Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1999-09-23
2003-09-23
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S518000, C436S523000, C436S525000, C436S532000, C436S538000, C436S501000, C436S823000, C436S807000, C436S824000, C435S005000, C435S006120, C435S007100, C435S007250, C435S007900, C435S091200, C435S261000, C209S214000, C209S217000, C209S223100, C209S232000, C210S094000, C210S095000, C210S222000, C210S223000, C210S695000, C536S024300
Reexamination Certificate
active
06623983
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to apparatus and methods for the enumeration, examination, and manipulation of magnetically labeled particulate entities, and especially biological particles, such as cells.
BACKGROUND OF THE INVENTION
A magnetic material or magnetic dipole will move in a magnetic field gradient in the direction of increasing magnetic field strength. Magnetic gradients employed in fluid separations are broadly divided into two categories. Internal magnetic gradients are formed by inducing magnetization in a susceptible material placed in the interior of a separation vessel. External gradients are formed by an externally positioned magnetic circuit.
In the case of a simple rectangular bar magnet, for example, field lines which form magnetic circuits conventionally move from North to South and are easily visualized with iron filings. From this familiar experiment in elementary physics it will be recalled that there is greater intensity of field lines nearest the poles. At the poles, the edges formed at the intersections of the sides and faces of the bar will display an even greater density or gradient. Thus, a steel ball placed near a bar magnet is first attracted to the nearest pole and next moves to the region of highest field strength, typically the closest edge. For magnetic circuits, any configuration which promotes increased or decreased density of field lines will generate a gradient. In opposing magnet designs, such as N-S-N-S quadrupole arrangements, opposing North poles or opposing South poles will have field lines such that in the center of such an arrangement there will be zero field. From the circuits that result from a North pole being opposite to each adjacent South pole, such arrangements generate radial magnetic gradients.
Internal high gradient magnetic separators have been employed for nearly 50 years for removing weakly magnetic materials from slurries such as in the kaolin industry, or for removing nanosized magnetic materials from solution. (See Kolm, Scientific American, November 1975). In an internal high gradient magnetic separator, a separation vessel is positioned in a uniform magnetic field. A ferromagnetic structure is positioned within the vessel in order to distort the magnetic field and to generate an “internal” gradient in the field. Typically, magnetic grade stainless steel wool is packed in a column which is then placed in a uniform magnetic field which induces gradients on the steel wool as in U.S. Pat. No. 3,676,337 to Kolm. Gradients as high as 200 kGauss/cm are easily achieved. The magnitude of the field gradient in the vicinity of a wire is inversely related to the wire diameter. The spatial extent of the high gradient region is proportionally related to the diameter of the wire. As will be explained below, collection of magnetic material takes place along the sides of the wire, perpendicular to the applied magnet field lines, but not on the sides tangent to the applied field. In using such a system, material to be separated is passed through the resulting magnetic “filter”. Then, the collected material is washed, and the vessel is moved to a position outside the field where magnetic materials are removed, allowing the collector to be reused.
Various attempts have been made to perform continuous (non-cycle) high gradient magnetic separation. Improvements include flow-through devices with fluctuating fields to separate magnetic material from non-magnetic material, as in U.S. Pat. No. 3,902,994 to Maxwell. Removable screens of ferromagnetic material are also well known in the art as in U.S. Pat. No. 4,209,394 to Kelland. Other flow-through devices are described in U.S. Pat. Nos. 4,261,815 and 4,663,029 to Kelland, U.S. Pat. No. 4,526,681 to Friedlaender, et al., and commonly owned U.S. patent application Ser. Nos. 08/424,271 and 08/482,652.
A method and apparatus for separating cells and other fragile particles is described by Graham, et al in U.S. Pat. No. 4,664,796. The apparatus contains a rectangular chamber within a cylinder. One pair of opposing sides of the chamber are made of non-magnetic material, while the other sides are made of magnetic material. The flow chamber is packed with a magnetically responsive interstitial separation matrix of steel wool. The material to be separated is passed through the chamber, which is positioned in a uniform magnetic field. During separation, the chamber is aligned in the magnetic field such that the magnetic sides of the chamber are parallel to the applied field lines, thus inducing a high gradient about the interstitial matrix in the chamber. When the chamber is in this position, magnetically labeled cells are attracted to the matrix and held thereon, while the non-magnetic components are eluted. The chamber is then rotated, so that the magnetic sides face magnets, which “shunts” or “short-circuits” the magnetic field, reclines the gradients in the flow chamber, and allows the particles of interest to be removed by the shearing force of the fluid flow.
Other internal magnetic separation devices are known. Commonly owned U.S. Pat. No. 5,200,084 discloses the use of thin ferromagnetic wires to collect magnetically labeled cells from solution. U.S. Pat. No. 5,411,863 to Miltenyi discloses the use of coated steel wool, or other magnetically susceptible material to separate cells. U.S. patent application Ser. No. 08/424,271 by Liberti and Wang discloses an internal HGMS device useful for the immobilization, observation, and performance of sequential reactions involving cells.
Turning to the magnetic particles used in such collection devices, over the last twenty years, superparamagnetic materials have become the backbone of magnetic separations technology in a variety of healthcare and bioprocessing applications. Such materials, ranging in size from 25 nm to 100 &mgr;m, are characterized in that they are only magnetic when placed in a magnetic field. Once the field is removed, they cease to be magnetic and can normally easily be dispersed into suspension. The basis for superparamagnetic behavior is that such materials contain magnetic cores smaller than 20-25 nm in diameter, which is estimated to be less than the size of a magnetic domain. A magnetic domain is the smallest volume in which a permanent magnetic dipole exists. Magnetically responsive particles can be formed about one or more such cores. The magnetic material of choice is magnetite, although other transition element oxides and mixtures thereof having appropriate particle size exhibit such superparamagnetic behavior.
Magnetic particles of the type described above have been used for various applications, particularly in health care, e.g. immunoassay, cell separation and molecular biology. Particles ranging from 2 &mgr;m to 5 &mgr;m are commercially available from Dynal. These particles are composed of spherical polymeric materials into which magnetic crystallites have been deposited. These particles because of their magnetite content and size, are readily separated in relatively low external gradients (0.5 to 2 kGauss/cm). Another similar class of materials are particles manufactured by Rhone Poulenc which typically are produced in the 0.75 &mgr;m range. Because of their size, they separate more slowly than the Dynal beads in equivalent gradients. Another class of particulate magnetic material is available from Advanced Magnetics. These particles are basically clusters of magnetite crystals, about 1 &mgr;m in size, which are coated with amino polymer silane to which bioreceptors can be coupled. These highly magnetic materials are easily separated in gradients as low as 0.5 kGauss/cm. Due to their size, both the Advanced Magnetics and Rhone Poulenc materials remain suspended in solution for hours at a time.
There is a class of magnetic particles which has been applied to bioseparations and which have characteristics which place them in a distinct category from those described above. These are nanosized colloids (see U.S. Pat. No. 4,452,773 to Molday; U.S. Pat. No. 4,795,698 to Owen, et al; U.S. Pat. No. 4,965,00
Rao Galla C.
Terstappen Leon W. M. M.
Chin Christopher L.
Dann Dorfman Herrell and Skillman
Do Pensee T.
Immunivest Corporation
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