CS228860B1 - Mouse lymphocytic hybridom producing an antisubstance a - Google Patents
Mouse lymphocytic hybridom producing an antisubstance a Download PDFInfo
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- CS228860B1 CS228860B1 CS593382A CS593382A CS228860B1 CS 228860 B1 CS228860 B1 CS 228860B1 CS 593382 A CS593382 A CS 593382A CS 593382 A CS593382 A CS 593382A CS 228860 B1 CS228860 B1 CS 228860B1
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- hybridoma
- antisubstance
- hybridom
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- 230000000527 lymphocytic effect Effects 0.000 title 1
- 210000004408 hybridoma Anatomy 0.000 claims description 15
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 claims description 7
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 claims description 7
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 239000012581 transferrin Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000003200 peritoneal cavity Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- 210000004754 hybrid cell Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102000007238 Transferrin Receptors Human genes 0.000 description 1
- 108010033576 Transferrin Receptors Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000002729 polyribosome Anatomy 0.000 description 1
- 239000011833 salt mixture Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Vynález se týká nového hybridomu, tj. hybridního jednobuněčného organismu, sestrojeného fúzí buňky myší myelomové linie P3-X63-Ag8.653 a myší slezinné lymfoidní buňky, produkující protilátku proti lidskému transferinu.The invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag8.653 and a mouse spleen lymphoid cell producing an antibody against human transferrin.
Doposud se protilátky proti transferinu vyrábějí tak, že je transferin opakovaně injikován jako antigen pokusným zvířatům, nejčastěji králíkům a prasatům. Sérum takto imunizovaných zvířat, odebírané po určité době působení antigenu, slouží jako zdroj protilátek, užívaných zejména pro kvantitativní stanovení plasmatického transferinu metodou jednoduché radiální imunodifúze. Tento postup, nazývaný konvenční imunizací, má několik nevýhod. V séru imunizovaných zvířat se nachází heterogenní směs protilátek, jejichž spektrum je v každém jednotlivém organismu různé a neopakovatelné. Organismus zpravidla vytvoří kromě protilátek vůči žádanému antigenu i protilátky proti nečistotám antigenního preparátu; ty je nutné ze sér odstraňovat vysycováním. Výrobní šarže konvenčních sér se proto dají těžko standardizovat a vycházejí z výroby v širokém rozmezí kvality. Pro výrobu Každé šarže je třeba připravit čistý imunizační antigen a další antigeny pro vy2 sycení balastních protilátek proti nečistotám.To date, anti-transferrin antibodies have been produced by repeatedly injecting transferrin as an antigen in test animals, most commonly in rabbits and pigs. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies, used in particular for the quantitative determination of plasma transferrin by a simple radial immunodiffusion method. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum varies and does not repeat in each individual organism. As a rule, the organism will produce antibodies to the impurities of the antigen preparation in addition to the antibodies to the antigen of interest; these must be removed from the sera by saturation. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. For the manufacture of each batch, a pure immunization antigen and other antigens are required to saturate the ballast antibodies against impurities.
Uvedené nedostatky výše zmíněného a dosud používaného postupu odpadnou, je-li k dispozici hybridom, produkující monoklonální protilátku proti lidskému transferinu, uložený ve Sbírce hybridomů Ústavu molekulární genetiky ČSAV v Praze 4, Vídeňská čísle 1083, pod označením IMG CZAS HTF-06.These drawbacks of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against human transferrin, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vienna No. 1083, is available IMG CZAS HTF-06.
Uvedený hybridom byl získán způsobem známým z odborné literatury (Fazekas de St. Groth, S., Scheidigger, D.: Production of monoclonal antibodies: Stratégy and tactics,The hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, S., Scheidigger, D., Production of monoclonal antibodies: Strategies and tactics,
J. Immunol. Meth., 35:1-21, 1980; Galfré, G., Howe, S. C., Milstein, C., Butcher, G. W., Howard, J. C.: Antibodies to major histocompatibility antigens produced by hybrid cell lineš, Nátuře 266 : 550, 1977 J klonováním souboru hybridních buněk, vzniklých fúzí buněk myší myelomové linie P3-X63-Ag8.653 a buněk, získaných ze sleziny myší kmene Balb/c, imunizovaných lidským transferinem.J. Immunol. Meth., 35: 1-21 (1980); Galfré, G., Howe, SC, Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977 J by cloning a set of hybrid cells resulting from fusion of myeloma mouse cells line P3-X63-Ag8.653 and cells derived from the spleen of Balb / c mice immunized with human transferrin.
Výhodou hybridomu je, že produkuje homogenní protilátku, tzv. protilátku monoklonální, která je schopna specificky reagovat s lidským transferinem. Hybridom HTF-06 je možné kultivovat in vitro v médiích vhodných pro živočišné buňky a je adaptovánThe advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with human transferrin. The HTF-06 hybridoma can be cultured in vitro in media suitable for animal cells and is adapted
228880 pro růst in vivo v peritoneální dutině myší kmene Balb/c. Z konzerv, uchovávaných v kapalném dusíku, je možné zahájit produkci protilátky bez dalšího antigenu. Protilátka, produkovaná hybridomem HTF-06, je specifická výhradně pro lidský transferin a není třeba se zbavovat protilátek balastních.228880 for in vivo growth in the peritoneal cavity of Balb / c mice. From cans stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the HTF-06 hybridoma is specific to human transferrin and does not need to be rid of ballast antibodies.
PříkladExample
Za účelem pomnožení hybridomových buněk in vivo bylo aplikováno 2 X 106 buněk do peritoneální dutiny myší. Aby došlo k lepšímu uchycení aplikovaných buněk, byla myš 14 dní před přenosem buněk hybridomu ovlivněna parafinovým olejem (0,5 ml intraperitoneálně]. Po 20 dnech růstu hybridomu v peritoneální dutině byla myš zabita a naprodukovaná ascitická tekutina odebrána. Celkem bylo získáno 4,0 ml ascitické tekutiny, která obsahovala 15 mg/ml imunoglobulinu. Protilátka reagovala se specifickým antigenem v enzymoimunologickém testu (při použití prasečí antimyší protilátky značené křenovou peroxidázouj až do ředění 1 : 107. Za pomoci nepřímé imunofluorescenční techniky byl takto získanou protilátkou detekován receptor pro transferin na lidských myelomových buňkách.To expand the hybridoma cells in vivo, 2 X 10 6 cells were injected into the peritoneal cavity of mice. For better attachment of the cells, mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells, and after 20 days of hybridoma growth in the peritoneal cavity, the mice were killed and the produced ascites fluid collected. ml ascitic fluid, which contained 15 mg / ml immunoglobulin. the antibody reacted with the specific antigen in the enzyme immunoassay (using porcine anti-mouse antibody labeled with horseradish peroxidázouj up to a dilution of 1: 10 7th using indirect immunofluorescence technique was thus obtained antibody detected transferrin receptors on human myeloma cells.
Buňky hybridomu HTF-06 mají ultrastrukturální obraz typických myelomových buněk, kde převažující organelou jsou volné a na membrány vázané polyrlbosomy. In vltro rostou jako polosuspenzní kultury. Základním kultivačním médiem je Eaglovo minimální esenciální médium s Hanksovou solnou směsí doplněné o neesenciální aminokyseliny, L-glutamin (3 mM), pyruvát sodný (1 mM). Toto médium (označované jako H-MEMd, Ústav molekulární genetiky ČSAV], je pro kultivaci hybridomu HTF-06 doplněno penicilinem, streptomycinem, gentamycinem, 2-merkaptoethanolem (0,05 mM) pufrem HEPES (10 mM) a inaktivovaným bovinním sérem (Bioveta, Ivanovice na Hané, 10%). Hybridom je kultivován při 37 °C. Střední generační čas je 19,7 hodin a 12 měsíců po sestrojení byl modální počet chromosomů 85. Produkovaná protilátka je monoklonální imunoglobulin podtřídy IgGl.HTF-06 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. In vitro they grow as semi-suspension cultures. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (referred to as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM) in HEPES buffer (10 mM) and inactivated bovine serum (Bioveta) (Ivanovice na Hané, 10%) The hybridoma is cultured at 37 DEG C. The mean generation time is 19.7 hours and 12 months after construction, the modal number of chromosomes was 85. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.
Monoklonální protilátka, produkovaná hybridomem HTF-06 reaguje specificky s lidským transferinem.The monoclonal antibody produced by the hybridoma HTF-06 reacts specifically with human transferrin.
Hybridom HTF-06 může být průmyslově využíván jako zdroj monoklonální protilátky proti transferinu v metodách analytických nebo preparativních.The HTF-06 hybridoma can be used industrially as a source of anti-transferrin monoclonal antibody in analytical or preparative methods.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS593382A CS228860B1 (en) | 1982-08-10 | 1982-08-10 | Mouse lymphocytic hybridom producing an antisubstance a |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS593382A CS228860B1 (en) | 1982-08-10 | 1982-08-10 | Mouse lymphocytic hybridom producing an antisubstance a |
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| Publication Number | Publication Date |
|---|---|
| CS228860B1 true CS228860B1 (en) | 1984-05-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CS593382A CS228860B1 (en) | 1982-08-10 | 1982-08-10 | Mouse lymphocytic hybridom producing an antisubstance a |
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| CS (1) | CS228860B1 (en) |
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1982
- 1982-08-10 CS CS593382A patent/CS228860B1/en unknown
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