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Title: Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment-D dimer
Authors: Vandamme, Anne-Mieke ×
Bulens, F
Bernar, Hilde
Nelles, Lucien
Lijnen, Roger
Collen, Desire #
Issue Date: Sep-1990
Publisher: Published by Springer-Verlag on behalf of the Federation of European Biochemical Societies
Series Title: European Journal of Biochemistry vol:192 issue:3 pages:767-75
Abstract: cDNA libraries in lambda phage were generated from the murine hybridoma secreting mAb-15C5, a monoclonal antibody directed against fragment-D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemostasis 61, 307-313], and clones encoding fragments of the heavy (gamma 1) and the light (kappa) chain were isolated. The kappa-chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The gamma 1-chain cDNA was reconstructed from the mAb-15C5 kappa-chain signal sequence, the mAb-15C5 gamma 1 variable-domain coding sequence and murine gamma 1-gene and gamma 1-chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb-15C5 was purified from the conditioned medium by chromatography on Zn-chelate - Sepharose, protein-A - Sepharose and insolubilized fragment-D dimer, with a yield of 50 micrograms/l and a recovery of 20%. SDS-gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light-chain band with identical and a heavy-chained band with a somewhat slower mobility than that of the natural mAb-15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb-15C5 for fibrin fragment-D dimer. Thus recombinant mAb-15C5, obtained by co-expression of the reconstructed cDNAs of the kappa and gamma 1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb-15C5. These recombinant mAb-15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.
ISSN: 0014-2956
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Laboratory of Clinical and Epidemiological Virology (Rega Institute)
Molecular and Vascular Biology
× corresponding author
# (joint) last author

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