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Weak affinity scFvs directed towards carbohydrate structures Lewis x and sialyl-Lewis x

Författare:
  • Reine Johansson
  • Mats Ohlin
  • Bo Jansson
  • Sten Ohlson
Publiceringsår: 2003
Språk: Engelska
Dokumenttyp: Konferensbidrag

Sammanfattning

The Lewis (Le) blood group antigens are a set of fucosylated glycan structures. They are present as N- and O-linked structures on glycoproteins and as glycolipid-based structures on cell surfaces. Depending on the exact structure, the Lewis group is divided into four subgroups; Lea, Leb, Lex and Ley. Lex and Lea can further be sialylated and/or sulfonated. Among these, Lex has been found to be implicated in tumour progression and possibly in the attachment of Helicobacter pylori whereas sialylated Lex (sLex), which is a natural ligand for the selectins, is implicated in leukocyte trafficking and in tumour progression.
We have characterised two human single chain Fv (scFv) antibody fragments, previously selected from the nCoDeR phage display library (BioInvent, Lund, Sweden). The two clones are designated LeX1 and sLeX10 and were selected against Lex and sLex, respectively. We were interested in finding out whether the scFvs have a low affinity and fast kinetics, making them candidates for future applications in multivalency based separation systems such as weak affinity chromatography (WAC), affinity capillary electrophoresis (ACE) and whole cell chromatography. The scFvs were characterised regarding affinity, kinetics and specificity by the means of surface plasmon resonance (SPR) on a Biacore X instrument. As expected, both LeX1 and sLeX10 showed low affinity, with a KD value in the vicinity of 5·10-5 M. Kinetic studies revealed the scFvs to be associated with fast dissociating rates, with kd values of approximately 0.8 s-1 and 2.2 s-1 for LeX1 and sLeX10, respectively. Specificity was determined by competitive analyses. Beside the Lewis structures Lex and sLex, we investigated the conformational isomers Lea and sLea together with GlcNAc, Gal, Fuc and sialic acid as they are the monosaccharide units of the Lewis structures. Both scFvs showed high specificity for their respectively carbohydrate.
To our knowledge, this is the first time that two recombinant antibody fragments specific for Lex and sLex have been described. Taken together, these new scFvs are reassuring for further development of multivalency based analysis strategies, to be used in our ongoing exploration of cell and glycoprotein carbohydrate diversity.

Disputation

Nyckelord

  • Chemistry

Övriga

Unpublished

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