CS272886B1 - Mouse lymphocyte hybridome blvgp 51 ueo-6a12/a7 - Google Patents
Mouse lymphocyte hybridome blvgp 51 ueo-6a12/a7 Download PDFInfo
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- CS272886B1 CS272886B1 CS152289A CS152289A CS272886B1 CS 272886 B1 CS272886 B1 CS 272886B1 CS 152289 A CS152289 A CS 152289A CS 152289 A CS152289 A CS 152289A CS 272886 B1 CS272886 B1 CS 272886B1
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Vynález sa týká nového myšieho lymfocytárneho hybrídómu BLVgp51UE0-6A12/A7 t.j. hybridně j bunečnej linie, ktorá bola zostrojená fúziou buniek myšej myelómovej linie Sp2/0 a myších lymfoidných buniek, produkujících protilátky namierené proti obalovému glykoproteínu gp51 virusu bovinnej leukémie (BLV). Obalový glykoproteín je doležitou súčastou virusu bovinnej leukémie, je zodpovědný za infektivitu virusu. Nález anti gp-protílátok u zvierat má zásadný význam pre diagnostiku leukémie hovádzieho dobytka.The invention relates to a novel murine lymphocyte hybridoma BLVgp51UE0-6A12 / A7 i.e. hybridoma cell line, which was constructed by fusing cells of the murine myeloma line Sp2 / 0 and murine lymphoid cells producing antibodies directed to 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 the diagnosis of bovine leukemia.
Doteraz sa protilátky proti antigénom BLV vyrábajú tak, že purífikovaný gp51 je opakované injikovaný produkčným zvíeratám, najčastejšie králikom. Sérum takto imunizovaných zvierat, odobrané pa určitej době půsohenia antigénu, slúži ako zdroj protilátok. Tento postup má mnohé nevýhody, pre každí imunizáciu je nutné izolovat znova glykoproteín gp51 z virusu, jeho purifíkácia je drahá a časové náročná. Okrem toho 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 odstraňovat vysycovaním. Z týchto dóvodov výrobně šarže sa značné odlišujú a kvalita získaných polyklonálnych protilátok nie je štandartná.To date, antibodies against BLV antigens have been produced by purified gp51 being injected repeatedly into production animals, most commonly rabbits. The serum of animals thus immunized, collected at a certain time of antigen challenge, 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, the sera of the immunized animals contain a heterogeneous mixture of antibodies, the spectrum of which is distinct and unique in each animal. The serum thus obtained also contains antibodies to the antigen preparation impurities, which must then be removed by saturation. For these reasons, the production batch differs considerably and the quality of the polyclonal antibodies obtained is not standard.
Uvedené nedostatky výše uvedeného postupu konvenčnej imunizácie odpadní, keď máme k dispozici! hybridónr, produkujúci monoklonálnu protilátku proti obalovému glykúproteínu BLV, ktorý je uložený v zbierke hybridómov oddelenia molekulárnej virológie, Ústavu experimentálně j onkológie SAV v Bratislavě, ul. Čsl. armády 21, pod označením UE0-6A12/A7.The above drawbacks of the above-mentioned conventional immunization wastes process, when available! hybridon, producing a monoclonal antibody against envelope glycoprotein BLV, which is deposited in the collection of hybridomas 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 UE0-6A12 / A7.
Uvedený hybridóm bol získaný v podstatě postupom známým v odbornej literatúre ako hybridómová technológia (Kohler, G., Milstein, C. z Continucts cultures of fused cells secreting antibody of prodefined specificity, Nátuře 256, 495, 1975), R. H. Burdon, Ρ. H. VanKnippenberg: Laboratory techniques in biochemístry and molecular 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, J. Immunoi. Methods, 115, 55-59, 1988).Said hybridoma was obtained essentially by a method known in the literature as hybridoma technology (Kohler, G., Milstein, C. of Continued Cultures of Fused Cell Secreting Antibody of Prodefined 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 hybridomas were prepared has been modified by us (Orlik, N. Altaner: Modifications of hybridoma technology yield of monoclonal antibody producing cells, J. Immunoi. Methods, 115, 55-59, 1988).
Výhodou hybrídómu je, že produkuje homogenně monoklonálne protilátky, ktoré reagují s obalovým glykoproteínom BLV. Hybridóm SLVgp51-6A12/A7, je možno kultivovat in vitro v médíách vhodných pře živočišné buňky a súčasne je adaptovaný pre rast in vivo v peritoneálnej dutině myšieho kmeňa 8ALB/c. Z konzerv, ktoré sí uchovávané v kapalnom dusíku, je možno zahájit produkciu protilátky bez ďalšieho antigénu. Protilátka produkovaná klónom BLVgp51-6A12/A7 reaguje specificky s determinantou obalového glykoproteínu 8LV a má neutralizačný charakter.The advantage of the hybridoma is that it produces homogeneously monoclonal antibodies that react with the BLV envelope glycoprotein. The hybridoma SLVgp51-6A12 / A7 can be cultured in vitro in media suitable for animal cells while being adapted for growth in vivo in the peritoneal cavity of the 8ALB / c mouse strain. From cans that are stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the clone BLVgp51-6A12 / A7 reacts specifically with the determinant of the envelope glycoprotein 8LV and has a neutralizing character.
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 ascitu, ktorý napomáhá rastu injikovaných hybridómových buniek. Po 14 dnách 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 imunoglobulínu. Monoklonálna protilátka reagovala specificky s glykoproteínom gp51-BLV a s desíntegrqvaným vírusom v rádíoprecipitačnom teste a rádioimunologickom teste. Z ascitickej tekutiny bol imunoglobulín izolovaný pomocou chromatografie na kolóne Protein A-Sepharese alebo inou vhodnou metodou. Takto purifikovaná monoklonálna protilátka je vhodná pre potahovanie polystyrénových materiálov (mikrotítračné doštičky, polystyrénové tyčinky) za účelom přípravy 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 hybridoma cell growth 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 immunoglobulin was obtained. The monoclonal antibody reacted specifically with the gp51-BLV glycoprotein and the disintegrated virus in the radio-precipitation and radioimmunoassays. Immunoglobulin was isolated from the ascites fluid by Protein A-Sepharese 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.
In vitro rastů buňky hybrídómu ako polosuspenzná kultúra. Základným kultivačným médíom je Dulbeccova modifikácia Eaglova minimálneho esenciálneho média doplněného 5 percentami koňského alebo iného séra vhodného pre tkáňové kultúry. Hybridóm je kultivovaný priIn 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
CS 272006 fll 2 °C v C02 termostate, jeho štandardná generační doba je přibližné 24 hodin. Produkovaná . protilátka je monoklonálny imunoglobulín podtriedy IgGl.CS 272006 f11 2 ° C in C0 2 thermostate, its standard generation time is approximately 24 hours. Produkovaná. the antibody is a monoclonal immunoglobulin of IgG1 subclass.
Hybridóm BLVgpUEO-6A12/A7 mĎže byí priemyslovo využívaný ako zdroj protilátky proti obalovému glykoproteínu gp51 BLV v diagnostických metodách, v purifikačných metodách a iných aplikáciach.The BLVgpUEO-6A12 / A7 hybridoma can be used industrially 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 imunoafinltnou chromatografiou, při vyhodnocovaní antigénnej variability virusového glykoproteínu, pre přípravu antiidiotypovej protilátky pre vakcinačné účely, ako zdroj protilátky jedinej podtriedy pře přípravu sér, pře detekciu virusového antigénu v infikovaných zvieratách, 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 of viral glycoprotein, in anti-idiotypic antibody virus antigen in infected animals prior to BLV neutralization.
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