Monosized magnetic particles and their use in selective cell separation

Ugelstad, J.; Berge, Arvid; Ellingsen, T.; Aune, Oddvar; Kilaas, Lars; Nilsen, Tom-Nils; Schmid, Ruth; Stenstad, Per; Funderud, Steinar; Kvalheim, Gunnar; Nustad, Kjell; Lea, T.; Vartdal, F.; Danielsen, Håvard E.

doi:10.1002/masy.19880170113

Cited by 53 publications

(20 citation statements)

References 21 publications

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“…The length of incubation of IH with UB25 did not affect the enrichment. Direct coupling of IH to UB25-coated Dynabeads gave a lower yield and purit>' (Pain, 1994), possibly due to poor accessibility of the IgG binding sites to their target epitopes (Ugelstad et at., 1988). Attempts were made to deplete tbe preparation of contaminating eel!…”

Section: Enrichment Of Intracellular Hyphae By Immunomagnetic Separationmentioning

confidence: 99%

“…In this way, up to 6 X 10'' IH of C. lindemuthianum could be obtained from 80 g of leaf tissue, which would be sufficient for the immunization of mice and screening of MAbs. IMS has been wideiy used in animal cell biology and microbiology for the purification of cells, bacteria and viruses from mixed cell populations (Sharpe, 1988;Ugelstad et al, 1988), but this is the first report of its use to purify fungal cells.…”

Section: Ulsct Ssion Purification Of Intracellular Hyphaementioning

confidence: 99%

“…One of tbese MAbs, UB25, recognized a set of ,V-linked glycoproteins present only on the surface ot IH. Here, we bave used this MAb for the selective enrithnnent of IH from the IPC fraction using magnetizable polystyrene beads (Ugelstad et al, 1988). We show that, using this method, IH can be recovered with high viability and purity.…”

Section: Introduction Plasma Membrane As With the Haustoria Of Obligatementioning

confidence: 99%

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Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody

et al. 1994

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SUMMARY A method is described for isolating intracellular hyphae (IH, i.e. infection vesicles and primary hyphae) of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected leaves‐of bean (Phaseolus vulgaris L.). 1H were recovered from homogenates of infected leaves after filtration through a 45 μm nylon mesh and isopyenic centrifugation on Percoll. 1H were then affinity‐purified by immunomagnetic separation using Dynabeads coated with monoclonal antibody UB25, specific for IH surface glycoproteins. The method yielded 7 × 104 IH g‐1 f. wt leaf tissue, with 27% purity and 62% viability, as judged by staining with fluorescein diacetate. The Viability of isolated IH was confirmed by their ability to grow in nutrient medium and by the normal ultrastructure of their cytoplasm. The host plasma membrane and matrix layer which surround IH in planta were absent from isolated IH. Staining with lectins. Calcofluor and aniline blue showed that the walls of IH contain N‐acetylgalactosamine. α‐linked mannose residues and β‐linked polysaccharides, including‐chitin and β‐1,3‐glucans. Potential uses of the isolated IH are discussed.

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Section: Enrichment Of Intracellular Hyphae By Immunomagnetic Separationmentioning

confidence: 99%

Section: Ulsct Ssion Purification Of Intracellular Hyphaementioning

confidence: 99%

Section: Introduction Plasma Membrane As With the Haustoria Of Obligatementioning

confidence: 99%

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Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody

et al. 1994

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“…Selective approaches to cell separation often involve the recognition of cell-surface antigens by ligands immobilized to some form of support matrix. Matrices that have been used include magnetic polystyrene particles (Ellingsen et al, 1993;Ugelstad et al, 1985Ugelstad et al, , 1988, magnetic colloids (Hancock and Kemshead, 1993), derivatized glass surfaces (Norden et al, 1994), and silica microparticles (Seeger et al, 1993). Affinity liquid perfluorocarbon emulsions have also been developed for the rapid depletion of certain cell types in batch adsorption (McCreath and Chase, 1996a;1996b) and in this study we derivatize a fluidizable solid perfluorocarbon particle for use in expanded beds.…”

Section: Introductionmentioning

confidence: 98%

Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon

Clemmitt

Chase

2003

Biotech & Bioengineering

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The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated. Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids. The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1). Adsorption kinetics were rapid with a time constant of

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“…An indirect method of positive selection was used for the IMS, in which the appressoria were first coated with primary antibody, MAb UB31, before being incubated with Dynabeads coated with a secondary antibody specific for the primary antibody isotype. We used this approach because antibodies in solution find their target epitopes more readily than if the antibody is bound directly to the surface of a bead (21). IMS with MAb UB31 enriched appressoria from the original IPC preparation, and final yields were 3 ϫ 10 5 appressoria g of leaf tissue Ϫ1 , with a purity of 91% Ϯ 1% (mean of four replicate experiments).…”

Section: Resultsmentioning

confidence: 99%

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

Hutchison

Perfect

O’Connell

et al. 2000

Appl Environ Microbiol

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We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.For many fungal plant pathogens, the appressorium is developmentally the first and most important infection structure formed in preparation for invasion of the host. Appressoria increase the area of contact and attachment between the fungus and host surface, provide the mechanical force and enzymes required for penetration, and can promote survival in adverse conditions (10). Species of Colletotrichum, the anthracnose fungi, produce melanized appressoria, and several mRNAs and proteins specific to these structures have been identified. For example, the cap20 gene in Colletotrichum gloeosporioides is induced by the surface wax of its host, avocado, and is expressed during appressorium formation (8). In Colletotrichum lindemuthianum, have been analyzed the cell surface of appressoria and other infection structures using lectins and monoclonal antibodies (MAbs) (12, 13). A plasma membrane-associated glycoprotein specific to appressoria has also been identified using an MAb (16).Our objective in this study was to purify C. lindemuthianum appressoria for further biochemical analysis of the cell surface and to compare appressoria with other fungal structures. To date, there have been no reports of the enrichment of appressoria from infected plant tissue, although appressoria produced on artificial substrata by species of Colletotrichum and Uromyces have been isolated by mechanical scraping (8,9,19). However, appressoria harvested in this way are contaminated with other fungal cell types, e.g., conidia and germ tubes, and there is evidence that appressoria formed in vitro do not have the same composition as appressoria formed on host surfaces (7).A variety of methods have been used to isolate the inter-and intracellular infection structures formed by fungal pathogens inside infected plant tissues. These include enzymatic maceration (2) and mechanical disruption followed by either density gradient centrifugation (1,3,20) or lectin affinity chromatography (4). Immunomagnetic separation (IMS) has been widely used in animal cell biology and microbiology for the purification of cells, bacteria, and viruses from mixed cell populations (18). Magnetic Dynabeads (Dynal) coated ...

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Monosized magnetic particles and their use in selective cell separation

Cited by 53 publications

References 21 publications

Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody

Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody

Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

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