A (1-]3,1-]4)-BETA-GLUCAN-SPECIFIC MONOCLONAL-ANTIBODY AND ITS USE INTHE QUANTITATION AND IMMUNOCYTO-CHEMICAL LOCATION OF (1-]3,1-]4)-BETA-GLUCANS

Monoclonal antibodies were raised against a (1-->3,1-->4)-beta-glucan- bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1-->3,1-->4)-beta-glucan, displaying no binding activity against a (1-->3)-beta-glucan-BSA conju gate and minimal binding against a cellopentaose-BSA conjugate. A rang e of oligosaccharides was prepared by enzymatic digestion of (1-->3,1- ->4)-beta-glucan, purified by size exclusion chromatography and charac terized by H-1-NMR and anion exchange chromatography. These (1-->3,1-- >4)-beta-oligoglucosides, together with (1-->3)-beta- and (1-->4)-beta -oligoglucosides were used to characterize the binding site of the mon oclonal antibody (BG1) by competitive inhibition. The monoclonal antib ody showed maximal binding to a heptasaccharide with the structure Glc (1-->3) Glc(1-->4) Glc(1-->4) Glc(1-->3) Glc(1-->4) Glc(1-->4) Glc and was determined to have an affinity constant of 3.8 x 10(4) M-1 for th is oligoglucoside. The monoclonal antibody (BG1) has been used to deve lop a sensitive sandwich ELISA for the specific quantitation of (1-->3 ,1-->4)-beta-glucans. The assay operates in the range 1-10 ng ml-1 and shows no significant cross-reaction with tamarind xyloglucan, wheat e ndosperm arabinoxylan or carboxymethylpachyman ((1-->3)-beta-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and vi ewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections o f immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1-->3)-b eta-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.