High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles
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Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.
KeywordsCarbohydrate microarrays Plant cell walls Monoclonal antibodies Hierarchical clustering
Carbohydrate microarrays provide a means of rapidly screening the interactions between glycans and other molecules [1, 2, 3, 4, 5, 6, 7]. Applications for this technology include the screening of protein–glycan interactions, characterization of carbohydrate-active enzymes and the analysis of the specificities of monoclonal antibodies (mAbs) and carbohydrate-binding modules [8, 9]. mAbs are powerful tools for investigating the biological roles of glycans but producing them is time consuming, labour-intensive and costly. Hybridoma-based mAb production involves the fusion of spleen cells from an immunized animal with myeloma cells. The resulting hybridoma cell lines are both immortal and secrete antibody into the cell supernatant . This procedure is conventionally performed in a one-by-one fashion, such that each animal is immunized with a single antigen and the resulting antibodies are screened for desired specificities using enzyme-linked immunosorbent assays (ELISAs). However ELISA-based screening is low throughput because only a limited amount (∼100 μl) of hybridoma supernatant is available for screening during the initial stages of mAb production and this is typically only sufficient to test each mAb against just one or two antigens. An alternative approach involves ‘shotgun’ immunisation with a mixture of antigens . This potentially results in the simultaneous generation of antibodies with a range of specificities, but the time limiting step then becomes the detailed retrospective screening of their specificities. However, microarrays offer a means of rapidly screening limited amounts of hybridoma supernatant against multiple antigens and therefore greatly increasing throughput in the identification of valuable cell lines. Here we report the use of shotgun immunisation followed by microarray-based screening of hybridoma supernatants in order to produce mAbs against plant cell wall glycans.
Plant cell walls are fibre composites that contain some of the most complex glycans known [12, 13]. In addition to their biological roles, many cell wall components have important industrial applications including as functional food ingredients, pharmaceuticals, nutriceuticals, fibres and increasingly, bio-fuels [14, 15, 16]. Cell wall glycans can be broadly grouped into cellulose, hemicelluloses, pectins and glycoproteins . Cellulose microfibrils are cross-linked by hemicelluloses such as xyloglucans, xylans and mixed linkage glucans forming a tough load-bearing matrix which is embedded in pectic polysaccharides . Pectins are the most complex and heterogeneous family of cell wall glycans and are comprised of a series of galacturonic acid-rich polymers including homogalacturonan (HG), rhamnogalacturonan I (RGI), rhamnogalacturonan-II (RG-II) and xylogalacturonans (XG). In addition, structurally complex arabinan, galactan and arabinogalactan polymers may be present as side chains to the galacturonan-rich backbone domains . The protein moieties of cell wall glycoproteins are often rich in hydroxyproline (Hyp) and these polymers, which are collectively referred to as Hyp-rich glycoproteins (HRGPs), include the extensins and arabinogalactan-proteins (AGPs) [19, 20, 21].
The fine structures and relative amounts of cell wall components vary greatly not only among plants, but also between organs, tissues, cells, and even between different micro-domains within a single cell wall. This complexity and heterogeneity presents a major barrier to detailed analysis and our understanding of many aspects of plant cell wall structure and function is far from complete. Several mAbs with specificities for diverse plant cell wall components have been developed and these are powerful tools for the analysis of cell walls . However, the repertoire of mAbs currently available covers only a small proportion of the glycan structures that have been identified and there is a pressing need for a wider range of mAbs to facilitate the further characterization of cell walls. We have developed a method of rapidly screening mAb specificities using microarrays of cell wall glycans including pectins with different degrees and patterns of methyl-esterification, pectic side chains (e.g. arabinan and galactan), hemicelluloses (e.g. xylans, mannans, and xyloglucans), HRGPs (e.g. AGP-rich gums), β-linked glucans (e.g. (1→3)(1→4)-β-glucan and (1→3)-β-glucan) and celluloses (e.g. hydroxylethyl cellulose and carboxylmethyl cellulose). A further 14 samples were sequentially extracted from A. thaliana using CDTA and NaOH which are known to predominately solublize pectic polymers and hemicelluloses respectively .
2 Experimental procedures
2.1 Immunisation and generation of monoclonal hybridomas
Cell wall polymers were isolated from 6 week old wild type A. thaliana plants, ecotype Col-0, grown in soil at 22°C with cycles of 10 h of light/14 h of darkness. One g dry weight of a mixture of leaves, stems and roots was homogenized to a fine powder in liquid nitrogen. The homogenate was incubated with 20 ml 50 mM 1,2-Diaminocyclohexanetetraacetic acid (CDTA; pH 7.5) for 3 h at 18°C and centrifuged for 20 min at 4,400 rpm. The supernatant was collected and dialyzed extensively against deionized water (dH2O) in dialysis tubing (6–8,000 kDa molecular weight cut off) to remove low molecular weight molecules and freeze dried. The material was dissolved in phosphate-buffered saline (PBS) to generate the immunogen. Rats were used for antibody production so as to make subsequent comparisons with existing mAbs, most of which were produced in rats, as valid as possible. The immunization of rats, hybridoma preparation and cloning procedures were as described previously . Briefly, two male Wistar rats were each injected subcutaneously with 250 μl of an emulsion of the isolated cell wall material at 1 mg/ml in PBS with an equal volume Freund’s complete adjuvant on day 0. On days 40 and 79 the injections were repeated using incomplete adjuvant. Tail bleeds were taken 10 days after injections to assess the immune response. On day 198 a pre-fusion boost was given to the selected rat and 3 days later, the spleen was removed and lymphocytes were isolated and fused with rat myeloma cell line IR983F  using standard polyethylene glycol fusion of lymphocytes and myeloma cells. Hybridoma lines were initially screened by ELISA with the immunogen coated onto microtitre plates (MaxiSorp, Nunc, Roskilde, Denmark) at 50 μg/ml.
2.2 Previously described monoclonal antibodies
Previously characterized monoclonal antibodies used to probe glycan arrays. HG, homogalacturonan
Un-esterified/Calcium ion cross-linked HG
Partially methyl-esterified HG
Partially methyl-esterified HG
2.3 Glycan samples used on the array
Samples included on the glycan arrays
Arabinan (sugar beet)
Homogalacturonan (sugar beet)
Pectin (lime) B15
Pectin (lime) B43
Pectin (lime) B71
Pectin (lime) 96
Pectin (lime) F11
Pectin (lime) F19
Pectin (lime) F43
Pectin (lime) F76
Pectin (lime) P16
Pectin (lime) P24
Pectin (lime) P32
Pectin (lime) P41
Pectin (lime) P46
Pectin (lime) P60
Pectin (lime) P76
RGII (A. thaliana)
MHR I (apple)
MHR II (carrot)
MHR III (potato)
MHR HS1 (apple)
MHR HS2 (apple)
AGP (P. patens)
Seed mucilage (A. thaliana)
Gum (locust bean)
Gum arabic (acacia)
Mannan (ivory nut)
CDTA extract (A. thaliana flowers)
CDTA extract (A. thaliana siliques)
CDTA extract (A. thaliana stem top)
CDTA extract (A. thaliana stem middle)
CDTA extract (A. thaliana stem base)
CDTA extract (A. thaliana leaves)
CDTA extract (A. thaliana roots)
NaOH extract (A. thaliana flowers)
NaOH extract (A. thaliana siliques)
NaOH extract (A. thaliana stem top)
NaOH extract (A. thaliana stem middle)
NaOH extract (A. thaliana stem base)
NaOH extract (A. thaliana leaves)
NaOH extract (A. thaliana roots)
2.4 Post-printing modification of glycans
Glycan samples on selected arrays were modified in situ after printing by enzymatic digestion. For the data in Fig. 6 selected arrays were digested with endo-α(1-5)-L-arabinanase or endo-β(1-4-)-galactanase (both from Aspergillus niger, Megazyme (Bray, Ireland) used at 1 U/ml in 200 mM sodium acetate pH 4.0.
2.5 Printing of arrays
Glycans were applied to nitrocellulose membrane (0.45 μm pore size, Schleicher and Schuell, Dassel, Germany) at two concentrations (0.2 and 0.04 mg/ml) and in duplicate such that each sample was represented by four spots. CDTA and NaOH extracted A. thaliana material was printed as extracted and as a five fold dilution, also in duplicate. Printing was performed using a microarray robot (Microgrid II, Genomic Solutions, Ann Arbor, MI, USA) equipped with split pins (MicroSpot 2500, Genomic Solutions). Pins were washed twice in dH2O after deposition of each sample.
2.6 Probing of arrays
Arrays were blocked by incubation for 1 h in PBS (140 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5) containing 5% w/v low fat milk powder (5%MPBS). Arrays were then probed for 2 h with antibodies diluted in 5%MPBS. All antibodies were used as 1/10 dilutions except CCRC-M1 which was used at 1/50, and BS-400-2, BS-400-3 and BS-400-4 which were used at 1/200. After washing with PBS, arrays were incubated for 2 h in either anti-rat or anti-mouse secondary antibody conjugated to alkaline phosphatase (Sigma, Poole, UK) diluted 1/5000 in 5%MPBS. After washing in PBS, arrays were developed using a substrate containing 5-bromo,4-chloro,3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) in BCIP/NBT buffer (100 mM NaCl, 5 mM MgCl2, 100 mM diethanolamine, pH 9.5).
2.7 Scanning and analysis
2.8 Indirect immunofluorescence labeling of plant materials
Resin embedded and fresh plant material was sectioned and labelled with antibodies as described previously [26, 27]. LM14 and LM13 hybridoma supernatants were used as 1/10 dilutions in 5%MPBS. Sections were counterstained for cellulose with Calcofluor white (fluorescent brightener 28, Sigma, Poole, UK) used as a 0.005% aqueous solution.
2.9 Competitive-inhibition ELISA assays
The ability of arabinose, galactose, rhamnose and oligoarabinosides to inhibit the binding of LM14 and LM13 to the immuogen in ELISAs was assessed as described previously .
2.10 Sugar composition analysis of modified pectic hairy regions (MHRs)
Sugar composition was determined using methanolysis as described previously . MHRs were treated with 2 N HCl in dry methanol for 16 h at 80°C, followed by 1 h of 2 M CF3CO2H (TFA) at 121°C. The released sugars were analysed using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) as described previously .
3.1 Production of microarrays of plant cell wall polymers
3.2 Probing of plant cell wall glycan arrays
Arrays were probed with a range of mAbs with previously defined specificities for epitopes occurring on the major classes of cell wall polymers. Details of the mAbs used are provided in Table 1. A total of 23 mAbs were tested and 5 representative examples are shown in Fig. 2a. The TMSVs for the binding of these mAbs to each sample (derived from three independent experiments) are shown as a heatmap in Fig. 2b, where spot signals are correlated to colour intensity. mAb binding profiles were in good agreement with the published results for the antibodies tested. For example, mAb PAM1 has specificity for HG with a low degree of methyl-esterification (DE)  and consistent with this, PAM1 bound to lime pectin samples with DEs of 11–19% (samples E1, A2 and B2). PAM1 did not bind above background to other pectin samples with higher DEs, or to any other cell wall glycans. mAb BS-400-2 is specific for (1→3)-β-glucan  and, of the defined samples, only bound to this polymer (sample A7) on the arrays. (1→3)-β-glucan is known to be extractable from plant cell walls using NaOH and consistent with this, BS-400-2 also bound to weakly to NaOH-solubilized extracts from A. thaliana (samples B8-H8). As expected, the epitopes recognized by some mAbs were detected on multiple samples and this was the case for mAbs LM5 (anti-(1→4)-β-galactan), JIM13 (anti-AGP) and JIM5. However, the binding profiles of these mAbs were also consistent with their known specificities. For example, JIM5 bound with greatest avidity to pectin samples that contained abundant HG domains (such as the lime pectins E1-C3), but not to pectic fragments lacking HG (such as the arabinan or galactan samples A1 and C1).
To generate a new series of cell wall-directed mAbs, rats were immunized with an immunogen consisting of a crude extract of cell wall material solubilized from A. thaliana using CDTA. 13 cell lines were selected by an initial ELISA analysis against the immunogen and these were subsequently probed against the cell wall polymer arrays and four representative examples are shown in Fig. 3. Antibodies that bound with very low avidity, or appeared to have identical binding profiles to previously generated antibodies were not selected for further analysis whilst the data for the remaining seven were subjected to hierarchical cluster analysis.
3.3 Hierarchical clustering of antibody binding profiles
3.4 Detailed characterization of the epitopes recognized by LM14 and LM13
3.5 Immunolocalization of the LM14 and LM13 epitopes
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.
The arrays we constructed were based on the non-covalent attachment of glycans to nitrocellulose. This approach has the advantage that molecules can be immobilized directly without the need to create functional groups, and nitrocellulose has been previously shown to be a effective substrate for the immobilization of diverse glyans [35, 36]. Non-covalent attachment has the potential drawback that variations in the effectiveness of immobilization may result from differences in the structural properties of the arrayed molecules . All of the arrayed samples used in this work were recognized to some extent by at least one of the mAbs used (as shown in Fig. 4), indicating that all were immobilized to some degree. Nevertheless, the spot signals obtained can only provide semi-quantitative information about mAb binding.
Two new probes, LM13 and LM14 were produced against cell wall polymers and both have distinctly different specificities to previously generated antibodies. Cluster analysis, ciELISA data immunoblotting and arabinanase sensitivity suggested that the epitope recognized by LM13 is an arabinan-containing structure that occurs as a side chain on pectic polymers. Pectic side chains typically consist of either galactan, arabinan or type I arabinogalactan chains in which arabinose usually occurs as a terminal sugar . It was of note that LM13 binding to arrays was reduced to some extent by galactanase digestion. However, the galactanase used had very low arabinanase side activity (<0.03 U/ml specific activity with an arabinan substrate compared to 780 U/ml specific activity with a galactan substrate). It is possible therefore that galactanase digestion resulted in the indirect loss of the LM13 epitope by cleavage of galactan to which an arabinan-containing epitope is attached. The fact that LM14 bound to both pectin-derived MHR samples and to AGP-like material on a blot suggests that this mAb binds to an epitope of type II arabinogalactan that may occur on both pectins and AGPs. The novel patterns of recognition on A. thaliana and other plant materials indicates that these are useful new probes for the analysis of cell wall glycans polymers and complex cell wall architectures.
Thanks to Mike Hahn for mAb CCRC-M1 and samples G4 and F4 and to Marie-Christine.Ralet and Jean-François Thibault for samples D1 and F3.
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