CS272888B1 - Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 - Google Patents
Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 Download PDFInfo
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- CS272888B1 CS272888B1 CS152489A CS152489A CS272888B1 CS 272888 B1 CS272888 B1 CS 272888B1 CS 152489 A CS152489 A CS 152489A CS 152489 A CS152489 A CS 152489A CS 272888 B1 CS272888 B1 CS 272888B1
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- 210000004698 lymphocyte Anatomy 0.000 title claims abstract description 5
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 4
- 210000004408 hybridoma Anatomy 0.000 claims description 19
- 101710121417 Envelope glycoprotein Proteins 0.000 claims description 5
- 102100021696 Syncytin-1 Human genes 0.000 claims description 5
- 101500007117 Bovine leukemia virus Surface protein Proteins 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 9
- 239000000427 antigen Substances 0.000 abstract description 7
- 102000036639 antigens Human genes 0.000 abstract description 7
- 108091007433 antigens Proteins 0.000 abstract description 7
- 241000283690 Bos taurus Species 0.000 abstract description 6
- 108090000288 Glycoproteins Proteins 0.000 abstract description 6
- 102000003886 Glycoproteins Human genes 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 208000032839 leukemia Diseases 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000006386 neutralization reaction Methods 0.000 abstract description 2
- 238000002255 vaccination Methods 0.000 abstract description 2
- 238000001261 affinity purification Methods 0.000 abstract 1
- 230000003302 anti-idiotype Effects 0.000 abstract 1
- 235000015278 beef Nutrition 0.000 abstract 1
- 108010011013 glycoprotein GP51 Proteins 0.000 abstract 1
- 241000714266 Bovine leukemia virus Species 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 101800001271 Surface protein Proteins 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 210000003200 peritoneal cavity Anatomy 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 101000920732 Bovine leukemia virus Envelope glycoprotein Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
(57) Riešenie sa- týká myšieho lymfocytárneho hybridómu produkujúceho neutralizačnú monoklonálnu protilátku proti obalovému glykoproteínu gp51 virusu bovinnej leukémie uloženého v zbierke hybridómov oddelenia molekulárnej virológie Ústavu experimentálnej onkológie SAV pod vyznačením UE0-25E11/2. Samotná monoklonálna protilátka tohoto hybridómu je vhodná hlavně na použitie pre výrobu imunodiagnostických súprav na detekciu hovádzleho dobytka infikovaného vírusom bovinnej leukémie a na detekciu virusového antigénu. Je taktiež vhodná pre účely preparatívnej ako imunofinitnej purifikácie virusového glykoproteínu, na přípravu antiidiotypovej protilátky pře vakoinačné účely a na neutralizáciu virusu.(57) The invention relates to a murine lymphocyte hybridoma producing a neutralizing monoclonal antibody against the envelope glycoprotein gp51 of the bovine leukemia virus deposited in the hybridoma collection of the Department of Molecular Virology of the Institute of Experimental Oncology of SAS under designation UE0-25E11 / 2. The monoclonal antibody of this hybridoma itself is particularly suitable for use in the manufacture of immunodiagnostic kits for the detection of bovine leukemia-infected cattle and for the detection of a viral antigen. It is also suitable for preparative as immunofinite purification of viral glycoprotein, for the preparation of an anti-idiotypic antibody for vaccination purposes and for virus neutralization.
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CS 272888 8181 272888 81
Vynález sa týká nového myšieho lymfocytárneho hybridómu BLVgp51UE0-25Ell/2, t.j. hybridnej bunečnej linie, ktorá bola zostrojená fúziou buniek myšej myelómovej. linie Sp2/0 a myších lymfaidných buniek, pradukujúcich protilátky namierené proti obalovému glykoproteínu gp51 virusu bovinnej leukémie (BLV). Obalový glykoproteín je důležitou súčasíou virusu bovinnej leukémie, je zodpovědný za infektivitu virusu. Nález anti-gp protilátok u zvierat má zásadný význam pre diagnostiku leukémie hovadzieho dobytka.The invention relates to a novel murine lymphocyte hybridoma BLVgp51UE0-25E11 / 2, i. hybrid cell line, which was constructed by fusing mouse myeloma cells. the Sp2 / 0 line and mouse lymphoma cells producing antibodies directed against the envelope glycoprotein gp51 of bovine leukemia virus (BLV). The envelope glycoprotein is an important component of the bovine leukemia virus and is responsible for the infectivity of the virus. The finding of anti-gp antibodies in animals is essential for diagnosing bovine leukemia.
Doteraz sa protilátky proti antigénom BLV vyrábajú tak, že purifíkovaný gp51 je opakované injikovaný predukčným zvieratám, najčastějšie králikom. Sérum takto imunizovaných zvierat, odobrané po určitej době působenia antigénu, slúži ako zdroj protilátok. Tento postup má mnohé nevýhody, pre každú imunizáciu je nutné izolovat znova glykoproteín gp51 z virusu, jeho purifikácia je drahá a časové náročná. Okrem toho v sére imunizovaných zvierat sa nachádza heterogénna zmes protilátok, ktorých sepktrum je v každom zvierati různé a neopakovatelné. Sérum takto získané obsahuje tiež protilátky voči nečistotám antígénového preparátu, ktoré je potom nutné odstraňovat vysycovaním. Z týchto důvodov výrobně šarže sa značné odlišujú a kvalita získaných polyklonálnych protilátok nie je Standardně.To date, antibodies against BLV antigens have been produced by purified gp51 being repeatedly injected into the production animals, most commonly rabbits. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies. This procedure has many disadvantages, for each immunization it is necessary to isolate the gp51 glycoprotein again from the virus, its purification is expensive and time consuming. In addition, there is a heterogeneous mixture of antibodies in the sera of the immunized animals, the septrum of which is different and unrepeatable in each animal. The serum thus obtained also contains antibodies to the impurities of the antigen preparation, which must then be removed by saturation. For these reasons, production batches vary widely and the quality of the polyclonal antibodies obtained is not standard.
Uvedené nedostatky výše uvedeného postupu konvenčnej ímunizácie odpadnú, ked máme k dispozícii hybridóm, produkujúci monoklonálnu protilátku proti obalovému glykoproteínu BLV, ktorý je uložený v zbierke hybridómov oddelenia molekulárnej virológie, Ústavu experimentálnej onkológíe SAV v Bratislavě, ul. Csl. armády 21, pod označením UE0-25E11/2.The aforementioned drawbacks of the above-mentioned conventional immunization process will be eliminated when we have a hybridoma producing a monoclonal antibody against the envelope glycoprotein BLV, which is stored in the hybridoma collection of the Department of Molecular Virology, Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. CSL. Army 21, under the designation UE0-25E11 / 2.
Uvedený hybridóm bol získaný v podstatě postupom známým v odbornej Iíteratúre ako hybridómová technológía (Kohler, G., Milstein, C.: Continous cultures of fused cells secreting antibody of prodefíned specificity, Nátuře 256, 495, 1975), R. H. Burdon, Ρ. H. VanKnippenberg: Laboratory techniques in biochemistry and molecular biology, Vol. 13 Monoclonal antibody Technology, Elsevier, Amsterdam 1984. Postup, ktorým holí hybridomy připravené bol námi modifikovaný (0. Orlík, Č. Altaner: Modifications of hybridóm technology yield of monoclonal antihody producing cells, J. Immunol. Methods, 115, 55-59, 1988).Said hybridoma was obtained essentially by a method known in the art as hybridoma technology (Kohler, G., Milstein, C .: Continous cultures of fused cell secreting antibody of predefined specificity, Nature 256, 495, 1975), R. H. Burdon, Ρ. H. VanKnippenberg: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13 Monoclonal Antibody Technology, Elsevier, Amsterdam 1984. The procedure by which the hybridoma prepared was modified by us (Orlík, Altaner No.: Modifications of hybridoma technology yielding monoclonal anti-producing cells, J. Immunol. Methods, 115, 55-59 (1988).
Výhodou hybridómu je, že produkuje homogénne monoklonálne protilátky, ktoré reagujú s obalovým glykoproteínom BLV. Hybridóm BLVgp51-25Ell/2, je možno kultivoval in vitro v médiách vhodných pre živočišné buňky a súčasne je adaptovaný pre rast in vivo v peritoneálnej dutině myšeiho kmeňa BALB/c. Z konzerv, ktoré sú uchovávané v kvapalnom dusíku, je možno zahájit produkciu protilátky bez áalšieho antigénu. Protilátka produkovaná klónom 8LVgp51-25Ell/2 reaguje Specificky s determinantou obalového glykoproteínu BLV a má neutralízačný charakter.An advantage of the hybridoma is that it produces homogeneous monoclonal antibodies that react with the BLV envelope glycoprotein. The hybridoma BLVgp51-25E11 / 2, can be cultured in vitro in animal cell media while being adapted for growth in vivo in the peritoneal cavity of the BALB / c mouse strain. From cans that are stored in liquid nitrogen, antibody production without further antigen can be initiated. The antibody produced by clone 8LVgp51-25E11 / 2 reacts specifically with the BLV envelope glycoprotein determinant and is neutralizing in nature.
PříkladExample
Za účelom pomnoženia hybridómových buniek in vivo bolo 2-10 x 10^ buniek injikované do peritoneálnej dutiny myší kmeňa BALB/c. Týmto myšiam bolo 14 dní predtým injikované do peritoneálnej dutiny 0,5 ml Přistanu alebo inkompletného Freudova adjuvans za účelom vytvorenia sterilného ascítu, ktorý napomáhá rastu injikovaných hybridómových buniek.To expand hybridoma cells in vivo, 2-10 x 10 6 cells were injected into the peritoneal cavity of BALB / c mice. These mice were injected 14 days prior to the peritoneal cavity with 0.5 ml of Landing or Incomplete Freud's Adjuvant to create sterile ascites that aided the growth of injected hybridoma cells.
Po 14 dnoch rastu hybridómových buniek v peritoneálnej dutině, myš bola usmrtená a neprodukovaná ascitická tekutina bola sterilně odobraná. Celkom sa získalo 8 ml ascitovej tekutiny, ktorá obsahovala 9 mg/ml imunoglobulínu. Monoklonálna protilátka reagovala Specificky s glykoproteínom gp51-BLV a s desintegrovaným vírusom v rádioprecipitačnom teste a rádioimunologickom teste. Z ascitickej tekutiny bol imunoglobulín izolovaný pomocou chromatografie na klone Protein A-Sepharose alebo inou vhodnou metodou. Takto purifikovaná monoklonálna protilátka je vhodná pre poíahovanie polystyrénových materiálov (mikrotitračné doštičky, polystyrénové tyčinky) za účelom přípravy diagnostických, súprav na detekciu BLV infikovaných zvierat.After 14 days of growth of hybridoma cells in the peritoneal cavity, the mouse was sacrificed and unproduced ascites fluid was harvested sterile. A total of 8 ml of ascites fluid containing 9 mg / ml immunoglobulin was obtained. The monoclonal antibody reacted specifically with the glycoprotein gp51-BLV and with the disintegrated virus in the radioprecipitation assay and the radioimmunoassay. Immunoglobulin was isolated from ascites fluid by chromatography on a Protein A-Sepharose clone or other suitable method. The monoclonal antibody thus purified is suitable for coating polystyrene materials (microtiter plates, polystyrene rods) in order to prepare diagnostic kits for detecting BLV infected animals.
In vitro rastů buňky hybridómu ako polosuspenzná kultúra. Základným kultivačným médiom je Dulbeccova modifikácia Eaglova minimálneho esencíálneho média doplněného 5 procentami koňského alebo iného séra vhodného pre tkáňové kultúry. Hybridóm je kultivovaný pri iIn vitro hybridoma cell growths as a semi-suspension culture. The basic culture medium is Dulbecco ' s modification of Eagle's minimum essential medium supplemented with 5 percent of horse or other tissue culture-appropriate serum. The hybridoma is cultured at i
'ί'ί
CS 272880 Bl °C v C02 termostate, jeho štandardná generačná doba je přibližné 24 hodin. Produkovaná protilátka je monoklonálny imunoglobulín podtriedy IgG2b.CS 272880 B1 ° C in CO 2 thermostate, its standard generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of IgG2b subclass.
Hybridóm BLVgp51UED-25Ell/2 může byt priemyslovo využívaný ako zdroj protilátky proti obalovému glykoproteínu gp51 BLV v diagnostických metodách, v purifikačných metodách a iných aplikáciách.The BLVgp51UED-25E11 / 2 hybridoma can be industrially used as a source of antibody to the gp51 BLV envelope glycoprotein in diagnostic methods, purification methods, and other applications.
Konkrétné má využitie: pri výrobě imunodiagnostických súprav na detekciu infekcie hovadzieho dobytka vírusom bovinnej leukémie, při purifikácii virusového glykoproteínu imunoafinitnou chromatografiou, při vyhodnocovaní antigénnej variability virusového glykoproteínu, pře přípravu antiidiotypovej protilátky pre vakcinačné účely, ako zdroj protilátky jedinej podtriedy pře přípravu sér, pře detekciu virusového antigénu v infikovaných zviera tách, pře neutralizáciu BLV.It has particular applications: in the production of immunodiagnostic kits for the detection of bovine leukemia virus infection in bovine leukemia virus, in the purification of viral glycoprotein by immunoaffinity chromatography, in the evaluation of antigenic variability in viral glycoprotein; virus antigen in infected animals, prior to neutralization of BLV.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS152489A CS272888B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS152489A CS272888B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 |
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| Publication Number | Publication Date |
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| CS152489A1 CS152489A1 (en) | 1990-06-13 |
| CS272888B1 true CS272888B1 (en) | 1991-02-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CS152489A CS272888B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 |
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| CS152489A1 (en) | 1990-06-13 |
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