CS272889B1 - Mouse lymphocyte hybridome blv p 24 ueo-25 e 11/8 - Google Patents
Mouse lymphocyte hybridome blv p 24 ueo-25 e 11/8 Download PDFInfo
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- CS272889B1 CS272889B1 CS152589A CS152589A CS272889B1 CS 272889 B1 CS272889 B1 CS 272889B1 CS 152589 A CS152589 A CS 152589A CS 152589 A CS152589 A CS 152589A CS 272889 B1 CS272889 B1 CS 272889B1
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- 210000004698 lymphocyte Anatomy 0.000 title claims abstract description 5
- 210000004408 hybridoma Anatomy 0.000 claims description 12
- 241000714266 Bovine leukemia virus Species 0.000 claims description 10
- 241001529936 Murinae Species 0.000 claims description 4
- 108010087302 Viral Structural Proteins Proteins 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 8
- 239000000427 antigen Substances 0.000 abstract description 5
- 102000036639 antigens Human genes 0.000 abstract description 5
- 108091007433 antigens Proteins 0.000 abstract description 5
- 241000283690 Bos taurus Species 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 208000032839 leukemia Diseases 0.000 abstract description 3
- 101710172711 Structural protein Proteins 0.000 abstract description 2
- 238000001261 affinity purification Methods 0.000 abstract 1
- 235000015278 beef Nutrition 0.000 abstract 1
- 230000003472 neutralizing effect Effects 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 101900319158 Bovine leukemia virus Capsid protein p24 Proteins 0.000 description 4
- 210000003200 peritoneal cavity Anatomy 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 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
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000011156 evaluation Methods 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
- 238000000338 in vitro Methods 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
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000001228 spectrum Methods 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
- 210000002845 virion Anatomy 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
Vynález sa týká nového myšieho lymfocytárneho hybridámu BLV p24UBO-25Ell/8 t. j. hybridnej bunečnej linie, ktorá bola zostrojená fáziou buniek myšej myelámovej linie Sp2/O a myších lymfoidných buniek, produkujácich protilátky namierené proti hlavnému Štrukturálnemu proteinu p24 virusu bovinnej leukémie (BLV). Protein p24 je základnou sáčastou viriónu virusu bovinnej leukémie. Nález antip24-protilátok u zvierat má význam pře diagnostiku leukémie hovadzieho dobytka.The present invention relates to a novel murine lymphocyte hybridoma BLV p24UBO-25E11 / 8t. j. a hybrid cell line that was constructed by a phase of murine myeloma line Sp2 / O cells and murine lymphoid cells producing antibodies directed against the major structural protein of bovine leukemia virus (BLV) p24. The p24 protein is an essential part of the bovine leukemia virus virion. Finding antip24-antibodies in animals is important for diagnosing bovine leukemia.
Doteraz sa protilátky proti antigénom BLV vyrábajú tak, že purifikovaný p24 je opakované injikovaný produkčným zvieratám, najčastejšie králikom. Sérum takto imunizovaných zvierat, odebrané po určitej době pSsobenia antigénu, sláži ako zdroj protilátok. Tento postup má mnohé nevýhody, pře každá imunizáciu je nutné izolovat’ znova protein p24 z virusu, jeho purifikáeia je drahá a časové náročná. Okrem toho v sére imunizovaných zvierat sa nachádza heterogénna zmes protilátok, ktorých spektrum je v každom zvierati rSzne a neopakovatelné. Sérum takto získané obsahuje tiež protilátky voči nečistotám antigénového preparátu, ktoré je potom nutné odstraňovat/ vysycovaním.To date, antibodies against BLV antigens have been produced by purified p24 being injected repeatedly into 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 drawbacks, for each immunization it is necessary to isolate the p24 protein again from the virus, its purification is expensive and time consuming. In addition, there is a heterogeneous mixture of antibodies in the serum of the immunized animals, the spectrum of which is different and non-repeatable in each animal. The serum thus obtained also contains antibodies to the impurities of the antigen preparation, which must then be removed / by saturation.
Z týchto dovodov výrobné šarže sa značné odliSujá a kvalita získaných polyklonálnych protilátok nie je štandardná.From these reasons, the production batches differ significantly and the quality of the polyclonal antibodies obtained is not standard.
Uvedené nedostatky hoře uvedeného postupu konvenčnej imunizácie odpadná, keS máme k dispozícii hybridám produkujúci monoKLonálnu protilátku proti štrukturálnemu proteinu p24 BLV, ktorý je uložený v zbierke hybridómov oddelenia molekulárnej virológie Ústavu experimentálnej onkolágie SAV v Bratislavě, ul. 5sl. armády 21, pod označením UEO-25E11/8.The above drawbacks of the above-mentioned conventional immunization procedure are avoided when we have hybrids producing a monoclonal antibody against the structural protein p24 BLV, which is deposited in the hybridoma collection of the Department of Molecular Virology of the Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. 5SL. Army 21, under the designation UEO-25E11 / 8.
Uvedený hybridám bol získaný v podstatě postupom známým v odbornéj literatuře ako hybridómová technolágia (Kohler, G,, Milstein, C.: Continuous cultures of fused cells secretxng antibody of prodefined speoificity, Nátuře 256, 495, 1975),(B.H.Burdon, P.H. VanKnippenberg: Laboratory techniques in biochemistry and molecular biology. Vol. 13, Monoelonal Antibody Technology, Elsevier, Amsterdam 1984). Postup, ktorým boli hýbridámy připravené bol námi modifikovaný (0,Orlík, ¢. Altaner: Modifications of hybridoma technology yield of monoelonal antibody producing cells, J. Immunol. Methods, 115,Said hybrids were obtained essentially by a method known in the literature as hybridoma technology (Kohler, G, Milstein, C .: Continuous Cultures of Fused Cells Secreted by Prodefined Speoificity, Nature 256, 495, 1975), (BH Burdon, PH VanKnippenberg) : Laboratory techniques in biochemistry and molecular biology, Vol. 13, Monoelonal Antibody Technology, Elsevier, Amsterdam 1984). The procedure by which the hybrids were prepared has been modified by us (Orlik, Alt. Altaner: Modifications of hybridoma technology yielding monoelonal antibody producing cells, J. Immunol. Methods, 115,
55-59, 1988). ’55-59, 1988). '
Výhodou hybridámu je, že produkuje homogénne monoklonálne protilátky, ktoré reagujá s proteínom p24 BLV, Hybridám BLVp240E0-25Ell/8, je možno kultivovat in vitro v médiách vhodných pre živočišné buňky a sáčasne je adaptovaný pre rast in vivo v peritoneálnej dutině myšieho kmeňa BALB/c. Z konzerv, ktoré sú uchovávané v kvapalnom dusíku, je možno zahájit produkciu protilátky bez áalšieho antigénu. Protilátka produkovaná KLánom BLVp24-25Ell/8 reaguje Specificky s determinantou proteinu p24 BLV,An advantage of the hybridoma is that it produces homogeneous monoclonal antibodies that react with the p24 BLV protein, BLVp240E0-25E11 / 8 hybrids, can be cultured in vitro in animal cell media, and is simultaneously adapted for growth in vivo in the peritoneal cavity of the BALB / mouse strain. c. From cans that are stored in liquid nitrogen, antibody production without further antigen can be initiated. The antibody produced by KLan BLVp24-25E11 / 8 reacts specifically with the determinant of the p24 BLV protein,
Příklad: Za áčelom pomnoženia hybridámových buniek in vivo bolo 2*10x10^ 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 Preudova adjuvans za áčelom vytvorenia sterilného ascitu, ktorý napomáhá· rastu injikovaných hybridámových buniek. Po 14 dňoch rastu hybridámových buniek v peritoneálnej dutině, myš bola usmrtená a naprodukovaná ascitická tekutina bola sterilně odobraná. Celkom sa získalo 8 ml ascitovej tekutiny, ktorá obsahovala 9mg/ml imunoglobulínu. Monoklonálna'protilátka reagovala Specificky s proteínom p24-BLV a s desintegrovaným vírusom v rádioprecipitačnom teste a rádioimunologickom teste. Z ascitickej tekutiny bol imunoglobulín izolovaný pomocou chromatografie na koláne'Protein’ A-Sepharose alebo inou vhodnou metádou. Takto purifikovaná monoklonálna protilátka je vhodná pre poťahovanie polystyrénových materiálov (mikrotitračné. doštičky, polystyrénové tyčinky) za áčelom přípravy diagnostických sáprav na detekciu BLV infikovaných zvierat.Example: In order to expand hybridoma cells in vivo, 2 * 10x10 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 an incomplete Preud's adjuvant to create sterile ascites to aid the growth of injected hybridoma cells. After 14 days of growth of the hybridoma cells in the peritoneal cavity, the mouse was sacrificed and the ascitic fluid produced was harvested sterile. A total of 8 ml of ascites fluid containing 9mg / ml immunoglobulin was obtained. The monoclonal antibody reacted specifically with the p24-BLV protein and with the disintegrated virus in the radioprecipitation assay and the radioimmunoassay. Immunoglobulin was isolated from ascites fluid by means of column chromatography 'Protein' A-Sepharose or other suitable metadata. The monoclonal antibody thus purified is suitable for coating polystyrene materials (microtiter plates, polystyrene rods) for the preparation of diagnostic kits for the detection of BLV infected animals.
Xn vitro rastů buňky hybridómu ako polosuspenzná kultúra, Základným kultivačným mé diom je Dulbeccova modifikácia Eaglova minimálneho esenoiálneho média doplněného 5 percentami koňského alebo iného séra vhodného pre tkáňové kultúry. Hybridóm je kultivovaný pri 37 °C v C02 termostate, jeho štandardná generačná doba je přibližné 24 hodin. Produ kovaná protilátka je monoklonálny imunoglobulín podtriedy IgGl.Xn vitro hybridoma cell growths as a semi-suspension culture. The basic culture diode is Dulbecco's modification of Eagle's minimal esenoial medium supplemented with 5 percent of horse or other tissue culture-appropriate serum. The hybridoma is cultured at 37 ° C in a CO 2 thermostate, its standard generation time being approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.
Hybridóm BLVp24UEO-25Ell/8 mdže byť priemyslovo využívaný ako zdroj protilátky pro ti štrukturélnerau proteinu p24 BLV v diagnostických metodách, v purifikačných metodách iných aplikáciach.The BLVp24UEO-25E11 / 8 hybridoma can be industrially used as an antibody source for three BLV p24 protein structures in diagnostic methods, in purification methods of other applications.
Konkrétné má využitie: pri výrobě imunodiagnostických súprav na detekciu infekcie hovádzieho dobytka vírusom bovinnej leukémie, pri purifikécii virusového proteinu p24 imunoafinitnou chromatografiou, pri vyhodnocovaní antigénnej variability virusového proteinu p24, ako zdroj protilátky jedinej podtriedy pre přípravu sér, pre detekciu virusového antigénu v infikovaných zvieratách.In particular, it has utility: in the manufacture of immunodiagnostic kits for the detection of bovine leukemia virus infection in bovine leukemia virus, in the purification of p24 virus protein by immunoaffinity chromatography, in the evaluation of antigenic variability of p24 virus protein
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS152589A CS272889B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blv p 24 ueo-25 e 11/8 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS152589A CS272889B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blv p 24 ueo-25 e 11/8 |
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| Publication Number | Publication Date |
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| CS152589A1 CS152589A1 (en) | 1990-06-13 |
| CS272889B1 true CS272889B1 (en) | 1991-02-12 |
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| CS152589A CS272889B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blv p 24 ueo-25 e 11/8 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116284352A (en) * | 2022-08-22 | 2023-06-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Bovine leukemia virus antibody and detection kit |
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1989
- 1989-03-11 CS CS152589A patent/CS272889B1/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116284352A (en) * | 2022-08-22 | 2023-06-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Bovine leukemia virus antibody and detection kit |
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| Publication number | Publication date |
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| CS152589A1 (en) | 1990-06-13 |
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