Chemical apparatus and process disinfecting – deodorizing – preser – Chemical reactor – With means applying electromagnetic wave energy or...
Reexamination Certificate
1999-08-18
2002-03-26
Le, Long V. (Department: 1641)
Chemical apparatus and process disinfecting, deodorizing, preser
Chemical reactor
With means applying electromagnetic wave energy or...
C422S091000, C422S105000, C422S105000, C422S255000, C422S261000, C422S264000, C422S267000, C436S526000, C436S531000, C436S534000, C436S538000, C436S173000, C435S002000, C435S007100, C435S007210, C435S007500, C435S007900, C435S007940, C435S308100, C209S214000, C209S223100, C210S222000, C210S695000
Reexamination Certificate
active
06361749
ABSTRACT:
FIELD OF THE INVENTION
The present inventions relates to the field of bioparticle isolation. More specifically, the invention provides novel magnetic separation devices and methods for isolating magnetically labeled substances of interest from a non-magnetic test medium by means of high gradient magnetic separation (HGMS).
BACKGROUND OF THE INVENTION
Magnetic separators and methods of separation of magnetic particles from non-magnetic media have been described for use in a variety of laboratory and clinical procedures involving biospecific affinity reactions. Such reactions are commonly employed in testing biological samples, such as bodily fluids like blood, bone marrow, leukapheresis products, spinal fluid or urine, for the determination of a wide range of target substances, especially biological entities such as cells, proteins, nucleic acid sequences, and the like.
As used herein, the term “target substance” refers to any member of a specific binding pair, i.e. a pair of substances or a substance and a structure exhibiting a mutual affinity of interaction and includes such things as cells, cell components, biospecific ligands and receptors. “Ligand” is used herein to refer to substances, such as antigens, haptens and various cell-associated structures, having at least one characteristic determinant or epitope, which is capable of being biospecifically recognized by and bound to a receptor. “Receptor” is used herein. to refer to any substance or group of substances having biospecific binding affinity for a given ligand, to the substantial exclusion of other substances. Among the receptors determinable via biospecific affinity reactions are antibodies (both polyclonal and monoclonal) , antibody fragments, enzymes, nucleic acids, C1q, peptides, lectins, protein A/G, single chain antibodies and the like. The determination of any member of a biospecific binding pair is dependent upon its selective interaction with the other member of the pair.
Various methods are available for determining the above-mentioned target substances based upon complex formation between the substance of interest and its specific binding partner. Means are provided in each instance whereby the occurrence or degree of target substance/binding partner complex formation is determinable.
Small magnetic particles have proved to be quite useful in analyses involving biospecific affinity reactions, as they are conveniently coated with biofunctional polymers, e.g., proteins, provide very high surface areas, and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents, having reasonably good suspension characteristics when mildly agitated. Insofar as is known, however, without some degree of agitation, all of the magnetic particles presently in commercial use settle in time and must be resuspended prior to use. This adds another step to any process employing such reagents.
Small magnetic particles, such as those mentioned above, generally fall into two broad categories. The first category, includes particles that are permanently magnetized; and the second comprises particles that become magnetic only when-subjected to a magnetic field. The latter are referred to herein as magnetically responsive particles. Materials displaying strong magnetically responsive behavior are sometimes described as paramagnetic. However, certain ferromagnetic materials, e.g., magnetic iron oxide, may be characterized as magnetically responsive when the crystal size is about 300A or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. See P. Robinson et al.,
Biotech Bioeng
. XV:603-06 (1973).
Magnetically responsive colloidal magnetite is known. See U.S. Pat. No. 4,795,698 to Owen et al., which relates to polymer-coated, sub-micron size magnetite particles that behave as true colloids.
The magnetic separation apparatus/method used for bound-free separations of target substance-bearing magnetic particles from test media will depend on the nature and particle size of the magnetic particle. Micron size ferromagnetic, i.e., permanently magnetized, particles are readily removed from solution by means of commercially available magnetic separation devices. These devices employ a single relatively inexpensive permanent magnet located externally to a container holding the test medium. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass., the DYNAL MPC-1 manufactured by DYNAL,Inc., Great Neck, New York and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass. A specific application of a device of this type in performing magnetic solid-phase. radioimmunoassay is described in L. Hersh et al.,Clinica Chemica Acta, 63: 69-72 (1975). A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass. is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.
Colloidal magnetic materials are not readily separable from solution as such, even with powerful electromagnets but, instead, require high gradient field separation techniques. See, R. R. Oder,
IEEE Trans. Magnetics
, 12: 428-35 (1976); C. Owen and P.Liberti,
Cell Separation: Methods and Selected Applications
, Vol. 5, Pretlow and Pretlow eds., Academic Press, N.Y., (1986); J. T. Kemshead and J. Ugelstad,
Magnetic Molecular and Cellular Biochem
., 67, 11-18 (1985). The gradient fields normally used to filter such materials generate huge magnetic forces. Another useful technique for performing magnetic separations of colloidal magnetic particles from a test medium, by various manipulations of such particles, e.g., addition of agglomerating agents, described in U.S. Pat. No. 5,108,933
High gradient magnetic separation (HGMS) is typically accomplished by using a device having a separation chamber in which a wad of magnetic stainless steel wire is disposed between the poles of a conventional electro- or superconducting magnet and serves to generate large field gradients around the wire which exert a strong attractive force on target substance-bearing magnetic particles. A commercially available high gradient magnetic separator of the type described immediately above is the MACS device made by Miltenyi Biotec GmbM,Gladbach, West Germany, which employs a column filled with a non-rigid steel wool matrix in cooperation with a permanent magnet. In operation, the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic test medium passes through and is removed from the column. Similar magnetic separators employing a steel wool matrix for separating colloidal size magnetic components from a slurry containing the same are also disclosed in U.S. Pat. Nos. 3,567,026, 3,676,337 and 3,902,994. In the last mentioned patent, the separator is provided with a magnetic wool matrix capable of movement into and out of the influence of a magnetic field as a continuously moving element.
It has been found that the steel wool matrix of such prior art HGMS devices often gives rise to non-specific entrapment of biological entities, other than the target substance, which cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance and multiple passages through the device. Moreover, the size of the column in many of the prior art HGMS devices requires substantial quantities of experimental materials, which limits their use in performing various important laboratory-scale separ
Dolan Gerald J.
Doyle Gerald V.
Liberti Paul A.
Terstappen Leon M. M.
Dann Dorfman Herrell and Skillman
Do Pensee T.
Immunivest Corporation
Le Long V.
Skillman Henry H.
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