Transiently binding antibody fragments against Lewis x and sialyl-Lewis x.
Biomolecular recognition is often characterised by low affinity where many weak interactions work either alone or in concert, resulting in an inherent dynamic situation. For example the well-studied weak binding of cell-cell interactions is predominantly based on a range of carbohydrates that interact with numerous (protein) ligands. Finding appropriate binders to these carbohydrate structures may pave the way for new analytical strategies based on low affinity, and recombinant antibody technology is a promising approach to the development of such reagents. We have in the present study characterised two low affinity human single chain antibody fragments (scFv) by surface plasmon resonance for use in such applications. The two clones, LeX1 and sLeX10, had been selected from a naive phage display library against Lewis x (Le(x)) and sialyl Le(x) (sLe(x)), respectively. Both LeX1 and sLeX10 showed low affinity, with K(D) values of 3.5+/-0.7 x 10(-5) M for Le(x) and 2.6+/-0.7 x 10(-5) M for sLe(x), respectively. Kinetic studies revealed the scFvs to be associated with fast dissociation rates, with Kd values higher than 0.1 s(-1) for both LeX1 and sLeX10. Apart from the Lewis structures Le(x) and sLe(x), we investigated the conformational isomers Lewis a and sialyl-Lewis a together with the monosaccharide units of the Lewis structures, and both scFvs showed high specificity for their respective carbohydrate. Taking these observations together we have demonstrated that scFv with fast reaction kinetics and low affinity have the necessary characteristics for further development as specific tools in analytical strategies, e.g. differentiation of cells based on the various configurations of carbohydrate epitopes.
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
Reine
R
Johansson
Mats
M
Ohlin
Bo
B
Jansson
Sten
S
Ohlson
D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D007700:Kinetics; D016256:Lewis X Antigen / Q000032:analysis* / Q000276:immunology; D009844:Oligosaccharides / Q000032:analysis* / Q000276:immunology; D019151:Peptide Library; D000080506:Sialyl Lewis X Antigen; D020349:Surface Plasmon Resonance
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D016256:Lewis X Antigen; D009844:Oligosaccharides; D019151:Peptide Library; D000080506:Sialyl Lewis X Antigen