A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres

Horton, Jeffrey K.; Evans, O.M.; Swann, Karl; Swinburne, S.

doi:10.1016/0022-1759(89)90357-8

Cited by 19 publications

(6 citation statements)

References 10 publications

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“…Horton et al [8] have described a method for selection and cloning of antibody-secreting hybridomas with antigencoated paramagnetic beads. After fusion and screening, they exposed the hybridomas from the positive colonies to antigencoated paramagnetic beads, thus using magnetic separation prior to subcloning.ln the present investigation we wanted to see if it was possible to use this separation technique7'«.sf after fusion, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…The cells were washed out of the bottles with PBS-G, and then eentrifuged and washed again twice with PBS-G. After the collagenase treatment the cells were incubated under agitation for 48 h at 37°C, to obtain a suspension of cells that expressed antibodies on the cell surface. The selection procedure chosen was a modification of the method of Horton [8]. The cells were washed twice with Hank's BSS (Flow Laboratories) to remove all unbound antibody, and then counted using a Coulter Counter (Coulter Electronics Ltd, Harpenden, Herts, UK).…”

Section: Methodsmentioning

confidence: 99%

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A Rapid Method for Selecting Specific Hybridoma Clones using Paramagnetic Dynabeads®

Bennick¹,

Brosstad²

1993

Scand J Immunol

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This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X-63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti-mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD. Mixing such beads with the fused cells allowed selective harvesting of cells with membrane-expressed anti-DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96-well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40-79% of the tested clones. It is concluded that it is possible to use antigen-coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Rapid Method for Selecting Specific Hybridoma Clones using Paramagnetic Dynabeads®

Bennick¹,

Brosstad²

1993

Scand J Immunol

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“…In contrast to auxotrophic mutants, the production of som atic cell hybrids that express lymphocyte cell surface molecules cannot be readily selected by differential growth. Expression of the appropriate membrane molecule must be positively selected by immunoasorbti on techniques including panning (38,39) and magnetic beads (40) or by cell sorting using flow cytometry. (25) Because the native expression of most functional membrane molecules is typically restricted to leukocytes, the fusion partners are generally T or B lymphocytes.…”

Section: Discussionmentioning

confidence: 99%

Generation of Sheep X (Sheep X Mouse) Heterohybridoma Cell Line Expressing the Beta-1 Integrin Membrane Molecule

He²,

West³

et al. 2000

Hybridoma

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Sheep are an important biological model in such diverse areas as immunology and reproductive biology. The limitation of sheep as an experimental model is the absence of reliable cell lines. To establish cell lines that express functional sheep membrane molecules, we produced a sheep x mouse heterohybridoma by fusion of sheep efferent lymph T cells with the murine myeloma cell line NS1. A cloned heterohybridoma fusion partner was selected by treatment with 8-azaguanine. The resulting cell line HL1/385 was selected for hypoxanthine/aminopterin/thymidine (HAT) sensitivity and growth efficiency. The HL1/385 cell line was used as a back-fusion partner into lectin-stimulated efferent T lymphocytes. The back-fusion approach produced more than 50 heterohybrid cell lines with high growth efficiency. The expression of physiological levels of the sheep beta-1 integrin cell surface molecule on the HT4/6 cell line was stable for months in culture. These results suggest that somatic heterohybrids may provide a reliable source of cell lines for sheep studies in vitro.

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“…The routine use of magnetic particles in research and clinical diagnostics is well established.These particles are often formed from a super-paramagnetic iron core with a polymer coating to which agents can be conjugated via a number of chemical attachment methods [5]. Examples of biological molecules attached to magnetic particles include anti-bodies [6], nucleic acids [7,8], peptides [9], carbohydrates and other polymers [10].…”

Section: Introductionmentioning

confidence: 99%

Magnetizable antibody‐like proteins

Dehal¹,

Livingston²,

Dunn³

et al. 2010

Biotechnology Journal

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International audienceUse of paramagnetic particles to isolate molecules or cells from complex media is well established. Typically, particles are manufactured and coated with a biological molecule which confers specific biorecognition. Incubation of particles with sample and exposure to magnetic fields isolates the species of interest. We designed, produced and assessed magnetized fusion proteins consisting of the antigen binding portion of an antibody (single chain Fv; scFv) fused to the heavy chain of the iron binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. The fusion proteins were chemically magnetized by introducing a paramagnetic iron core. The resultant fusion protein was shown to be magnetizable as well as capable of binding target antigens. These “organic” magnetizable particles possess a number of theoretical advantages over traditional inorganic particles

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A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres

Cited by 19 publications

References 10 publications

A Rapid Method for Selecting Specific Hybridoma Clones using Paramagnetic Dynabeads®

A Rapid Method for Selecting Specific Hybridoma Clones using Paramagnetic Dynabeads®

Generation of Sheep X (Sheep X Mouse) Heterohybridoma Cell Line Expressing the Beta-1 Integrin Membrane Molecule

Magnetizable antibody‐like proteins

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