The location of (1?3)-?-glucans in the walls of pollen tubes of Nicotiana alata using a (1?3)-?-glucan-specific monoclonal antibody

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Abstract
The location of the (1→3)-β-glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (1→3)-β-glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (1→3, 1→4)-β-glucan-BSA conjugate. Binding was inhibited by (1→3)-β-oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (1→3)-β-linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 104M−1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (1→3)-β-glucan in the inner wall layer of thin sections of the N. alata pollen tubes.