John Ugelstad

Swelling of oligomer-polymer particles. New methods of preparation

Abstract The stabilization of polydisperse aqueous emulsions towards degradation by molecular diffusion is discussed. A new method for preparation of stable monomer emulsions by diffusion, based on the physicochemical principles underlying said stabilization, is described. After homogenization of a highly water insoluble compound with water and emulsifier, the dispersed phase is swollen with more than 200 times its own volume of monomer. With stable, finely dispersed monomer emulsions, the monomer droplets may become center for particle initiation even in case one applies a water soluble initiator. The kinetics of the polymerization of such emulsions is discussed in terms of the Smith-Ewart theory, taking into account that the monomer concentration at the reaction center decreases from the start of reaction. Polymer particles consisting of pure polymer are only capable of absorbing 0.5 to 5 times their own volume of low molecular weight compounds. Calculations based upon an extended Flory-Huggins equation predict that if the particles contain a substantial amount of an oligomer which is water insoluble and has a relatively low molecular weight, one may expect an enormous increase in the swelling capacity of such particles for monomer. Methods are described where the polymer particles have been swollen with monomer up to a v/v ratio of > 1000. The principles outlined above have been used to prepare monodisperse particles with very large sizes, including monodisperse, porous particles.

Kinetics and Mechanism of Emulsion Polymerization

Abstract The course of emulsion polymerization may be considered as involving three intervals: Interval I, where particle formation takes place. The end of this interval is not dependent upon the degree of conversion, but on the total amount of polymer formed. With usual recipes, it ends at about 1-5% conversion. Interval II lasts from the end of Interval I until monomer disappears as a separate phase. In this interval, the particle number is usually found to be constant, the particle volume increases proportional to conversion, the monomer concentration in the particles is approximately constant, and therefore the termination is also constant within the particles. Interval III starts when the monomer disappears as a separate phase. The transition from Intervals II to III is determined by the degree of conversion and differs for different monomers. In cases where the solubility of monomer in the water phase is low, the monomer present in the aqueous phase may usually be neglected compared to the monomer p...

Isolation of pure functionally active CD8+ T cells positive selection with monoclonal antibodies directly conjugated to monosized magnetic microspheres

A monoclonal antibody of the IgM isotype, ITI-5C2, which binds with high affinity to CD8 molecules, was directly conjugated to the monosized magnetic microspheres M-450. This permits selective removal of the CD8+ T cell subset (T8) from peripheral blood mononuclear cell suspensions in a rapid one-step procedure. With a low ratio of microspheres to cells (2:1), functionally active T8 cells can be recovered. In vitro experiments involving such positively selected T8 cells or recombinations of isolated T8 and T4 subsets, demonstrate that the presence of M-450 microspheres coated with ITI-5C2 do not interfere with the immunological functions of the positively selected cells. The method has possible application in the isolation of all cell populations where high avidity mAbs of appropriate specificity are available.

Monodisperse magnetic polymer particles: new biochemical and biomedical applications

The method of activated swelling of polymer particles developed by the authors allows the preparation of monodisperse spherical beads of predictable size from 1 to 100 microns in diameter. The polymer particles may be prepared from a number of different monomeric materials and with various morphologies including macroporous structures. The porous beads form the basis for magnetizable monodisperse polymer particles which have magnetic iron oxides distributed as small grains all through the volume of the beads. The magnetic particles are being used extensively for selective cell separation and for immunomagnetic separation within microbiology and molecular biology. A review of recent work within these fields is given. New methods for positive cell separation are announced.

Monosized stationary phases for chromatography

Abstract Polymer particles with a highly monodisperse particle size distribution were produced by a two-step microsuspension method. This process is based on the activation of monosized polymer seed particles by the introduction of a low-molecular-weight material, which leads to a large increase in the monomer swelling capacity of the seed particles. The versatility of the process allows the preparation of polymer monosized compact or macroporous particles of predetermined particle size in the range 1–100 μm and with application of a wide selection of polymeric materials. Underivatized, rigid, porous particles were developed for size-exclusion chromatography in organic solvents. The uniform packing that may be achieved with monosized particles has resulted in chromatographic columns with unusual efficiency and separation capacity. By coating the particle surface with a hydrophilic cross-linked polymer, supports for aqueous phase ion-exchange and size-exclusion chromatography may be produced.

Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T-lymphocyte-specific monoclonal antibodies.

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene micro-spheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43–74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.

Cell labeling and magnetic separation by means of immunoreagents based on polyacrolein microspheres

Polyacrolein (PA) microspheres were synthesized by means of ionizing radiation and shown to contain aldehyde groups which form covalent bounds with amino compounds and proteins. PA microspheres made fluorescent after reaction with fluorescein-labeled antibodies were found to specifically label sensitized sheep red blood cells (SRBC). PA microspheres could also be grafted onto a variety of polymeric spheres of different sizes and composition by ionizing radiation. These hybrid spheres, i.e., preformed polymeric spheres with PA microspheres grafted on their surfaces could bind antibodies which retained specificity of reaction with cell surface receptors. Purification of sensitized SRBC from a mixture containing chicken red blood cells (CRBC) by means of hybrids magnetic spheres in a magnetic field was demonstrated.

Monosized polymer particles in size-exclusion chromatography : I. Toluene as solvent

Abstract Monosized macroporous poly(styrene-divinylbenzene) particles were prepared by a multi-step swelling process. The preparation of particles of different sizes (5, 10 and 20 μm) with similar pore-size distributions allowed a selective evaluation of the effect of particle size on column efficiency. All three particle sizes yielded columns with similar calibration graphs, which was to be expected because of their nearly identical pore-size distributions. The uniform packing which may be achieved with monosized particles resulted in columns with high efficiency and separation capacity. With 5-μm particles more than 50 000 theoretical plates were obtained in a 30-cm column (HETP ≈ 0.006 mm). The resolution, as measured by the parameter Rsp, was good throughout the experiments and especially high for the 5-μm particles. The theoretical prediction that HETP is proportional to the square of the particle diameter was confirmed by the experimental data.

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

Immunoisolation Using Magnetic Solid Supports: Subcellular Fractionation for Cell-Free Functional Studies

Publisher Summary In the endocytic pathway, the different steps of membrane traffic remain poorly understood and controversial. The experimental strategy to study membrane traffic in endocytosis has been to reconstitute a specific step of the pathway in a cell-free system using defined subcellular fractions. This chapter develops immunoisolation that uses the specificity of antibodies, as a subcellular fractionation method. The chapter uses monodisperse magnetic solid supports that efficiently and rapidly isolate the fraction of interest. The chapter describes the methods used in experimental approach. How immunoisolated fractions are used in assays of vesicle fusion are also described in the chapter. Immunoisolated fractions were used to study the in vitro reconstitution of endocytic-vesicle fusion. These fractions correspond to different times of G protein internalization. The G molecules move from the plasma membrane to an “early” tubulovesicular endosome located at the cell periphery and then to “late” acid phosphatase-positive endosomal structures with a complex morphology in the perinuclear region.