704A NO 772704 A NO772704 A NO 772704A NO 772704 L NO772704 L NO 772704L
- Authority
- NO
- Norway
- Prior art keywords
- tetanus
- product
- tetanus toxin
- fragment
- toxin
- Prior art date
Links
- 108010055044 Tetanus Toxin Proteins 0.000 title claims description 33
- 229940118376 tetanus toxin Drugs 0.000 title claims description 33
- 230000002163 immunogen Effects 0.000 title claims description 11
- 239000012634 fragment Substances 0.000 claims description 38
- 108091005804 Peptidases Proteins 0.000 claims description 8
- 102000035195 Peptidases Human genes 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 235000019833 protease Nutrition 0.000 claims description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 229960005486 vaccine Drugs 0.000 claims description 6
- 206010043376 Tetanus Diseases 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 230000008105 immune reaction Effects 0.000 claims description 4
- 238000005194 fractionation Methods 0.000 claims description 3
- 238000004255 ion exchange chromatography Methods 0.000 claims description 3
- 239000002808 molecular sieve Substances 0.000 claims description 3
- 231100000252 nontoxic Toxicity 0.000 claims description 3
- 230000003000 nontoxic effect Effects 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- 229960002766 tetanus vaccines Drugs 0.000 claims description 3
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims description 2
- 125000001931 aliphatic group Chemical group 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 229960005367 tetanus antitoxin Drugs 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims 2
- 239000000047 product Substances 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000243 solution Substances 0.000 description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- 108090000526 Papain Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 229940055729 papain Drugs 0.000 description 7
- 235000019834 papain Nutrition 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 208000003217 Tetany Diseases 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 231100000668 minimum lethal dose Toxicity 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 206010041415 Spastic paralysis Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- CWGJBBRZFIPXNZ-UHFFFAOYSA-N [C-]#N.Br Chemical compound [C-]#N.Br CWGJBBRZFIPXNZ-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- -1 diethylaminoethyl cellulose ion Chemical class 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 229960005030 other vaccine in atc Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
Atoksisk, immunogent produkt av tetanus-toksin.Atoxic, immunogenic product of tetanus toxin.
Oppfinnelsen vedrører et atoksi.sk, immunogent produkt som kan fåes av tetanus-toksin ved hjelp av en proteinase. Den vedrører videre en tetanus-vaksine inneholdende det nye produkt. The invention relates to an atoxic, immunogenic product which can be obtained from tetanus toxin by means of a proteinase. It further relates to a tetanus vaccine containing the new product.
Tetanus-toksin er som kjent, høy-toksisk overfor pattedyr og også mennesker og må for dets anvendelse som immu-niserende stoff avgiftes. Det er kjent at tetanus-toksin er å avgifte ved at det behandles med formaldehyd. Det er videre kjent at et atoksisk, immunogent produkt kan fåes ved behandling av tetanus-toksin med en proteinase. Dette atoksiske, immunogene produkt betegnes i den vitenskapelige litteratur som fragment C av tetanus-toksinet. As is known, tetanus toxin is highly toxic to mammals and also to humans and must be detoxified for its use as an immunizing substance. It is known that tetanus toxin can be detoxified by treating it with formaldehyde. It is further known that an atoxic, immunogenic product can be obtained by treating tetanus toxin with a proteinase. This atoxic, immunogenic product is referred to in the scientific literature as fragment C of the tetanus toxin.
Det er nå overraskende funnet at ved behandling av tetanus-toksin med en proteinase, fortrinnsvis med papain, opp-står et ytterligere kjemisk entydig karakteriserbart stoff med spesielt fordelaktige egenskaper som kan isoleres.etter kjente biokjemiske fremgangsmåter. It has now surprisingly been found that by treating tetanus toxin with a proteinase, preferably with papain, a further chemically unambiguously characterizable substance with particularly advantageous properties is produced which can be isolated according to known biochemical methods.
Til de spesielt fordelaktige egenskaper av det som fragment Bo betegnede produkt hører dels manglende toksisitet og dets immunogenitet. Disse egenskaper gjør produktet egnet som bestanddel av vaksiner mot tetanus. The particularly advantageous properties of the product known as fragment Bo include its lack of toxicity and its immunogenicity. These properties make the product suitable as a component of vaccines against tetanus.
Oppfinnelsens gjenstand er følgelig et atoksisk, immunogent produkt oppnådd fra tetanus-toksin ved dets behandling med en proteinase, fortrinnsvis papain, idet produktet erkarakterisert vedfølgende parametre: The object of the invention is consequently an atoxic, immunogenic product obtained from tetanus toxin by its treatment with a proteinase, preferably papain, the product being characterized by the following parameters:
1. Sedimentasjonskonstant S°ow5,66-0,341. Sedimentation constant S°ow5.66-0.34
2. Immunologisk reaksjon med tetanus-antitoksin:2. Immunological reaction with tetanus antitoxin:
partielt identisk med det av tetanus-toksin.partially identical to that of tetanus toxin.
3. Immunologisk reaksjon med fragment C: ikke identisk.3. Immunological reaction with fragment C: not identical.
4. Molekylvekt, bestemt ved gel-elektroforese i natriumdodecylsulfat, 95.000 - 5000. 5. Ved reduksjon spaltbart i en underenhet med en molekylvekt på 45.000 - 2.500 4. Molecular weight, determined by gel electrophoresis in sodium dodecyl sulfate, 95,000 - 5,000. 5. Cleavable by reduction into a subunit with a molecular weight of 45,000 - 2,500
og en med det kjente derivat av de lette kjeder av tetanus-toksin immunologisk identisk underenhet med and one with the known derivative of the light chains of tetanus toxin immunologically identical subunit with
en molekylvekt på 48.000 2.500 (hver gang bestemta molecular weight of 48,000 2,500 (each time determined
i natrium-dodecylsulfat-elektroforese).in sodium dodecyl sulfate electrophoresis).
De i parametrene gjengitte svingningsbredder beror på feilgrenser innen bestemmelsesmetodene. The fluctuation widths given in the parameters are based on error limits within the determination methods.
Sedimentasjonskonstantene ble bestemt i en vandig opp-løsning av 0,2M NaCl, 0,02M Na2H P04, 0,03M Na H2P04ved en pH The sedimentation constants were determined in an aqueous solution of 0.2M NaCl, 0.02M Na2HPO4, 0.03M NaH2PO4 at a pH
på 6,8 ifølge T. Svendberg og K.O. Pedersen "The Ultra-centrifuge", The Clarendon Press, Oxford, 1940, i en oversiktningscelle av en analyttisk ultrasentrifuge i tilknytning til Vinograd, Proc.Acad. Sei. USA, 49, 902 (1963). Oversiktningen av den prøveoppløsning of 6.8 according to T. Svendberg and K.O. Pedersen "The Ultra-centrifuge", The Clarendon Press, Oxford, 1940, in an overview cell of an analytical ultra-centrifuge associated with Vinograd, Proc.Acad. Pollock. USA, 49, 902 (1963). The overview of the sample solution
som inneholder produktet ifølge oppfinnelsen foregikk på 1 M Nacl-oppløsning av pH-verdi 7,0. containing the product according to the invention took place in 1 M NaCl solution of pH value 7.0.
Den immunologiske reaktivitet ble undersøkt under anvendelse av antisera, som ble dannet ved immunisering av kaniner med tetanus-toksid eller fragment C ifølge oppfinnelsen. Det ble anvendt den kjente dobbelt-diffusjonsteknikk. Ved hjelp av denne teknikk kan det vises at det tidligere omtalte fragment C at-skiller seg entydig fra fragment B ifølge oppfinnelsen. Som det videre kunne vises er fragment B å oppdele i to spaltstykker hvorav det ene viser det samme immunologiske forhold som deri-vatet av den lette kjede som er omtalt i tysk patent nr. (tysk søknad nr. P 25 57-047 /HOE 74/B 028). The immunological reactivity was investigated using antisera, which were formed by immunization of rabbits with tetanus toxide or fragment C according to the invention. The known double diffusion technique was used. With the help of this technique, it can be shown that the previously mentioned fragment C is distinctly different from fragment B according to the invention. As could be further shown, fragment B can be divided into two fragments, one of which shows the same immunological relationship as the derivative of the light chain which is mentioned in German patent no. (German application no. P 25 57-047 /HOE 74 /B 028).
Gel-elektroforesen i en 7%-ig polyakrylamidgel under anvendelse av natriumdodecylsulfat som bestanddel av elektroforese-pufferen ble gjennomført ifølge K. Weber og M. Osborn, J.Biol. Chem., Vol. 244, 4406 - 4412 (1969). Før gjennomføringen av gel-elektrof oresen ble prøvene som skulle analyseres i vandig oppløs-ning blandet med natriumdodecylsulfat inntil en konsentrasjon på The gel electrophoresis in a 7% polyacrylamide gel using sodium dodecyl sulfate as a component of the electrophoresis buffer was carried out according to K. Weber and M. Osborn, J.Biol. Chem., Vol. 244, 4406-4412 (1969). Before carrying out the gel electrophoresis, the samples to be analyzed in aqueous solution were mixed with sodium dodecyl sulfate until a concentration of
1% og holdt 1 minutt i kokende vann. Beregningen av molekylvekten foregikk ved sammenlikning med standardstoffer for molekylvekt-bestemmelser (aldolase og katalase, oppnådd gjennom firmaet Boehringer, Mannheim, Kat. Nr. 15 575 - "Eichproteine Grosse II"). Beregningsmetoden er angitt i ovennevnte arbeid av Weber og Osborn. 1% and held for 1 minute in boiling water. The calculation of the molecular weight took place by comparison with standard substances for molecular weight determinations (aldolase and catalase, obtained through the company Boehringer, Mannheim, Cat. No. 15 575 - "Eichproteine Grosse II"). The calculation method is indicated in the above-mentioned work by Weber and Osborn.
Det nye atoksiske, immunogene som fragment B av tetanus-toksin betegnede produkt er oppnåelig etter den i tysk søknad P 25 10 987 (HOE 73/B 018) omtalte fremgangsmåte ved behandling av tetanus-toksin med en proteinase. The new atoxic, immunogenic product designated as fragment B of tetanus toxin can be obtained by the method described in German application P 25 10 987 (HOE 73/B 018) by treating tetanus toxin with a proteinase.
I en foretrukket utførelsesform oppnås fragment B ved behandling av tetanus-toksin, som kan fremstilles fra kultur-filtrater av Clostrium-Tetani ved ioneutvekslingskromatografi med papain, fortrinnsvis i bærebundet form i nærvær av et redu-serende stoff. In a preferred embodiment, fragment B is obtained by treating tetanus toxin, which can be prepared from culture filtrates of Clostrium Tetani by ion exchange chromatography with papain, preferably in carrier-bound form in the presence of a reducing substance.
Det har da vist seg nyttig å velge inkubasjonstempera-turen høyere enn vanlig, d.v.s. mellom 30 og 60°C, fortrinnsvis mellom 45 og 55°C. Ved denne forholdsregel kan utbyttet av produktet ifølge oppfinnelsen økes. Etter den proteolyttiske inn-virkning på tetanus-toksinet fjernes det bærebundne enzym fra reaksjonsblandingen hensiktsmessig ved sentrifugering, og den gjengivende oppløsning underkastes en molekylsiktfraksjonering. Fordelaktig hardet derved vist seg anvendelsen av med epiklorhy-(R) It has then proved useful to choose the incubation temperature higher than usual, i.e. between 30 and 60°C, preferably between 45 and 55°C. With this precaution, the yield of the product according to the invention can be increased. After the proteolytic action on the tetanus toxin, the carrier-bound enzyme is removed from the reaction mixture appropriately by centrifugation, and the resulting solution is subjected to molecular sieve fractionation. Advantageously, the application of with epichlorohy-(R)
drin-nettdannet dextran, eksempelvis "Sephadex G 100 fra firmaet Pharmacia, Uppsala eller"Ultrogel(R)" fra LKB, Bromma drin-netted dextran, for example "Sephadex G 100 from the company Pharmacia, Uppsala or "Ultrogel(R)" from LKB, Bromma
(R) (R)
eller "Bio-Gel p fra Bio-Rad Laboratories, Richmond, Calif.or "Bio-Gel p from Bio-Rad Laboratories, Richmond, Calif.
I tilfellet at det anvendes et oppløselig enzymprepar-at skjer atskillelsen av enzymet ved gjennomføring av molekyl-siktf raksjoneringen. In the event that a soluble enzyme preparation is used, the separation of the enzyme takes place by carrying out the molecular sieve fractionation.
Så snart fragment B en gang.er oppnådd på denne måten, lar det seg dermed fremstille et antiserum ved immunisering av forsøksdyr med fragment B, med hvis hjelp det på vanlig måte kan fremstilles en immunadsorbens. Denne er igjen egnet til å utvinne fragment B i spesielt ren form fra inkubasjonsblandingen av nevnte fermentasjonsoppløsning. As soon as fragment B has once been obtained in this way, it is thus possible to prepare an antiserum by immunizing experimental animals with fragment B, with the help of which an immunoadsorbent can be prepared in the usual way. This in turn is suitable for extracting fragment B in a particularly pure form from the incubation mixture of said fermentation solution.
Også fjerning av fragment C eller tetanus-toksin fra en blanding med fragment B, lar seg oppnå immunadsorptivt. Dertil fremstilles i første rekke et antiserum mot det kjente fragment C. Med dette antiserum kan det på vanlig måte fremstilles et immunadsorbens. Dette er igjen egnet til å fjerne rester av fragment C og av tetanus-toksin fra fragment B, da såvel fragment C som også tetanus-toksin reagerer med anti-fragment C-serum, imidlertid„;.ikke fragment B. ■ Also, removal of fragment C or tetanus toxin from a mixture with fragment B can be achieved immunoadsorptively. In addition, an antiserum against the known fragment C is first prepared. With this antiserum, an immunoadsorbent can be prepared in the usual way. This is again suitable for removing residues of fragment C and of tetanus toxin from fragment B, as both fragment C and tetanus toxin react with anti-fragment C serum, but not fragment B.
Andre isoleringsf remgangsmåter som o står til disposi-sjon for fagfolk til atskillelse av protein av forskjellig .elek- trisk ladning fører likeledes til resultat. Det er spesielt elek-troforetisk og ioneutvekslings-kromatografi-fremgangsmåter. Other isolation methods that are available to professionals for the separation of proteins of different electrical charge likewise lead to results. It is particularly electrophoretic and ion exchange chromatography methods.
Dette på denne måten oppnådde rene fragment B av tetanus-toksin viser seg sammenliknet med tetanus-toksin som ikke toksisk. Mens man ved tetanus-toksin pr. mg påviser 30 x 10g minimal dødelig dose, har fragment B 5 minimal dødelig dose pr.mg. Fragment B viser seg med hensyn til den toksiske foreteelse helt forskjellig til tetanus-toksin, den viser ikke de ved spastisk paralyse karakteriserte foreteelser. På tross av nedsettelse av toksisiteten til 1 : 6 x 10 er det hensiktsmessig som omtalt i det følgende å behandle fragment B med et aldehyd. This thus obtained pure fragment B of tetanus toxin proves to be non-toxic compared to tetanus toxin. While with tetanus toxin per mg shows 30 x 10g minimal lethal dose, fragment B has 5 minimal lethal dose per mg. Fragment B is completely different from tetanus toxin with regard to the toxic phenomenon, it does not show the phenomena characterized by spastic paralysis. Despite reducing the toxicity to 1:6 x 10, it is appropriate, as discussed below, to treat fragment B with an aldehyde.
For denne behandling behandles det ved hjelp av proteinase fremstilte produkt etter dets isolering med en proteinkonsen-trasjon på ca. 1 mg protein pr. ml. eller mindre enn en puffer-oppløsning på pH-verdi 6,0 - 8,5, fortrinnsvis 7,8, og en molaritet av pufferoppløsningen på 0,01 - 0,2 M med 0,015 - 0,3 M av et alifatisk mono- eller dialdehyd med 1-6 karbonatomer, fortrinnsvis formaldehyd i 14 - 28 dager ved 20 - ^37°C. Produktet kan hvis ønsket underkastes en 10 - 20, fortrinnsvis 15 timers dialyse mot en fysiologisk tålbar oppløsning som 0,15 molar natriumklorid og/eller sterilfiltreres. For fremstilling av en vaksine blandes produktet hensiktsmessig dessuten med et adju-vans, f.eks. aluminiumhydroksyd. Ved fortynning med en fysiologisk tålbar oppløsning som 0,15 molar natriumkloridoppløsning inn-stilles konsentrasjonen på det ønskede antigeninnhold. For this treatment, the product produced by proteinase is treated after its isolation with a protein concentration of approx. 1 mg of protein per ml. or less than a buffer solution of pH 6.0 - 8.5, preferably 7.8, and a molarity of the buffer solution of 0.01 - 0.2 M with 0.015 - 0.3 M of an aliphatic mono- or dialdehyde with 1-6 carbon atoms, preferably formaldehyde for 14 - 28 days at 20 - ^37°C. If desired, the product can be subjected to a 10 - 20, preferably 15 hour dialysis against a physiologically tolerable solution such as 0.15 molar sodium chloride and/or sterile filtered. For the production of a vaccine, the product is suitably mixed with an adjuvant, e.g. aluminum hydroxide. When diluting with a physiologically tolerable solution such as 0.15 molar sodium chloride solution, the concentration is set to the desired antigen content.
Denne vaksine kan anvendes alene, men også i kombina-sjon med andre vaksiner. Til kombinasjonen egner det seg spesielt difterietoksoid, pertussisimmunogen, poliomyelittvira eller meslinger-vira. Ved adsorpsjonsforsøk kan man vise at om-trent halvparten av alle mot tetanus-toksin rettede beskyttende antilegemer er rettet mot fragment B. This vaccine can be used alone, but also in combination with other vaccines. Diphtheria toxoid, pertussis immunogen, poliomyelitis viruses or measles viruses are particularly suitable for the combination. In adsorption experiments, it can be shown that approximately half of all protective antibodies directed against tetanus toxin are directed against fragment B.
Oppfinnelsen skal forklares nærmere ved hjelp av noen eksempler. The invention will be explained in more detail with the help of some examples.
Eksempel 1.Example 1.
Clostrium-Tetani fermenteres i et latam-medium. Det derved dannede toksin absorberes på dietylaminoetylcellulose-ioneut-veksler og elueres ved hjelp av en gradientøkende molaritet av en Clostrium-Tetani is fermented in a latam medium. The toxin thus formed is absorbed on a diethylaminoethyl cellulose ion exchanger and eluted using a gradient increasing molarity of a
fosfatpuffer av pH-verdi 7 fra 0,01 mol til 0,4 mol. Fraksjonene phosphate buffer of pH value 7 from 0.01 mol to 0.4 mol. The factions
.som inneholder toksinet oppdeles igjen ved en kromatografi på en .which contains the toxin is divided again by a chromatography on a
søyle som er fylt med "Sephadex(R)" G 100 og fraksjoner som inneholder toksinet utvinnes og forenes. column packed with "Sephadex(R)" G 100 and fractions containing the toxin are recovered and combined.
2,5 g tetanus-toksin i en oppløsning med en sluttkonsen-trasjon på 15 mg tetanus-toksin/ml av en 0,1 mol fosfatpuffer pH 6,5 inneholdende 0,01 mol Na2EDTA og 0,001 mol cystein HC1 blandes med 40 mg papain. Papainet inneholder 30 enheter enzym-atrisk aktivitet/mg stoff. I første rekke holdes blandingen i en time ved 45°C, deretter ytterligere i 2 timer ved 55°C. 2.5 g of tetanus toxin in a solution with a final concentration of 15 mg tetanus toxin/ml of a 0.1 mol phosphate buffer pH 6.5 containing 0.01 mol Na2EDTA and 0.001 mol cysteine HCl are mixed with 40 mg papain . Papain contains 30 units of enzymatic activity/mg substance. First, the mixture is held for one hour at 45°C, then for a further 2 hours at 55°C.
For spaltningen av tetanus-toksinet kan det fortrinnsvis også anvendes bærebundet papain. Carrier-bound papain can preferably also be used for the cleavage of the tetanus toxin.
Hertil sammenbringer en mot 0,1 mol Na^O^-puffer av pH-verdi 10,0 dialysert oppløsning av 500 ml papain med 50 mg/ml med en ved hjelp av cyanbromid-aktivert agarosegel og hensettes i 24 timer ved 4°C. Det flere ganger med en 4 mol urinstoff og 0,5 mol NaCl pr. liter holdige oppløsning vaskede reaksjonspro-dukt anvendes analogt til det oppløselige enzym for den proteolyttiske spaltning av tetanus-toksinet. For this, a dialyzed solution of 500 ml of papain with 50 mg/ml against 0.1 mol of Na^O^ buffer of pH value 10.0 is combined with an agarose gel activated by means of cyanide bromide and left for 24 hours at 4°C . It several times with a 4 mol urea and 0.5 mol NaCl per liters of solution containing washed reaction product are used analogously to the soluble enzyme for the proteolytic cleavage of the tetanus toxin.
Etter blandingens avkjøling inndampes volumet ved hjelp av ultrafilter og deretter påføres på en 10 x 100 cm lang After the mixture has cooled, the volume is evaporated using an ultrafilter and then applied to a 10 x 100 cm long
(R) (R)
søyle, fylt med "Sephadex " G 100. Elueringen foregår med 0,1 M trishydroksymetylaminometan-saltsyrepuffer av pH-verdi 8,0, inneholdende 1 mol NaCl. column, filled with "Sephadex" G 100. The elution takes place with 0.1 M trishydroxymethylaminomethane hydrochloric acid buffer of pH value 8.0, containing 1 mol of NaCl.
Under elueringen foregår kontinuerlig måling av adsorp-sjonen i området til bølgelengdene på 280 nm. Fraksjonene som i dette området viser en adsorpsjon, oppfanges atskilt. Det opp-står 4 fraksjoner, idet den første danner en dobbelttopp. During the elution, continuous measurement of the adsorption takes place in the range of wavelengths of 280 nm. The fractions that show adsorption in this area are collected separately. There are 4 fractions, the first forming a double peak.
Ved en gjentatt kromatografi under de samme betingelser trekkes dobbelttoppen fra hverandre og endelig isoleres 2. topp. Denne inneholder det ifølge oppfinnelsen atoksiske, immunogene produkt, fragment B av tetanus-toksin. In a repeated chromatography under the same conditions, the double peak is pulled apart and finally the 2nd peak is isolated. This contains the atoxic, immunogenic product according to the invention, fragment B of tetanus toxin.
Spesielt enkelt lar fragment B ifølge oppfinnelsen seg utvinne når man binder antiserum rettet mot det kjente fragment C (10 ml med 1000 IU/ml) på vanlig måte til Br-CN-aktivert agarose og deretter sammenblander et ved gel-kromatografi partielt renset preparat av fragment B med et slikt immunadsorbens. Etter 60 minutters omrøring atskilles gelen de dertil bundne forurens-ninger og fragment B utvinnes ved hjelp av en gjentatt gel-kromatograf i . Fragment B according to the invention can be recovered particularly easily when you bind antiserum directed against the known fragment C (10 ml with 1000 IU/ml) in the usual way to Br-CN-activated agarose and then mix together a preparation partially purified by gel chromatography of fragment B with such an immunoadsorbent. After 60 minutes of stirring, the gel is separated from the contaminants bound to it and fragment B is recovered with the aid of a repeated gel chromatograph in .
Det er også mulig å nøytralisere forurensningene ved tilsetning av antifragment C-antiserum (IgG fraksjon) og å at-skille immunkomplekset ved gel-kromatografi fra fragment B. It is also possible to neutralize the contaminants by adding antifragment C antiserum (IgG fraction) and to separate the immune complex from fragment B by gel chromatography.
Endelig lar fragment B ifølge oppfinnelsen seg også fremstille når det ved hjelp av et engang fremstillet fragment B over et antiserum fremstilles et immunadsorbens for fragment B. Man kan derved betjene seg av kjente fremgangsmåter. Ved hjelp av immunadsorbenset adsorberes fragment B selektivt fra blandingen med andre reaksjonsprodukter fra proteinasebehandlingen av tetanus-toksinet selektivt, hvoretter det kan elueres selektivt fra immunadsorbens. Finally, according to the invention, fragment B can also be prepared when an immunoadsorbent for fragment B is prepared with the help of a once-prepared fragment B over an antiserum. One can thereby make use of known methods. Using the immunoadsorbent, fragment B is selectively adsorbed from the mixture with other reaction products from the proteinase treatment of the tetanus toxin, after which it can be selectively eluted from the immunoadsorbent.
Eksempel 2.Example 2.
Fragment B fortynnes med 0,1 molar fosfatpufferoppløs-ning av pH-verdi 6,5 til 200 yug pr. ml og blandes med 0,06% formaldehyd og hensettes ved 37 oC i 21 dager. Deretter dialyseres mot et flere ganger volum 0,15 natriumkloridoppløsning i 16 timer og forarbeides på vanlig måte til en vaksine. Fragment B is diluted with 0.1 molar phosphate buffer solution of pH value 6.5 to 200 yug per ml and mixed with 0.06% formaldehyde and left at 37 oC for 21 days. It is then dialysed against a several times volume of 0.15 sodium chloride solution for 16 hours and processed in the usual way into a vaccine.
Claims (8)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2634584A DE2634584C3 (en) | 1976-07-31 | 1976-07-31 | Atoxic, immunogenic product from tetanus toxin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NO772704L true NO772704L (en) | 1978-02-01 |
Family
ID=5984461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO772704A NO772704L (en) | 1976-07-31 | 1977-07-29 | ATOXIC, IMMUNOGENIC PRODUCT OF TETANUS TOXIN |
Country Status (18)
| Country | Link |
|---|---|
| JP (1) | JPS5326319A (en) |
| AT (1) | AT362498B (en) |
| AU (1) | AU516191B2 (en) |
| BE (1) | BE857373A (en) |
| CA (1) | CA1099635A (en) |
| DE (1) | DE2634584C3 (en) |
| DK (1) | DK342577A (en) |
| EG (1) | EG12732A (en) |
| FI (1) | FI58500C (en) |
| FR (1) | FR2360315A1 (en) |
| GB (1) | GB1570958A (en) |
| IE (1) | IE45539B1 (en) |
| IL (1) | IL52617A (en) |
| IT (1) | IT1085660B (en) |
| LU (1) | LU77874A1 (en) |
| NL (1) | NL7708279A (en) |
| NO (1) | NO772704L (en) |
| SE (1) | SE7708693L (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8516442D0 (en) * | 1985-06-28 | 1985-07-31 | Wellcome Found | Cloned antigen |
-
1976
- 1976-07-31 DE DE2634584A patent/DE2634584C3/en not_active Expired
-
1977
- 1977-07-26 NL NL7708279A patent/NL7708279A/en not_active Application Discontinuation
- 1977-07-27 EG EG446/77A patent/EG12732A/en active
- 1977-07-27 AU AU27380/77A patent/AU516191B2/en not_active Expired
- 1977-07-28 FI FI772305A patent/FI58500C/en not_active IP Right Cessation
- 1977-07-28 SE SE7708693A patent/SE7708693L/en not_active Application Discontinuation
- 1977-07-29 GB GB31968/77A patent/GB1570958A/en not_active Expired
- 1977-07-29 IE IE1590/77A patent/IE45539B1/en unknown
- 1977-07-29 DK DK342577A patent/DK342577A/en not_active Application Discontinuation
- 1977-07-29 JP JP9213677A patent/JPS5326319A/en active Pending
- 1977-07-29 IL IL52617A patent/IL52617A/en unknown
- 1977-07-29 NO NO772704A patent/NO772704L/en unknown
- 1977-07-29 LU LU77874A patent/LU77874A1/xx unknown
- 1977-07-29 CA CA283,802A patent/CA1099635A/en not_active Expired
- 1977-07-29 AT AT563477A patent/AT362498B/en not_active IP Right Cessation
- 1977-07-29 IT IT26354/77A patent/IT1085660B/en active
- 1977-08-01 FR FR7723600A patent/FR2360315A1/en active Granted
- 1977-08-01 BE BE179821A patent/BE857373A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FI58500C (en) | 1981-02-10 |
| FR2360315B1 (en) | 1980-04-11 |
| IE45539B1 (en) | 1982-09-22 |
| DE2634584B2 (en) | 1979-03-22 |
| DK342577A (en) | 1978-02-01 |
| DE2634584C3 (en) | 1979-11-08 |
| GB1570958A (en) | 1980-07-09 |
| JPS5326319A (en) | 1978-03-11 |
| AU516191B2 (en) | 1981-05-21 |
| IL52617A0 (en) | 1977-10-31 |
| EG12732A (en) | 1979-09-30 |
| IE45539L (en) | 1978-01-31 |
| NL7708279A (en) | 1978-02-02 |
| FI772305A (en) | 1978-02-01 |
| SE7708693L (en) | 1978-02-01 |
| DE2634584A1 (en) | 1978-02-02 |
| BE857373A (en) | 1978-02-01 |
| IT1085660B (en) | 1985-05-28 |
| CA1099635A (en) | 1981-04-21 |
| LU77874A1 (en) | 1978-02-02 |
| FI58500B (en) | 1980-10-31 |
| AU2738077A (en) | 1979-02-01 |
| IL52617A (en) | 1980-12-31 |
| ATA563477A (en) | 1980-10-15 |
| AT362498B (en) | 1981-05-25 |
| FR2360315A1 (en) | 1978-03-03 |
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