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|Antigen/Gene or Protein Targets||β-(1→4)-manno-oligosaccharides from DP2 to DP5.|
How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail.
It has been shown that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields.
Direct fluorescence imaging using a mannan‐specific carbohydrate‐binding module and sequential enzyme treatments with an endo‐β‐mannanase has confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro‐environments.
|Immunogen||Neoglycoprotein immunogen, prepared by coupling of mannopentaose (Man5) and digalactosylmannopentaose (Gal2Man5) oligosaccharides to bovine serum albumin (BSA) by reductive amination.|
This antibody recognises β-linked mannan polysaccharides of plant cell walls. It has no known cross-reactivity with other polymers and can recognise heteromannan polysaccharides in several species.
LM21 displays a wide recognition of mannan, glucomannan and galactomannan polysaccharides.
Rat monoclonal antibody derived subsequent to immunization of rat with neoglycoprotein immunogen, prepared by coupling of mannopentaose (Man5) and digalactosylmannopentaose (Gal2Man5) oligosaccharides to bovine serum albumin (BSA) by reductive amination (Roy et al., 1993). Briefly, 30 mg of oligosaccharide were dissolved in 1.0 ml sodium borate buffer, pH 8.5, followed by addition of 20 mg BSA and then 30 mg sodium cyanoborohydride. The mixture was maintained at 50°C for 24 h, and then the pH was adjusted to 4.0 using acetic acid. The materials were then dialysed against distilled water. The resulting neoglycoproteins were designated Man5–BSA and Gal2Man5–BSA. Rat immunization, hybridoma preparation and cloning procedures were performed as described previously (Willats et al., 1998). For each conjugate, two male Wistar rats were injected with 100 μg neoglycoprotein in complete Freund’s adjuvant administered subcutaneously on day 0, and the same amount was administered with incomplete Freund’s adjuvant on two subsequent occasions for Man5–BSA and three for Gal2Man5–BSA. Pre‐fusion boosts of 100 μg neoglycoprotein in 1 ml PBS were given by intraperitoneal injection 3 days before splenectomies leading to lymphocyte isolation and fusion with rat myeloma cell line IR983F (Bazin, 1982). Antibodies were selected by ELISA using the neoglycoproteins as antigens. Subsequent characterization was by means of immunochemical assays, oligosaccharide microarrays and immunofluorescence analysis of binding to plant materials.
|Research Area||Plant Science|
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