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Details for Patent: 4,628,037

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Details for Patent: 4,628,037

Title: Binding assays employing magnetic particles
Abstract:A process is provided for the preparation of magnetic particles to which a wide variety of molecules may be coupled. The magnetic particles can be dispersed in aqueous media without rapid settling and conveniently reclaimed from media with a magnetic field. Preferred particles do not become magnetic after application of a magnetic field and can be redispersed and reused. The magnetic particles are useful in biological systems involving separations.
Inventor(s): Chagnon; Mark S. (Lowell, MA), Groman; Ernest V. (Brookline, MA), Josephson; Lee (Arlington, MA), Whitehead; Roy A. (Hingham, MA)
Assignee: Advanced Magnetics, Inc. (Cambridge, MA)
Filing Date:Jun 13, 1985
Application Number:06/744,351
Claims:1. A method for determining the concentration of a ligate in a solution which comprises:

(a) reacting the solution, a known amount of labeled ligate, and magnetically-responsive particles to which a ligand specific for the ligate in solution is covalently coupled, to form ligand/ligate complexes;

(b) magnetically separating the magnetically-responsive particles from the reaction solution;

(c) measuring the amount of label associated with the magnetically-responsive particles or remaining free in solution; and

(d) relating the amount of label measured in step (c) to a standard curve to determine ligate concentration,

wherein the magnetically-responsive particles of step (a) individually comprise a magnetic metal oxide core generally surrounded by a coat of polymeric silane, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninty-five-percent-turbidity-decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

2. A method for determining the concentration of a ligate in a solution which comprises:

(a) reacting the solution, a known amount of labeled ligate, and magnetically-responsive particles to which a ligand specific for the ligate in solution is covalently coupled, to form ligand/ligate complexes;

(b) magnetically separating the magnetically-responsive particles from the reaction solution;

(c) measuring the amount of label associated with the magnetically-responsive particles or remaining free in solution; and

(d) relating the amount of label measured in step (c) to a standard curve to determine ligate concentration,

wherein the magnetically-responsive particles of step (a) individually comprise a superparamagnetic iron oxide core generally surrounded by a coat of polymer silane, the iron oxide core including a group of crystals of iron oxide, an individual particle having a mean diameter as measured by light scattering between about 0.1.mu. and about 1.5.mu. and a surface area as measured by nitrogen gas adsorption of at least about 100 m.sup.2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninety-five-percent-turbidity-decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

3. A method for determining the concentration of a ligate in a solution which comprises:

(a) reacting the solution, a known amount of labeled ligate, and magnetically-responsive particles to which a ligand specific for the ligate in solution is covalently coupled, to form ligand/ligate complexes;

(b) magnetically separating the magnetically-responsive particles from the reaction solution;

(c) measuring the amount of label associated with the magnetically-responsive particles or remaining free in solution; and

(d) relating the amount of label measured in step (c) to a standard curve to determine ligate concentration,

wherein the magnetically-responsive particles of step (a) individually comprise a ferromagnetic metal oxide core generally surrounded by a coat of polymeric silane, the metal oxide core including a group of crystals of metal oxide, an individual particle having a mean diameter as measured by light scattering between about 0.1.mu. and about 1.5.mu. and a surface area as measured by nitrogen gas adsorption of at least about 100 m.sup.2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninety-five-percent-turbidity-decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

4. The method of claim 1, 2 or 3 wherein the ligand is an antibody.

5. The method of claim 1, 2 or 3 wherein the antibody is selected from the group consisting of anti-thyroxine, anti-triiodothyronine, anti-thyroid stimulating hormone, anti-thyroid binding globulin, anti-thyroglobulin, anti-digoxin, anti-cortisol, anti-insulin, anti-theophylline, anti-vitamin B.sub.12, anti-folate, anti-ferritin, anti-human chorionic gonadotropin, anti-follicle stimulating hormone, anti-progesterone, anti-testosterone, anti-estriol, anti-estradiol, anti-prolactin, anti-human placental lactogen, anti-gastrin and anti-human growth hormone antibodies.

6. The method of claim 1, 2 or 3 wherein the ligate is selected from the group consisting of hormones, peptides, pharmacological agents, vitamins, cofactors, hematological substances, virus antigens, nucleic acids and nucleotides.

7. The method of claim 2 or 3 wherein the ligate is thyroxine and the ligand is an anti-thyroxine antibody.

8. The method of claim 2 or 3 wherein the ligate is theophylline and the ligand is an anti-theophylline antibody.

9. The method of claim 2 or 3 wherein the ligate is vitamin B.sub.12 and the ligand is vitamin B.sub.12 binding protein.

10. The method of claim 2 or 3 wherein the ligate is triiodo-thyronine and the ligand is an anti-triiodothyronine antibody.

11. The method of claim 2 or 3 wherein the ligate is a thyroid stimulating hormone and the ligand is an anti-thyroid stimulating hormone antibody.
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