Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance.
Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.
Aström
Eva
E
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
Ohlson
Sten
S
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry*; D002417:Cattle; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification; D017912:Inovirus / Q000737:chemistry*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D002241:Carbohydrates; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D012710:Serum Albumin, Bovine