Production and characterization of a phage-display recombinant antibody against carrageenans: evidence for the recognition of a secondary structure of carrageenan chains present in red algae tissues.
We report the isolation, for the first time by phage display, of a scFv recombinant antibody called B3 directed against carrageenans, the major sulphated polysaccharides of red seaweeds. Immunoassays were used to characterize the binding of B3 antibodies toward the three main carrageenan forms (iota, kappa, and lambda) differing by their sulfonic ester content and the presence of 3,6-anhydrogalactose. In enzyme-linked immunoadsorbent assay (ELISA), B3 soluble scFv showed a high reactivity towards iota-carrageenan at any titer but, at high titer only, recognized also the highly sulfated lambda-form. Surface-adsorbed kappa-polymers were only recognized in presence of poly-L-lysine (PLL). The replacement of Na+ ions by K+ in the buffers had no effect on kappa-polymer detection but increased the binding of B3 antibodies toward both iota- and lambda-carrageenans, whereas addition of Ca2+ decreased sharply the recognition of the iota-form. In competitive assays, low titer B3 soluble scFv showed a iota>kappa>lambda selectivity and recognized a mixture of iota-oligomers with degrees of polymerization between 4 and 18 but not sub-fractions of 4 or 6 residues long. We suggest therefore that the B3 epitope could consist of a helical conformation of carrageenan chains. Immunofluorescence microscopy showed that, amongst other red algae, Chondrus gametophyte (containing iota-chains) was strongly recognized by B3 scFv whereas sporophytic tissues rich in lambda-carrageenans were not, assessing the preference of this probe for iota-carrageenans in situ. The high potential of the B3 recombinant probe is discussed.
Liners
Françoise
F
URBV, University of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium.
Helbert
William
W
Van Cutsem
Pierre
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D002236:Carbohydrate Conformation; D002351:Carrageenan / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000461:Rhodophyta / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D002351:Carrageenan