A22 ve O1 Tipi Şap Virüsü Aşı Suşlarının 146S 12S Partüküllerine Karşı Monoklonal Antikor Üretimi

Yıl 1995, Cilt: 8 Sayı: 1, 56 - 68, 01.12.1995

S. İsmet Gürhan Burhan Gürhan Gülhan Aynagöz Nilay Ünal Gülnur Ünver Sinan Aktaş

Öz

Şap hastalığına yönelik serolojik ve immunolojik testlerde kullanmak üzere,
Türkiye'de uygulana •. şap virüsü aşı suşlarının (01 Man 69 ve A22 Mah 65)
146S ve 12S partiküllerine karşı monoklonal antikorlar üretildi.
Burada, standard immunizasyon protokoluna göre immunize edilen Balb/c
farelerin dalak hücreleri ile NSO myelome hücrelerine PEG4000 varlığında
fuzyon uygulandı. Oluşan hibridoma kolonileri indirekt ELISA yöntemi ile kontrol
edildi. Seçilen pozitif klonlar in vitro çoğaltıldı. Sonuçta A22 Mah65 ve O1 Man69
şap virüsü suşlarının 146S ve 12S partiküllerine karşı monoklenal antikor
salgılayan toplam 170 adet hibridoma klonu, (12 adet A22 146S, 10 adet A22
12S, 17 adet 0 1 146S ve 113 adet 0 1 12S) elde edildi ve sıvı azot içerisinde
(-196°C)de dondurularak stoklandı.

Anahtar Kelimeler

Şap, Şap Virüsü, Şap Aşısı, 146 S Partikülü, 12 S Partikülü, Monoklonal Antikor Üretimi, S. İsmet GÜRHAN, Burhan GÜRHAN, Gülhan AYNAGÖZ, Nilay ÜNAL, Gülnur ÜNVER, Sinan AKTAŞ, 1995, Etlik Veteriner Mikrobiyoloji Dergisi

Production of monoclonal antibodies against 146S and 12S particles of A22 and O1 types vaccine strains of FMDV

Öz

The aim of this study is to produce MAbs against 146S and 12S particles of
FMDV vaccine strains ( A22 Mah 65 and O1 Man 69) to be used in serological
and immunological testsfor FMD.
The spleen cells of Balb/c mice, immunized in accordance with the standard immunization protacal and NSO mouse myeloma cells were fused in the
presence of PEG 4000 as fusing agent. Hybridoma colonies were screened for
antigen specificity by indirect enzymelinked immunosorbant assay (ELISA).
Selected positive clones were produced in vitro. As a result, totally 170 MAb
producing hybridoma clones against 1468 and 12S particles of A22Mah 65 and
O1 Man 69 (12 for A22 1468, 10 for A22 128, 17 for O1 Man 69 1468 and 113 for
O1 Man 69 12S) were produced and stored in liquid nitragen (-196°C)

Kaynakça

• Antibodies, A Laboratory Manual, Eds. Ed Harlow, David Lane, Cold Spring Harbor Lab. Perss. ISBN-D-87969-374-6, 1988.
• Art to Science (in Tissue Culture). Vol.3. No 3 Sep 1984 (Newsletter of High Clone Laboratories) Taggart Hybridoma Technology (THT™) revolotionizes hybridoma research .
• Bolton, B.J., Morssi, C.B., and Mowles, J.M. (1993) Longterm cultures and cell-lines, Methds. Imm. Analys, 3:103-120.
• Butchaiah, G., Rao, B.V. (1989): Hibridoma cell-lines seereting Mabs to FMDV type Asia.l Acta Viral. 33:121-130.
• Butcher, R.N ., Obi. T.U . and Mc Cullough. K.C. (1991): Rapid isolation of Monocianal hybridoma cultures by a "Fusion-cloning" method: The requirement for aminopterin. Biologicals, 19: 171-175.
• Crowter, J.R. (1993): The use of Mabs in the malecular typing of animal viruses. Rev. Sci. Tech. Off. Int. Epiz. 12 (2): 369~383.
• Crowter, J.R. and Sammuel, A. (1987): Monocianal antibodies and FMD. Rep. Sess. Res. Gr. Stand. Tech. Comm. EC. Cont. FMD. Lyons, France, FAO, Rame, App 13,88-102.
• Crowter, J.R. and Sammuel, A. (1990): Monocianal antibodies and FMD. Eur. Comm. Cont. FMD. Res. Gr. Stand. Tech Comm. Lindholm.
• Doel T. and Mowat, G.N. (1985): An International collaborative study on FMD assay methods. 2. Quantifıcation of 146S particles. J. Biol. Stand. 13:335- 344.
• Fazekas, S.and Scheidegger . D. (1980) : Production of Mabs:Strategy and Tactics. J.lmm. Meths:35:1-21
• Galfre, G. and Milstein. C. (1982) : Preparation of Mabs: Strategies and procedures, Proc. Symp. Properties of The Mabs produced by hybridoma technology and their application to the study of diseases, Singapour 19-23. Oct. 1981 . UNDP/World Bank/WHO, Geneva, Switzerland.
• Gupta, R.K. Mısra , C.N. Gupta. C. K. and Saxena. 8.(1991): Growth of allogenic hybridoma cells in non-histocompatible mice with the help of EhrlichLetter ascites tum or ·ce lls. Biologicals. 18:243-245
• Jaspert, R. , Geske. T. , Teichmann. A. , Ka~ner , Y. , Kerzchmar. K. , L'Age-Stehr, J (1995) : Laboratory scale production af M absina tumbling chamber, J. lmmunol, Methds., 178: 77-87
• Jıskoot , W . . BloemendaL M., Haeringen. B., Grondelle., Beuvery E.C., Herron. J. and Crommelin, J.A. (1991) : Non-randam canfırmation of mouse lgG2a M ab at law pH. Eur.J. Biochm. 201 :223-232
• Köhler, G., Milstein, M. (1975) :Continuous cultures offused cells secreting antibody of predefıned specifıty . Nature, 256:495-497
• Lu, G.Z. , Thompson, B.G., Suresh, M.R. and Murray, R.G. (1995): Cultivation of hybridoma cells in an inclined bioreactor, Biotech and Bioeng, 45(2) : 176-186
• Marx, U. , Merz, W. , Koch, S. , Nagel, A. , Schlöf M. , Schlag, P.M. Liebriecn, W., Lübbe. L. , von Baehr.R. (1994): Cultivation of hybridomas, human cancer and human primary cells using the miniaturized hallaw fibre bioreactor TECHNOMOUSE. Anim.Cell. Tech: Basic and Applied Aspects.Voi,6 .Proc.6th.lnt.Meeting on the JAACT, Nagoya, Japan, Nov. 1993,171-175.
• Mc.Cullough,K.C. and B.utcher., R(1982) Monocianal antibodies against FMDV 1468 and 128 particles.; Arch.Virol.74:1-9.
• Mc.Cul!ough, K.C., Butcher.R.and Parkinson,D (1983). Hybridoma celllines seereting MAbs•against FMDV.II. Cloning conditions.J.Bioi.Stand.ll: 183- 194.
• Mc.Cullough, K C.,Crowther, J.P.and Butcher, R.N.(1985): Alteration in antibody reactivity with FMDV 1468 antigen before and after binding to a solid phase or complexing with specifıc antibody .J.Imm. Mth.82:91-100
• Michael, M.G.(1987) : Chinese hamsterovary cells. Meths. Enzym, 151 :3-8
• Monocianal Antibodies: Principle and Practice, Production and Application of Mabs in Cell Biology,Biochemistry and lmmunology (Ed), J .W. Goding, Acad. Press, London. 1983
• Rosevear, A.and Lambe,C.(1988) : Downstread processing of animal cell culture products. Recent developments.Anim.Ceii.Biotech.,3.397-440.
• Samuel, A.R., Knowles,N.J., Samuei,G.D.and Crowther ,J .R.(1991) : Evatatian of a trapping ELI SA and for the differentitation of FMDV strains using MAbs.Biological, 19 :299-31 O
• Samuei,A.R., Ouldridge,E .J.,Arrowsmith,A.E., Kitching,R .P. and Knowles,N.J.(1990) : Antigenic analysis of serotype O FMDV isolates from Middle East, 1981 to 1988.Vaccine, 8: 390-396
• Shen,B.,Greenfıeld,P . and Reid,S,(1994) : Calcium alginate immobilized hybridomas grown using a fluidized -bed perfusion system with a protein-free medium.Cytotechnoogy 14,109-117
• Shiral, Y., Yamaguehi,M ., Kobayashi,A.,Nishi.A.,Nakamura,H.and Murakami, H.,(1994):Change in growth kinetics of hybridoma cells entrapped in collagen gel effects by alkaline supply. Cytotechnology ,4:129-146
• Smith,G.J.,McFarland,R.D., Reisner,H .M. and Hudson.G.S. (1984) : Lymphoblastoid cell-produced immunoglobulins: Purification from culture. medium by hydroxylapatite chromatography,Anal.Biochem. , 141:432-436
• Twomey,T. ,Fiance.LL.,Hassard.S.,Burrage, T.G., Newman,J.F.and Brown,F.(1995) : Characterization of an acid-resistant mutant of FMDV. Virology, 206: 69-75.
• Van Maanen,C and Terpstra,C.(1991): Quantifıcation ofintact 146S FMD antigen for vaccine production by a DAS-ELISA using M abs. Rep. Europ. Com m. Cont. FMD. Sess. Res. Gr. Stand. Tech. Comm., Lindholm. , Denmark, 1990, App.ll
• Westerwoudt,R.J.(1986) : Factors affecting production of MAbs. Methds. Enzym.121:3-18