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Design of recombinant antibodies against tick-borne encephalitis virus
lnt,J. Med. Microbiol. 291. Suppl. 33. 174 (2002) © Urban & Fischer Verlag hltp://www.urbanfischer.defjournalslijmm
Abstract Design of recombinant antibodies against tick-borne encephalitis virus N. Tikunova *, G. Nikolenko, T. Un, E. Protopopova, S. Konovalova, L. Karpenko, V. Loktev, A. lIyichev State Research Centre of Virology & Biotechnology Vector, Novosibirsk, Russia
A murine single-chain antibody fragment (scFv), with variable heavy (Vh) and light (Vk) domains are derived from the mouse monoclonal antibody MabE6B specific for the glycoprotein E of tick-borne encephalitis (TBE) virus, and was expressed in Escherichia coli under the control of T7 bacteriophage late promoter. The ability of obtained scFv antibodies to bind its target antigen was shown by ELISA, Western blot and competition analysis. The recombinant antibodies were active in the hemagglutination inhibition reaction. To increase the avidity of scFv antibody for its antigen, the especial linear epitope, containing two cystein residues necessary for forming of disulfide bridges, was
fused to C-terminal end of scFv. As a result, stable dimeric scFv molecules were produced by E. coli cells. Both constructs carried six C-terminal histidine residues for purification. Both monomeric and dimeric scFv antibodies demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis with recombinant E protein and TBE virus. Affinity measurements carried out by competitive irnmunoassays showed that covalently linked (scFv')z have binding constants quite close to those of the parental monoclonal antibodies and three-fold higher than scFv monomers.
,. Corresponding author: Nina Tikunova, State Research Centre of Virology & Biotechnology Vector, Koltsovo, Novosibirsk Region 630559, Russia, Phone: 0073832366570, E-mail: [email protected] 1438-4221/01/291/8-000 $ 15.00/0
Abstract Design of recombinant antibodies against tick-borne encephalitis virus N. Tikunova *, G. Nikolenko, T. Un, E. Protopopova, S. Konovalova, L. Karpenko, V. Loktev, A. lIyichev State Research Centre of Virology & Biotechnology Vector, Novosibirsk, Russia
A murine single-chain antibody fragment (scFv), with variable heavy (Vh) and light (Vk) domains are derived from the mouse monoclonal antibody MabE6B specific for the glycoprotein E of tick-borne encephalitis (TBE) virus, and was expressed in Escherichia coli under the control of T7 bacteriophage late promoter. The ability of obtained scFv antibodies to bind its target antigen was shown by ELISA, Western blot and competition analysis. The recombinant antibodies were active in the hemagglutination inhibition reaction. To increase the avidity of scFv antibody for its antigen, the especial linear epitope, containing two cystein residues necessary for forming of disulfide bridges, was
fused to C-terminal end of scFv. As a result, stable dimeric scFv molecules were produced by E. coli cells. Both constructs carried six C-terminal histidine residues for purification. Both monomeric and dimeric scFv antibodies demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis with recombinant E protein and TBE virus. Affinity measurements carried out by competitive irnmunoassays showed that covalently linked (scFv')z have binding constants quite close to those of the parental monoclonal antibodies and three-fold higher than scFv monomers.
,. Corresponding author: Nina Tikunova, State Research Centre of Virology & Biotechnology Vector, Koltsovo, Novosibirsk Region 630559, Russia, Phone: 0073832366570, E-mail: [email protected] 1438-4221/01/291/8-000 $ 15.00/0