A (1-->3,1-->4)-beta-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical 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 conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1-->3,1-->4)-beta-glucan, purified by size exclusion chromatography and characterized by 1H-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 monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody 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 this oligoglucoside. The monoclonal antibody (BG1) has been used to develop 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 endosperm arabinoxylan or carboxymethylpachyman ((1-->3)-beta-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region.(ABSTRACT TRUNCATED AT 250 WORDS)
Meikle
P J
PJ
Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia.
Hoogenraad
N J
NJ
Bonig
I
I
Clarke
A E
AE
Stone
B A
BA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005936:Glucans / Q000032:analysis* / Q000276:immunology; D006001:Glycoconjugates / Q000737:chemistry; D007150:Immunohistochemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D047071:beta-Glucans
D000911:Antibodies, Monoclonal; D005936:Glucans; D006001:Glycoconjugates; D047071:beta-Glucans; C067858:beta-glucan, (1-3)(1-4)-