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- Inventor Info
|Antigen/Gene or Protein Targets||Arabinogalactan-protein|
Plant cell walls are fibre composites that contain some of the most complex glycans known. 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. 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.
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.
Compounding this, 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.
|Immunogen||A. thaliana cell wall polymer emulsified in PBS.|
Isolated from a HTP screen of antibodies generated subsequent to immunization with a pectic fraction. Recognizes arabinogalactan-proteins and will also bind to larch arabinogalactan. It can recognise AGPs in several species.
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.
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 at 50 μg/ml.
|Research Area||Plant Science|
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