Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment-D dimer.
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.
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
A M
AM
Vandamme
F
F
Bulens
H
H
Bernar
L
L
Nelles
R H
RH
Lijnen
D
D
Collen
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D000915:Antibody Affinity; D001483:Base Sequence; D002999:Clone Cells / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis* / Q000096:biosynthesis; D005260:Female; D005337:Fibrin / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA