Fig 1.
Heat map showing the relative binding of the BAM1 to BAM4, LM7 and LM23 MAbs to a range of brown algal, sulfated, red algal and land plant polysaccharides.
Binding was determined by ELISA with 50 μg/ml of polysaccharide samples and 25-fold dilutions of antibody hybridoma supernatants. S indicates polysaccharides containing sulfate residues. The colour scale in relation to absorbance values is shown top left. Values shown are means of 4 replicates and in all cases standard deviations (SDs) were <0.1 absorbance units.
Fig 2.
Sulfate assay and de-sulfation.
(A) Azure A absorbance spectra changes in the presence of differing levels (0 to 50 μg) of sulfated dextran, dextran and FS28. (B) Effect of de-sulfation on BAM MAb recognition of FS28. Samples of FS28 were collected over 4 time points (0, 1, 2 and 3 h) during a de-sulfation treatment with chlorotrimethylsilane and assayed using ELISA. Absorbance values are means of 4 replicates and error bars indicate SD. Sulfate levels as equivalents to native FS28 (w/w).
Fig 3.
Effect of Azure A inhibition and sodium metaperiodate treatment on BAM antibody recognition of fucoidan.
ELISA plates were coated with fucoidan and (A) pre-incubated with Azure A for 1 h, or equivalent control plates with buffer only, or (B) exposed to sodium metaperiodate for 0 to 24 h at 4°C in the dark. Absorbance values are means of 4 replicates and error bars indicate SD. LM11 binding to non-sulfated xylan was used as a control for Azure A inhibition.
Fig 4.
Epitope detection chromatographic (EDC) anion-exchange analysis.
EDC analysis of A, sulfated (DS0) and B, de-sulfated (DS3) derivatives of FS28 using BAM1, BAM2, BAM4 and LM23 as detection tools. The elution gradient was 0 to 4 M NaCl from 26 ml to 80 ml elution volume. Blue arrows indicate shift in peak of BAM1 epitope elution associated with de-sulfation. Green arrows indicate loss of BAM4 epitope peak height but no shift in elution time. EDC profiles shown are representative of two chromatographic runs.
Fig 5.
Indirect immunofluorescence analysis of F. vesiculosus receptacles.
(A) Bright field image of Toluidine Blue O (TBO) stained resin-embedded section and fluorescence imaging of the BAM1 to BAM4 epitopes in equivalent sections. E, epidermis; M, meristoderm; OC, outer cortex; IC, Inner cortex; Me, Medulla. Arrowheads indicate outer surface. (B) Combined bright field/fluorescence of BAM epitopes in relation to hyphal cells (H) and filament cells (F) of the medulla region. Scale bars = 30 μm.
Fig 6.
Indirect immunofluorescence analysis of F. vesiculosus female conceptacles.
Bright field images of TBO-stained sections and fluorescence imaging of the BAM2 and BAM3 epitopes in equivalent sections. (A) Conceptacles prior to ostiole opening and (B) conceptacles after ostiole opening. Asterisks indicate oogonia and arrowheads indicate paraphyses. Scale bars = 100 μm.
Fig 7.
Analysis of Ectocarpus subulatus.
Analysis of E. subulatus grown in seawater (100%) or in 20-fold diluted seawater (5%). (A) Indirect immunofluorescence detection of the BAM4 epitope at the surface of whole mount preparations, scale = 25 μm. (B) ELISA analysis of BAM4 epitope levels in extracts of AIR. C = CaCl2 extract, N = Na2CO3 extract, K = KOH extract. (C) Sulfate levels (FS28 equivalents w/w) in C, N and K extracts of AIR. Error bars indicate SD of 4 replicates.
Fig 8.
Heat map of antibody binding to cell wall extracts of a range of brown algae.
Heat map of relative BAM1 to BAM4, LM7 and LM23 epitope levels as determined by ELISA absorbance obtained from 25-fold dilution of MAbs with 50 μg/ml of extracts from 8 species of the Fucales (F), 6 species of Laminariales (L) and 1 species of Ectocarpales (E). Fucale and Laminariale species are listed in relation to approximate occurrence from upper to lower regions of intertidal zones. Extracts of AIR with CaCl2 (C), Na2CO3 (N) and KOH (K) after dialysis and freeze-drying. The colour scale in relation to absorbance values is shown top left. Values shown are means of 4 replicates and in all cases SDs were <0.1 absorbance units.