Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2000-11-28
2003-11-18
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
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Reexamination Certificate
active
06649419
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and apparatus for the manipulation of proteins and peptides by reversibly binding proteins and peptides to beads having a coating capable of binding with native proteins and peptides. More particularly, the invention is directed to methods and apparatus for capturing native proteins and peptides on magnetically responsive beads.
BACKGROUND OF THE INVENTION
Magnetically responsive beads or particles of small sizes are well known and have been used for a number of separation and diagnostic purposes. Small magnetic beads have been used to bind to cells and other particles in order to manipulate and separate them. See, for example, U.S. Pat. Nos. 4,230,685, 3,970,518, 5,508,164, 5,567,326 and 4,018,886.
Magnetic beads have been prepared from a variety of polymers with metal particles therein, including those prepared by gelatin, colloidal metal and other agents. An example of this type of bead is disclosed in U.S. Pat. No. 4,582,622. Chemical coupling of proteins to reactive moieties on magnetic beads has been proposed using various linking groups as disclosed in U.S. Pat. No. 4,628,037. While certain bonds may be cleavable, the protein binding is not disclosed as being reversible.
Magnetic beads coated with streptavidin have been used for immobilizing biotinylated substances. Magnetic beads of a polysaccharide (dextran) with a cleavable linker to protein A to immobilize antibodies and bind to a protein have been proposed in U.S. Pat. No. 4,452,773. Protein A coated magnetic beads have also been proposed in U.S. Pat. No. 5,158,871. U.S. Pat. No. 4,297,337 discloses magnetic beads made of porous glass.
U.S. Pat. No. 5,900,481 to Lough et al discloses using coated beads to bind to DNA and thereby manipulate the DNA. Other disclosures of manipulating various molecules bound to beads include Girault et al, Analytical Chemistry 68(13): 2122-6 (1996), and Tang et al, Nucleic Acids Research 23(16): 3126-31 (1995).
A number of magnetic separators for small beads are commercially available, which can readily remove micron size ferromagnetic particles from solution by employing relatively inexpensive permanent magnets. Examples of such magnetic separators include those manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass., the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass., U.S.A., the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y., U.S.A., and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass., U.S.A.
U.S. Pat. No. 5,834,197 to Parton discloses a method of capturing a species from a liquid using coated magnetic beads. The beads have a selective affinity for an antigen. A labeled antibody is added to sandwich the antigen and provide a detectable label bound to the magnetic beads for easy detection and recovery of the antigen.
One method for separation and purification of many different proteins in a sample is by using two-dimensional gel electrophoresis. Electrophoreses separation procedures are routinely applied to complex mixtures of proteins to resolve individual molecular species. Two-dimensional electrophoresis utilizes two orthogonal separations to produce a highly resolved pattern of protein spots, many of which may be effectively homogeneous protein samples. Examples include: O'Farrel, J. Biol. Chem. 250: 4007-4021, (1975), Anderson, et al, Anal. Biochem. 85: 3311-340, (1978), Anderson, et al, Anal. Biochem. 85: 341-354, (1978), Anderson, et al, Anal. Biochem. 93: 312-320, (1979) and Giometti, et, al, Anal. Biochem. 102: 47-58, (1980).
Methods have been described for the recovery of isolated proteins from such spots, generally for the purpose of characterization by microchemical Edman sequencing, amino acid analysis or mass spectrometry, or for preparation of an antigen for the immunization of animals to produce antisera.
It is current practice to identify an isolated protein found in these gels by excising a spot containing the protein of interest. Trypsin is added for cleaving the protein to generate peptides that are then identified by mass spectrometry (MS). One example is disclosed in Rosenfeld et al, Analytical Biochemistry 203:173-179 (1992). From the pattern of peptide molecular weights, or their fragments, one can then deduce the identity of the original protein by comparisons against a suitable sequence database.
One of the difficulties of this practice is that the quantity of these peptides is small and they diffuse freely from the gel into the surrounding liquid and interact with the walls of the container. The peptides in dilute solutions are recovered for use in MS analysis by centrifugal vacuum concentration (Speedvac system) or by lyophilization of a small volume of the surrounding liquid. This recovery step is fraught with difficulties, as the dilute peptides are exposed to plastic and other surfaces upon which they may be captured and thus lost to the investigator.
Likewise, when one wishes to isolate an intact protein from a spot on the gel, a recovery method is used. One method is electroelution, in which the protein is caused to move out of the excised gel spot under the influence of an electric field. In most procedures, the eluted protein is recovered in a small fluid volume by electrophoresing it against a membrane that is impermeable to protein of the expected molecular mass. Devices designed to accomplish this procedure are sold by BioRad, Pharmacia Biotech, Millipore and others. Such techniques suffer from low quantitative recovery probably due to the imperfect recovery of protein off the barrier membrane.
Other published procedures include adsorbing proteins into small C
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reverse phase chromatography columns for the HP G1000A protein sequencer. The Hewlett Packard protein sequencer may use sample cartridges that are small two-part disposable columns with an upper half containing C
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chromatography support, and a lower half containing an ion exchange support. As different aqueous and organic solvents flow through the cartridge, the direction of flow can be arranged so that the applied protein or peptide is always immobilized on the “downstream” support to prevent loss of material. It has been attempted to apply the protein to the C
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half of the column by electroeluting gel spot protein through the column. However, gas bubbles frequently form in the column and block the flow of current. Once successfully immobilized, the protein is then chemically modified and degraded for Edman sequencing directly.
Another technique is electroblotting, in which a gel is sandwiched against a protein-binding membrane, and an electric field is applied using suitable electrodes and buffers to cause the proteins to migrate out of the gel and through the membrane, where they are then immobilized. One, such example using a nitrocellulose blotting sheet is disclosed in Towbin, et al, Proc. Nat. Acad. Sci. USA 1979, 76, 4350-4354. The gel-separated proteins retain their spatial relationships while they move into the membrane, so that the pattern of separated proteins bound to the membrane is generally the same as the pattern of proteins resolved in the gel. When used with proteins this procedure is generally called “Western blotting”. A drawback of the blotting approach is the difficulty of subsequently removing the blotted proteins from the membrane for subsequent manipulation in a different format.
Polyvinylidene difluoride (PVDF) is very hydrophobic and binds proteins well. Polyvinylidene difluoride membranes (Immobilon®, Milipore) have been used for recovery of proteins from two-dimensional gels and for using in situ digestion and transfer of peptides from two-dimensional gels. Kennedy et al, Proc. Natl. Acad. Sci. U.S.A. 85(18):7008-12 (1988) and Kamps et al, Anal. Biochem, 176(1):22-7 (1989). Membranes bearing charged groups have also been used (Millipore cationic membranes). In cases, where the resolved protein is small, or the gel medium has very large pores (as with agarose gels), it is possible to recover some of the
Chin Christopher L.
Do Pensee T.
Large Scale Proteomics Corp.
Robbins John C.
Tarcza John E.
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