Magnetic-polymer particles

• US 4,795,698 A
• Filed: 09/16/1986
• Issued: 01/03/1989
• Est. Priority Date: 10/04/1985
• Status: Expired due to Term

• Current Assignee: Janssen Diagnostics, LLC (Johnson & Johnson)
• Original Assignee: Immunicon Corporation
• Inventors: Silvia, John C., Liberti, Paul A., D'Angelo, Louis, Owen, Charles S.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/906,521
• Time in Patent Office: 840 Days
• Field of Search: 435/4, 435/181, 435/7, 436/526, 436/528, 436/533, 427/2, 428/402, 530/363, 530/402, 530/420, 252/62, 252/56
• US Class Current: 435/4
• CPC Class Codes: A61K 49/1818 particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
• CPC Class Codes: A61K 9/5094 Microcapsules containing magnetic carrier material, e.g. ferrite for drug targeting
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier
• CPC Class Codes: G01N 2333/765 Serum albumin, e.g. HSA
• CPC Class Codes: G01N 2446/20 the magnetic material being present in the particle core
• CPC Class Codes: G01N 2446/30 the magnetic material being dispersed in the polymer composition before their conversion into particulate form
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/5434 using magnetic particle immunoreagent carriers which constitute new materials per se
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

A magnetic-polymer particle, useful in immunoassay techniques and various other biological/medical applications is produced by coprecipitation of transition metals in the presence of a polymer having available coordination sites. These particles are capable of forming stable aqueous suspensions and may be easily resuspended after agglomeration.

Claims

1. A process for the preparation of magnetic-polymer particles comprising the steps of:

(a) combining a first aqueous solution of at least two species of transition metal ions capable of reacting with each other to form a magnetic precipitate and a polymer having available coordination sites in proportions adapted to produce a resuspendable product;

(b) reacting said transition metal ions in the presence of said polymer to form a magnetic precipitate comprising magnetic-polymer particles;

(c) recovering said magnetic-polymer particles.

2. The process of claim 1 further including the step of:

(d) resuspending said magnetic-polymer particles in a second aqueous solution.

3. The process of claim 2 wherein said second aqueous solution is of low ionic strength adapted to allow the formation of a stable suspension.

4. The process of claim 1 further including the step of:

(d) filtering the suspension through a filter having pores of at most 0.44 um diameter.

5. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with a bifunctional compound specific to said polymer.

6. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with a bifunctional compound adapted to form primarily intra-particulate bonds.

7. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with a bifunctional compound adapted to form primarily intra-particulate bonds, said bifunctional compound having terminal groups selected from the group consisting of;

aryl nitrenes, imido-esters, N-hydroxysuccinimide-esters, 2-diazo-3,3,3-trifluoropropionate, maleimides, pyridyl-disulfides, halo-nitrobenzenes, isothiocyanates, halo-sulfonates, active halogens, and active aldehydes.

8. The process of claim 1 further including the steps of:

(d) reacting said magnetic-polymer particles with a bifunctional compound; and

(e) reacting the product particle of step (f) with a biofunctional ligand.

9. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with an activating agent; and

(e) reacting the product particles of step (f) with a biofunctional ligand.

10. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with both a bifunctional compound adapted to form primarily extra-particulate bonds, said bifunctional compound having terminal groups selected from the group consisting of;

aryl nitrenes, imido-esters, N-hydroxysuccinimide-esters, 2-diazo-3,3,3-trifluoropropionate, maleimides, pyridyl-disulfides, halo-nitrobenzenes, isothiocyanates, halo-sulfonates, active halogens, and active aldehydes and a biofunctional ligand selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, staphloccal Protein A (SPA), enzymes, serum proteins, Clq, complement proteins, and rheumatoid factor.

11. The process of claim 1 further including the step of:

(d) reacting said magnetic-polymer particles with both an activating agent selected from the group consisting of water-soluble carbodiimides, glutaraldehyde, cyanogen halides, periodates, and tannic acid and a biofunctional ligand selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, staphloccal Protein A (SPA), enzymes, serum proteins, Clq, complement proteins, and rheumatoid factor.

12. The process of claim 1 wherein the transition metal ions in solution comprise a mixture of Fe(II) and Fe(III).

13. The process of claim 1 wherein the weight ratio of metal to polymer in solution is between 1000:

1 and 1;

10.

14. The process of claim 1 wherein said first aqueous solution is sonicated during said reaction step (b).

15. The process of claim 2 wherein said second aqueous solution is sonicated during said resuspension step (d).

16. The process of claim 3 wherein said second aqueous solution is sonicated during said resuspenstion step (d).

17. The process of claim 16 wherein less than 40 mM of anions is added to the solution during resuspension (step d).

18. The process of claim 1 wherein said polymer is selected from the group of:

synthetic proteins, natural proteins, poly-amino acids, carboxy-poly-alkyls, alkoxy-poly-alkyls, amino-poly-alkyls, hydroxy-poly-alkyls, sulfoxy-poly-alkyls, carboxy-poly-alkylenes, alkoxy-poly-alkylenes, amino-poly-alkylenes, hydroxy-poly-alkylenes, sulfoxy-poly-alkylenes, poly-silanes, poly-phosphines, and copolymers thereof.

19. The process of claim 17 wherein said anions are selected from the group of phosphate, borate, or sulfate.

20. The process of claim 1 wherein said polymer contains oxy-acid functional groups having available coordination sites.

21. The process of claim 1 wherein said polymer is a protein having available coordination sites.

27. The process of claim 3 further including the steps of:

(e) adding a salt to said suspension to increase its ionic strength to a molarity adapted to cause agglomeration and precipitation of said magnetic-polymer particles;

(f) separating said agglomerated precipitated particles;

(g) resuspending said magnetic-polymer particles in an aqueous solution of low ionic strength adapted to allow the formation of a stable suspension.

31. A stable aqueous suspension of magnetic-polymer particles prepared by the process of claim 3.

37. The process of claim 1 wherein said transition metal ions are chosen from the group consisting of those which form coprecipitates having at least one unpaired electron, and having spinel or inverse spinel structure.

38. The process of claim 1 wherein said transition metal ions comprise a pair of ions selected from the group of:

Co(II)+Ga(III)

Ga(III)+Er(III)

Co(II)+Ru(III)

Ga(III)+Ru(III)

Co(II)+Mn(II)

Ga(III)+Mn(II)

Ga(III)+V(III)

Co(II)+V(III)

Ga(III)+Mo(V)

Ga(III)+Fe(III)

V(III)+Fe(III)

Mn(II)+Ru(III)

V(III)+Mn(II)

Co(II)+Mo(V)

Cr(III)+Ga(III)

Cr(III)+Mn(II)

Er(III)+Ru(III)

Er(III)+Co(II)

Mn(II)+Er(III)

Cr(III)+Fe(II).

39. The process of claim 1 wherein said transition metal ions comprise Fe(II) and a transition metal ion having an electromotive potential insufficient to oxidize Fe(II) to Fe(III).

22. A resuspendable magnetic-polymer particle comprising a particulate, magnetic reaction product of a water-soluble form of iron and a water-soluble polymer having available coordination sites.

23. The magnetic-polymer particle of claim 22 wherein the iron component of said particle comprises coprecipitated ferrous and ferric iron.

24. The magnetic-polymer particle of claim 22 wherein said polymer is selected from the group of:

synthetic proteins, natural proteins, poly-amino acids, carboxy-poly-alkyls, alkoxy-poly-alkyls, amino-poly-alkyls, hydroxy-poly-alkyls, sulfoxy-poly-alkyls, carboxy-poly-alkylenes, alkoxy-poly-alkylenes, amino-poly-alkylenes, hydroxy-poly-alkylenes, sulfoxy-poly-alkylenes, poly-silanes, poly-phosphines, and copolymers thereof.

25. The magnetic-polymer particle of claim 22 further including a bifunctional compound bonded to said polymer .

26. The magnetic-polymer particle of claim 22 further including a bifunctional compound bonded to said polymer and a biofunctional ligand bonded to said bifunctional compound.

28. A process for the preparation of ferromagnetic-polymer particles comprising the steps of:

(a) preparing an aqueous solution of iron capable of reacting to form a magnetic precipitate and a polymer having available coordination sites;

(b) adding a stoichiometric excess of a strong base;

(c) agitating said solution;

(d) allowing the magnetic-polymer particles to precipitate;

(e) recovering said magnetic-polymer particles.

29. The process of claim 28 wherein the addition of base (step b) is performed by titrating the solution with base until the solution reaches a slightly basic pH.

30. The process of claim 28 wherein said polymer is a protein, and the pH of said solution is maintained below that required to denature said protein.

32. A process for the assay of a predetermined species comprising the steps of:

(a) preparing an aqueous solution of transition metal ions capable of reacting to form a magnetic precipitate and a polymer having available coordinate sites in proportions adapted to produce resuspendable particles;

(b) reacting said metal ions in the presence of said polymer to form a magnetic precipitate comprising magnetic-polymer particles;

(c) recovering said magnetic-polymer particles;

(d) resuspending said magnetic-polymer particles in an aqueous solution;

(e) reacting said magnetic-polymer particles with both a bifunctional compound adapted to form primarily extra-particulate bonds, and a biofunctional ligand, said biofunctional ligand being specific in its ability to bond with said predetermined species;

(f) exposing a mixture containing an unknown quantity of said predetermined species to a suspension of said magnetic-polymer particles containing said biofunctional ligand, thereby causing said biofunctional ligand to bond to said predetermined species;

(g) passing said mixture through a magnetic filter having a magnetic field, said filter adapted to retain said magnetic-polymer particles;

(h) removing said magnetic field from said filter and eluting said retained magnetic-polymer particles;

(i) analyzing said eluted magnetic-polymer particles to provide desired data relative to said predetermined species.

33. The assay process of claim 32 wherein:

step d includes resuspending said magnetic-polymer particles in an aqueous buffer solution having low ionic strength and filtering said suspension through a filter having pores of at most 0.44 um diameter so as to sterilize said suspension.

34. The assay process of claim 32 wherein:

said bifunctional compound having terminal groups selected from the group consisting of;

aryl nitrenes, imido-esters, N-hydroxysuccinimide-esters, 2-diazo-3,3,3trifluoropropionate, maleimides, pyridyl-disulfides, halo-nitrobenzenes, isothiocyanates, halo-sulfonates, active halogens, and active aldehydes.

35. The assay process of claim 32 wherein:

said biofunctional ligand is selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, staphloccal Protein A (SPA), enzymes, serum proteins, Clq, complement proteins, and rheumatoid factor.

36. The assay process of claim 32 wherein:

said biofunctional ligand is selected to provide half of a binding pair selected from the group of;

antigens and their antibodies, haptenes and their antibodies, hormones and their receptors, vitamins and their receptors, toxins and their receptors, drugs and their receptors, and enzymes and their cofactors.

40. The assay of process of claim 32 wherein said suspension is further treated with an agglomerating agent to promote agglomeration of said magnetic-polymer particles.

41. The assay process of claim 40 wherein said agglomerating agent is a salt of a Group II metal.

42. The assay process of claim 40 wherein said agglomerated particles are filtered by capturing said particles using a hand-held permanent magnet.

43. The assay process of claim 40 wherein the amount of agglomeration caused by said agglomerating agent is sufficient to allow capture of said agglomerated particles by a hand-held permanent magnet in 3 minutes from 1 ml. of suspension, but insufficient to cause said agglomerated particles to settle out of suspension in less than 1 hour.

44. A process for the preparation of magnetic-polymer particles comprising the steps of:

(a) combining a first aqueous solution comprising at least two species of transition metal ions capable of reacting with each other to form a magnetic precipitate, and a polymer having biofunctional activity and having available coordination sites in proportions adapted to produce a resuspendable precipitate product having biofunctional activity;

(b) reacting said transition metal ions in the presence of said polymer to form a magnetic precipitate comprising a complex of said metal and said biofunctional polymer;

(c) recovering said magnetic precipitate from said first solution.