Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies

“…As a spacer for the spatial separation of two affi nity centers of the chimeric protein (CBD and ScFv) a sequence of 13 amino acids was introduced [5,6]. Professor Y. Shoham (Israel) kindly provided the plasmid vector pCBD with the cellulose binding domain (CBD) sequence from Clostridium thermocellum for our research.…”

In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b

“…were determined. An interesting point was that some producers randomly selected out of more than 30 isolated positive clones invariably differed between each other in the production level of the ScFv's (0.6 to 3.5 mg/L) and their accumulation rate, ScFv's sensitivity to proteolysis, ScFv's affini ty (1.96 · 10 -8 M to 1.69 · 10 -9 M), and ScFv's bind ing specificity with native and denatured rhIFN β1b [15].…”

“…In previous publica tions, we described generation of an immune com binatorial cDNA library of mouse immunoglobu lin V genes and isolation of a panel of single chain antibodies specific to rhIFN β1b [14,15]. For some isolated ScFv's such characteristics as bind ing specificity to native and denatured antigen, level of production in E. coli by secretion, stability etc.…”

“…For analysis, we used the four sequences of ScFv's against rhIFN β1b iso lated from the immune library, a sequence of ScFv against rhIFN β1b preliminarily obtained from a naїve library [15], as well as a sequence of ScFv's against rhIFN α2b [17]. According to the criteri on of homology for their DNA, all six ScFv's were provisionally divided into four groups (Figure).…”

“…It is a matter of fact that single aminoacid substitutions in the central part of CDRH3 may dramatically affect antibody's affinity and speci ficity. The aforementioned substitutions in CDR IMGT may explain the differences in the panel's ScFv's antigen binding properties studied in the previous work [15]. We suppose the identified amino acid residues are promising targets for fur ther ScFv's mutagenesis.…”