CS272887B1 - Mouse lymphocyte hybridome blvgp 30 ueo-94 c 11/2/e11 - Google Patents
Mouse lymphocyte hybridome blvgp 30 ueo-94 c 11/2/e11 Download PDFInfo
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- CS272887B1 CS272887B1 CS152389A CS152389A CS272887B1 CS 272887 B1 CS272887 B1 CS 272887B1 CS 152389 A CS152389 A CS 152389A CS 152389 A CS152389 A CS 152389A CS 272887 B1 CS272887 B1 CS 272887B1
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- 210000004698 lymphocyte Anatomy 0.000 title claims abstract description 6
- 241000700605 Viruses Species 0.000 claims abstract description 8
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 7
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 7
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 101710081103 Cuticular glutathione peroxidase Proteins 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 210000004408 hybridoma Anatomy 0.000 claims description 18
- 241000714266 Bovine leukemia virus Species 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 101710121417 Envelope glycoprotein Proteins 0.000 claims description 6
- 102100021696 Syncytin-1 Human genes 0.000 claims description 6
- 241001529936 Murinae Species 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 4
- 230000003612 virological effect Effects 0.000 claims description 4
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 101000920732 Bovine leukemia virus Envelope glycoprotein Proteins 0.000 claims description 2
- 101500007118 Bovine leukemia virus Transmembrane protein Proteins 0.000 claims 1
- 230000000890 antigenic effect Effects 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000002405 diagnostic procedure Methods 0.000 claims 1
- 238000011156 evaluation Methods 0.000 claims 1
- 230000009385 viral infection Effects 0.000 claims 1
- 241000283690 Bos taurus Species 0.000 abstract description 6
- 208000032839 leukemia Diseases 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000001261 affinity purification Methods 0.000 abstract 1
- 230000003302 anti-idiotype Effects 0.000 abstract 1
- 235000015278 beef Nutrition 0.000 abstract 1
- 238000002255 vaccination Methods 0.000 abstract 1
- 210000004027 cell Anatomy 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
- 238000005516 engineering process Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
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- 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
- 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
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 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
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
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- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography 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
- 238000000338 in vitro Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
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- 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
- 238000001228 spectrum Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
(57) Riešenie sa týká myšieho lymfocytárneho hybridómu produkujúceho monoklonálnu protilátku proti obalovému glykoproteínu gp30 virusu bovinnej leukémie uloženého v zbierke hybridómov oddelenia molekulárnej virelógie Ústavu experimentálnej onkológie SAV pod značením UE0-94C11/2/E11. Samotná monoklonálna protilátka tohoto hybridómu je vhodná hlavně na 'použitie pre výrobu imunodiagnostických súprav na detekciu hovádzieho dobytka infikovaného vírusom bovinnej leukémie a na detekciu virusového antigénu. Je taktiež vhodná pre účely preparatívnej ako imunoafinitnej purifikácie virusového glykoproteínu a na přípravu antiidiotypovej protilátky pre vakcinačné účely.(57) The invention relates to a murine lymphocyte hybridoma producing a monoclonal antibody against the envelope glycoprotein gp30 of bovine leukemia virus deposited in the hybridoma collection of the Department of Molecular Virology of the Institute of Experimental Oncology of SAS under the designation UE0-94C11 / 2 / E11. The monoclonal antibody of the hybridoma itself is particularly suitable for use in the manufacture of immunodiagnostic kits for the detection of bovine leukemia-infected bovine animals and for the detection of a viral antigen. It is also suitable for preparative as immunoaffinity purification of viral glycoprotein and for the preparation of an anti-idiotypic antibody for vaccine purposes.
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Vynález sa týká nového myšieho lymfocytárneho hybridómu BLVgp30UE0-94Cll/2/Ell, t.j. hybridnej bunečnej linie, ktorá bola zostrojená fúziou buniek myšej myelómovej linie Sp2/0 a myších lymfoidných buniek, produkujúcích protilátky namierené proti obalovému glykoproteínu gp30 virusu bovinnej leukémie (BLV). Obalov.ý glykoproteín je důležitou súčastou virusu bovinnej leukémie, je spolu zodpovědný za infektivitu virusu. Nález anti-gp protilátok u zvierat má zásadný význam pre diagnostiku leukémie hovadzieho dobytka.The present invention relates to a novel murine lymphocyte hybridoma BLVgp30UE0-94C11 / 2 / E1, i.e., a cell-bound hybridoma. a hybrid cell line that was constructed by fusing the murine myeloma cell line Sp2 / 0 and murine lymphoid cells producing antibodies directed against the gp30 envelope glycoprotein of bovine leukemia virus (BLV). The envelope glycoprotein is an important component of the bovine leukemia virus and is jointly responsible for the infectivity of the virus. The finding of anti-gp antibodies in animals is essential for diagnosing bovine leukemia.
Ooteraz sa protilátky proti antigénom BLV vyrúbajú tak, že purifikovaný gp30 je opakované injikovaný produkčným zvieratám, najčastejšle králikom. Sérum takto imunizovaných zvierat, odobrané po určitej době působenia antigénu, slúži ako zdroj protilátok. Tento postup má mnohé nevýhody, pre každé imunizáciu je nutné izolovaí znova glykoproteín gp30 z virusu, jeho purifikácia je drahá a časové náročná. Okrem toho v séru imunizovaných zvierat sa nachádza heterogénna zmes protilátok, ktorých spektrum je v každom zvierati rožne a neopakovatelné. Sérum takto získané obsahuje tiež protilátky voči nečistotám antigénového preparátu, ktoré je potom nutné odstraňoval vysycovaním. Z týchto dóvodov výrobné šarže sa značné odlíšujú a kvalita získaných polyklonálnych protilátok nie je štandartná.Meanwhile, antibodies against BLV antigens are produced by purified gp30 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 disadvantages, for each immunization it is necessary to isolate the gp30 glycoprotein from the virus again, 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 distinct and unique 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, the production batches differ considerably and the quality of the polyclonal antibodies obtained is not standard.
Uvedené nedostatky hoře uvedeného postupu konvenčněj imunizácie odpadnú, keď máme k dispozícii hybridom, produkujúci mcnoklonálnu protilátku proti obalovému glykoproteínu BLV, ktorý je uložený v zbierke hybridómov oddelenia molekulárnej virológie, Ústavu experimentálnej onkológie SAV v Bratislavě, ul. Čsl. armády 21, pod označením UE0-94C11/E11.These shortcomings of the above-mentioned conventional immunization procedure are eliminated when we have hybridomas producing a monoclonal antibody against the envelope glycoprotein BLV, which is deposited in the hybridoma collection of the Department of Molecular Virology, Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. CAA. Army 21, under the designation UE0-94C11 / E11.
Uvedený hybridom bol získaný v podstatě postupom známým v odbornej literatúre ako hybridómová technológia (Kohler, G., Milstein, C. iGoátinuous. - cultures of fused cells secreting antibody of pradefined specificity, Nátuře 256, 495, 1975 , R. H. Burdon, Ρ. H. VanKnippenberg: Laboratory techniques in biochemistry and moleoular biology, Vol. 13 Monoclonal Antibody Technology, Elsevier, Amsterdam 1984). Postup, ktorým boli hybridómy připravené bol námi modifikovaný (0. Orlík, Č. Altaner: Modifications of hybridoma technology yield of monoclonal antibody producing cells, 3. Immunol. Methods, 115, 55-59, 1988).Said hybrid was obtained essentially by a method known in the literature as hybridoma technology (Kohler, G., Milstein, C. iGoátinuous. - Cultures of fused cells secreting antibody of pradefined specificity, Nature 256, 495, 1975, RH Burdon, H. VanKnippenberg: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13 Monoclonal Antibody Technology, Elsevier, Amsterdam 1984). The procedure by which the hybridomas were prepared has been modified by us (Orlik, Altaner No.: Modifications of hybridoma technology yield of monoclonal antibody producing cells, 3. Immunol. Methods, 115, 55-59, 1988).
Výhodou hybridómu je, že produkuje homogénne monoklonálne protilátky, ktoré reagujé s obalovým glykoproteínom BLV. Hybridom BLVgp30-94Cll/Ell, je možno kultivoval in vitro v médiách vhodných pre živočišné buňky a súčasne je adaptovaný pre rast in vivo v peritoneálnej dutině myšieho kmeňa BALB/c. Z konzerv, ktoré sú uchovávané v kvapalnom dusíku, je možno zahájil produkciu protilátky bez Salšieho antigénu. Protilátka produkovaná klónom BLV gp30-94Cll/2/Ell reaguje specificky s determinantou obalového glykoproteínu BLV.An advantage of the hybridoma is that it produces homogeneous monoclonal antibodies that react with the envelope glycoprotein BLV. The BLVgp30-94C11 / EII hybridoma can be cultured in vitro in animal cell media while being adapted for growth in vivo in the peritoneal cavity of the mouse BALB / c strain. From cans that are stored in liquid nitrogen, antibody production without Saler antigen can be initiated. The antibody produced by the BLV clone gp30-94C11 / 2 / EI reacts specifically with the BLV envelope glycoprotein determinant.
PříkladExample
Za účelom pomnoženia hybridómových buniek in vivo bolo 2-10 x 10^ buniek injikované do peritoneálnej dutiny myší kemňa BALB/c. Týmto myšiam bolo 14 dní predtým injikované do peritoneálnej dutiny 0,5 ml Přistanu alebo inkompletného Freudova adjuvans za účelom vytvorenia sterilného ascitu, ktorý napomáhá rastu injikovaných hybridómových buniek. Po 14 dňoch rastu hybridómových buniek v peritoneálnej dutině, myš bola usmrtená a naprodukovaná ascitická tekutina bola sterilně odobraná. Celkom sa získalo 8 ml ascitovej tekutiny, ktorá obsahovala 9 mg/ml imunoglobulinu. Monoklonálna protilátka reagovala Specificky s glykoproteínom gp30-BLV a s desintegrovaným vírusoum v rádioprecipitačnom teste a rádioimunologickom teste. Z ascitickej tekutiny bol imunoglobulín izolovaný pomocou chromatografie na kelóne Protein A-Sepharose alebo lnou vhodnou metodou. Takto purifikovaná monoklonálna protilátka je vhodná pre poíahovanie polystyrénových materiálov (mikrotitračné doštičky, polystyrénové tyčinky) za účelom pripravy diagnostických súprav na detekciu BLV infikovaných zvierat.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. After 14 days of growth of 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 9 mg / ml of immunoglobulin was obtained. The monoclonal antibody reacted specifically with the glycoprotein gp30-BLV and with the disintegrated virus in the radioprecipitation and radioimmunoassays. Immunoglobulin was isolated from ascites fluid by Protein A-Sepharose column chromatography 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.
Iri vitro rastů buňky hybridómu ako polosuspenzná kultúra. Základným kultivačným médiom je Oulbeccova modifíkácia Eaglova mínimálneho esencíálneho média doplněného 5 percentami koňského alebo iného séra vhodného pře tkáňové kultúry, Hybridom je kultivovaný pri Iri vitro hybridoma cell growth as a semi-suspension culture. The basic culture medium is Oulbecco's Modification of Eagle's Minimum Essential Medium supplemented with 5 percent of horse or other serum of a suitable tissue culture.
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| Application Number | Priority Date | Filing Date | Title |
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| CS152389A CS272887B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 30 ueo-94 c 11/2/e11 |
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| CS152389A CS272887B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 30 ueo-94 c 11/2/e11 |
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| Publication Number | Publication Date |
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| CS152389A1 CS152389A1 (en) | 1990-06-13 |
| CS272887B1 true CS272887B1 (en) | 1991-02-12 |
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| CS152389A CS272887B1 (en) | 1989-03-11 | 1989-03-11 | Mouse lymphocyte hybridome blvgp 30 ueo-94 c 11/2/e11 |
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| CS152389A1 (en) | 1990-06-13 |
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