Liners Françoise, Helbert William, Van Cutsem Pierre
URBV, University of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium.
Glycobiology. 2005 Sep;15(9):849-60. doi: 10.1093/glycob/cwi072. Epub 2005 May 4.
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.
我们首次通过噬菌体展示技术分离出一种名为B3的单链抗体片段（scFv）重组抗体，该抗体针对角叉菜胶，即红藻的主要硫酸化多糖。免疫测定用于表征B3抗体与三种主要角叉菜胶形式（ι型、κ型和λ型）的结合情况，这三种形式在磺酸酯含量和3,6 - 脱水半乳糖的存在上有所不同。在酶联免疫吸附测定（ELISA）中，B3可溶性scFv在任何滴度下对ι型角叉菜胶都表现出高反应性，但仅在高滴度时也能识别高度硫酸化的λ型。表面吸附的κ型聚合物仅在聚-L-赖氨酸（PLL）存在时才能被识别。缓冲液中用K⁺替代Na⁺离子对角叉菜胶聚合物的检测没有影响，但增加了B3抗体与ι型和λ型角叉菜胶的结合，而添加Ca²⁺则急剧降低了对ι型的识别。在竞争测定中，低滴度的B3可溶性scFv表现出ι>κ>λ的选择性，并且识别聚合度在4至18之间的ι型寡聚体混合物，但不识别4或6个残基长的亚组分。因此，我们认为B3表位可能由角叉菜胶链的螺旋构象组成。免疫荧光显微镜显示，在其他红藻中，B3 scFv强烈识别含有ι型链的角叉菜配子体，而富含λ型角叉菜胶的孢子体组织则不被识别，这在原位评估了该探针对角叉菜胶的偏好。文中还讨论了B3重组探针的高潜力。
