Introduction 1 7 8 9 10 11 12 13 14 16 17 18 18 19 21 22 e.g. e.g. e.g. e.g. e.g. A. thaliana 23 Experimental procedures Immunisation and generation of monoclonal hybridomas A. thaliana 2 24 25 Previously described monoclonal antibodies 1 Table 1 Previously characterized monoclonal antibodies used to probe glycan arrays. HG, homogalacturonan Specificity Name Ref. Un-esterified HG PAM1 31 Un-esterified/Calcium ion cross-linked HG 2F4 34 Partially methyl-esterified HG JIM5 33 Partially methyl-esterified HG JIM7 33 (1→4)-β-galactan LM5 37 (1→5)-α-arabinan LM6 24 Fucosylated xyloglucan CCRC-M1 38 Non-fucosylated xyloglucan LM15  (1→4)-β-mannan/galacto-(1→4)-β-mannan BS-400-4 39 (1→4)-β-xylan LM10 40 (1→3)(1→4)-β-glucan BS 400-3 41 (1→3)-β-glucan BS 400-2 32 (1→4)-β-xylan/arabinoxylan LM11 40 Arabinogalactan-protein JIM4 43 Arabinogalactan-protein LM2 42 Arabinogalactan-protein MAC207 43 Arabinogalactan-protein JIM8 44 Arabinogalactan-protein JIM13 43 Arabinogalactan-protein JIM16 43 Arabinogalactan-protein JIM14 43 Extensin LM1 45 Extensin JIM19 46 Extensin JIM20 46 Glycan samples used on the array 2 2 2 2 w v A. thaliana 2 Table 2 Samples included on the glycan arrays Alphanumerical codes Samples A1 Arabinan (sugar beet) B1 Pectin (apple) C1 Galactan (lupin) D1 Homogalacturonan (sugar beet) E1 Pectin (lime) B15 F1 Pectin (lime) B43 G1 Pectin (lime) B71 H1 Pectin (lime) 96 A2 Pectin (lime) F11 B2 Pectin (lime) F19 C2 Pectin (lime) F43 D2 Pectin (lime) F76 E2 Pectin (lime) P16 F2 Pectin (lime) P24 G2 Pectin (lime) P32 H2 Pectin (lime) P41 A3 Pectin (lime) P46 B3 Pectin (lime) P60 C3 Pectin (lime) P76 D3 RGI (soybean) E3 A. thaliana F3 Xylogalacturonan (pea) G3 MHR I (apple) H3 MHR II (carrot) A4 MHR III (potato) B4 MHR HS1 (apple) C4 MHR HS2 (apple) D4 Xylogalacturonan (apple) E4 (P. patens) F4 A. thaliana G4 Xyloglucan/mannan (tomato) H4 Glucomannan (konjac) A5 Gum (guar) B5 Gum (locust bean) C5 Gum arabic (acacia) D5 Gum (karaya) E5 Gum (tragacanth) F5 AGP (larch) G5 Arabinoxylan (wheat) H5 β A6 Mannan (ivory nut) B6 Xyloglucan (tamarind) C6 Glucuronoarabinoxylan (maize) D6 Hydroxyethyl cellulose E6 β F6 Carboxymethyl cellulose G6 Alginic acid H6 β A7 β B7 β C7 A. thaliana D7 A. thaliana E7 A. thaliana F7 A. thaliana G7 A. thaliana H7 A. thaliana A8 A. thaliana B8 A. thaliana C8 A. thaliana D8 A. thaliana E8 A. thaliana F8 A. thaliana G8 A. thaliana H8 A. thaliana Alphanumerical codes refer to the position of samples on arrays. Source organisms are in parentheses RGI RGII MHR AGP Post-printing modification of glycans in situ 6 α β Aspergillus niger Printing of arrays A. thaliana 2 Probing of arrays 2 4 2 4 w v 2 Scanning and analysis 1 1 2 5 http://bbc.botany.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi http://ep.ebi.ac.uk/EP/EPCLUST/ Fig. 1 a b b Arabidopsis thaliana 2 c d red zone green zone e g R 2  Fig. 2 a b black boxes  Indirect immunofluorescence labeling of plant materials 26 27 Competitive-inhibition ELISA assays 28 Sugar composition analysis of modified pectic hairy regions (MHRs) 29 3 2 30 Results Production of microarrays of plant cell wall polymers 1 1 2 1 1 2 3 R 2 1 Fig. 3 Probing of cell wall glycan arrays with newly produced mAbs. Glycan arrays were probed with mono- or multiclonal antibodies generated following shotgun immunisation with plant cell wall polymers. Four representative examples are shown. Arrays were scanned and converted into 16 bit greyscale TIFFs  Probing of plant cell wall glycan arrays 1 2 2 31 32 A. thaliana A. thaliana 3 Hierarchical clustering of antibody binding profiles 4 33 31 34 Fig. 4 red diamonds black boxes 1 2 3 4 1  Detailed characterization of the epitopes recognized by LM14 and LM13 5 6 24 6 6 6 6 6 α α Fig. 5 a b black boxes  Fig. 6 a d dotted line e α α  35 A. thaliana 7 Fig. 7 Arabidopsis thaliana  Immunolocalization of the LM14 and LM13 epitopes in planta A. thaliana 8 in planta A. thaliana 8 A. thaliana in planta A. thaliana 8 A. thaliana 8 A. thaliana 8 8 Fig. 8 A. thaliana blue b A. thaliana Arrowheads a b c a d a e A. thaliana f arrowheads g mx h a d g h g e f  Discussion The work presented here demonstrates the potential of glycan microarrays for overcoming a major bottleneck in anti-glycan mAb production. Specifically, the use of microarrays enabled 50 μl of hybridoma supernatant to be screened rapidly and simultaneously against >60 potential epitope-bearing target molecules. A novel aspect of this work was the use of cluster analysis of array data to rapidly predict antibody specificities by comparison with previously defined mAbs. Subsequent detailed analyses of the specificities of the new mAbs LM13 and LM14 indicated they bound to pectic and AGP class of polymers respectively, as was predicted by the cluster analysis. These results indicate that if a relatively large set of probes with defined specificities are available to serve as references, this is an effective method for high-throughput initial mAb screening. One potential limitation of shotgun immunisation could be immuno-dominance, such that an immune response is elicited against a limited subset of the injected antigens. In this study two mAbs, LM13 and LM14 were selected with specificity for two different classes of molecule. However, of the seven mAbs selected for cluster analysis of binding profiles, six clustered with mAbs with specificity for AGPs and one with mAbs to pectic side chains, suggesting that AGPs were the immuno-dominant antigens in this case. It is therefore likely that such a multi-antigen approach towards cell wall polymers may be most effective to obtain a panel of mAbs with a range of specificities within a single class of polymer. 35 36 8 4 18 A. thaliana