title: Isolation of monocytes from human peripheral blood using immuno-affinity expanded-bed adsorption.
abstract: A novel technique for the separation of monocytes from human peripheral blood preparations has been developed. The technique is based on the use of expanded-bed adsorption and a solid perfluorocarbon derivatized with avidin or streptavidin for the indirect positive or negative capture of cells labeled with biotinylated monoclonal antibodies. The perfluorocarbon support was prepared and characterized and the contactor design and operating conditions, that enable cells to be selectively isolated, were investigated. Experiments consisted of applying an immunolabeled pulse of 1 x 10(8) peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, directly onto a refrigerated expanded bed. The major cell types remaining were T-lymphocytes, B-lymphocytes, and monocytes. Monocytes could be positively adsorbed, following labeling with anti-CD14 mAb, with a clearance of up to 89% and a depletion factor of 7.6. They could also be "eluted" using mechanical shear, with a 77% yield of the applied cells at a purity of 90% and >/= 65% viability. Negative isolation of monocytes, following labeling of the other cells present with anti-CD2, CD7, CD16, CD19, and CD56 mAbs, resulted in lymphocyte depletions of up to 81% with a monocyte enrichment factor of 3.8 and purity of 71%. The monocyte viability in the flowthrough was assessed to be > 95%. This combination of expanded-bed adsorption and fluidizable affinity supports shows significant potential for the intensification of cell separations.
authors.affiliation: Cobra BioManufacturing, Stephenson Building, Keele University Science Park, Keele, Staffordshire, ST5 5SP, United Kingdom.
authors.forename: L B
authors.initials: LB
authors.lastname: Ujam
authors.forename: R H
authors.initials: RH
authors.lastname: Clemmitt
authors.forename: S A
authors.initials: SA
authors.lastname: Clarke
authors.forename: R A
authors.initials: RA
authors.lastname: Brooks
authors.forename: N
authors.initials: N
authors.lastname: Rushton
authors.forename: H A
authors.initials: HA
authors.lastname: Chase
mesh_terms: D002469:Cell Separation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D006801:Humans; D007163:Immunosorbent Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology