Magnetic-polymer particles.

The invention relates to particles containing certain polymers in combination with magnetic metals, to compositions including such particles and to processes for producing and using such particles and compositions. In particular, the invention relates to particles in which the polymer has a biochemical function.

Biologically active magnetic particles can be used in a variety of preparative and diagnostic techniques. These include the magnetic steep gradient separation (EGMS), which uses the magnetic field to separate magnetic particles from a suspension. In cases where those particles are attached to interesting biological materials (e.g. Cells, drugs) are bound, that material of interest can be separated in this way from other materials not bound to magnetic particles.

Various properties are important if a magnetic particle is to be useful in biological, therapeutic and diagnostic systems. First, the particle must have the biological activity, affinity, or reactive character that it needs to perform its function. Secondly, for release reasons, the particles must be suspendable in an aqueous medium in a biological or reactive system. It may also be desirable that the suspension of the particles is stable (ie not settled or agglomerated). Finally, a small particle size may be desirable in order to be able to sterilize the suspension of the magnetic particles by means of conventional methods using filters.

Many techniques for making magnetic particles or organomagnetic materials have been proposed in the prior art. US-A-4 001 288 (Gable et al.) Discloses the preparation of organo-iron magnetic compounds which are both water-soluble and highly magnetic. In the preparation according to that of Gable et al. The teaching taught is treated with divalent iron in solution to precipitate an iron oxide compound with hydrogen peroxide and then with ammonium hydroxide. This compound is then oxidized with peroxide, treated with a hydroxycarboxylic acid and reacted with an alkali substance to form a soluble product.

Gable et al. also discloses the introduction of proteins or "protein degradation products" into the divalent iron solution, which is then reacted to precipitate the iron oxides. The only material exemplified is the hydrolysis product of gelatin treated with hydrogen peroxide.

US - A - 4 452 773 (Molday) discloses "colloidal size range" iron oxide particles coated with a polysaccharide (exemplified in that patent using dextran and dextran derivatives). Although the Molday particles are made in a manner somewhat similar to the present invention, it is believed that they are substantially different from the present inventive particles.

US Pat. No. 4,454,234 (Czerlinski) teaches the production of coated magnetic particles (generally by suspension polymerization on a magnetic particle substrate) which are reversibly suspendable in solution. The suspension parameters mediated by Czerlinski cause the Curie temperature effect of its magnetic particles, which alternately makes the particles magnetic or non-magnetic. Once the particles are magnetic, they tend to agglomerate, but can be resuspended after heating the particles to a temperature above the Curie temperature of the magnetite contained therein.

US - A - 4 230 685 (Senyei et al.) Describes the production of microspheres containing magnetite, albumin and protein A. The teaching taught by Senyei does not include the precipitation of magnetite in the presence of those other components, but it does rather towards coating prefabricated magnetic particles.

Other patents of interest that may be considered include US-A-4 152 210 (Robinson et al.), US-A-4 335 094 (Mosbach), 4 018 886 (Giaever) and 4 070 246 (Kennedy et al. ). While all of these patents per se disclose the manufacture or use of magnetic-biological particles, it can be considered that none of these particles is similar to the particles of the present invention.

The terms used in this document are explained below.

Active halogen: A halogen atom linked to a carbon atom which is either attached to or next to a carbon linked to an electron withdrawing group (for example: iodoacetamide, iodoacetate).

Available coordination site: A coordination site that is not sterically hindered (freely accessible) and designed to coordinate a metal atom in metal compounds (e.g. Fe 3 O 4).

Biofunctional ligand: A molecule with a biological effect or with a special affinity in a biological system which can be linked to the ferromagnetic polymer particles according to the invention, so that special biological properties are imparted to those particles.

Coordination sites: An atom in a molecular structure that has a "free" electron pair capable of forming a coordination bond with a transition metal atom.

Denatured proteins: Proteins that have lost a specific biological activity (e.g. enzyme, antigen or antibody activity etc.) due to a chemical or structural change. In the sense of use according to Gable et al .: Following the cleavage of some amide bonds with a molecular weight of <10,000 remaining protein residues.

Extra-particulate bonds: Linking a functional group of a bifunctional compound to a site on a ferromagnetic polymer particle, while the other functional group is linked to a site on another molecule or (typically a ligand).

Ferromagnetic: Permanent magnetic iron compositions (with a pure magnetic moment).

Intra-particulate bonds: Linking both functional groups of a bifunctional compound with different sites on the same magnetic polymer particle.

Low ionic strength: Aqueous solution with almost neutral pH and a total cation concentration of <40mM (usually seen from the buffer).

Protein: Amide bonds linked amino acid polymers (peptide bonds) with a molecular weight of> 10,000.

Resuspendable: Material that is resuspendable after agglomeration (e.g. by centrifugation, EGMS or flocculation) to achieve a stable suspension.

Sound treatment: exposure to high intensity ultrasound.

Stable suspension: A non-settling or otherwise agglomerating suspension, provided that it is left to stand for 2 days at standard temperature and pressure.

The present invention relates to a process for producing magnetic polymer particles, in which:

(a) an aqueous solution of transition metal ions capable of reacting to form a magnetic precipitate and producing a polymer having accessible coordination sites, in proportions adapted to the manufacture of a resuspendable product, with the proviso that the polymer is not a polysaccharide or derivative thereof;

(b) reacting the metal ions in the presence of the polymer to form a magnetic precipitate containing magnetic polymer particles; and

(c) recovering the magnetic polymer particles.

The invention also relates to resuspendable magnetic polymer particles containing the reaction product of a water-soluble form of iron and a water-soluble polymer which has accessible coordination sites, with the proviso that the water-soluble polymer is not a polysaccharide or derivative thereof.

The invention further relates to a method for producing ferromagnetic polymer particles, in which:

(a) an aqueous solution of iron capable of reacting to form a magnetic precipitate and producing a polymer having accessible coordination sites, provided that the polymer is not a polysaccharide or derivative thereof;

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

(c) stirring the solution;

(d) precipitates the magnetic particles; and

(e) recovering the magnetic polymer particles.

The invention also extends to a method for determining a given species, in which:

(a) an aqueous solution of transition metal ions capable of reacting to form a magnetic precipitate and producing a polymer having accessible coordination sites, in proportions adapted to the manufacture of a resuspendable product, with the proviso that the polymer is not a polysaccharide or derivative thereof;

(b) containing the metal ions in the presence of the polymer to form a magnetic precipitate that reacts magnetic polymer particles; and

(c) recovering the magnetic polymer particles;

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

(e) reacting the magnetic polymer particles both with a bifunctional compound which is suitable for binding predominantly extraparticulate bonds and with a biofunctional ligand, the binding property of the biofunctional ligand being specific to the predetermined species;

(f) contacting a mixture containing an unknown amount of the predetermined species with a suspension of the magnetic polymer particles containing the biofunctional ligand, thereby causing the biofunctional ligand to bind to the predetermined species;

(g) passing the mixture through a magnetic filter with a magnetic field, the filter being designed to retain the magnetic polymer particles;

(h) removes the magnetic field from the filter and elutes the retained magnetic polymer particles; and

(i) Analyze the eluted magnetic polymer particles using a preselected analytical method designed to provide the desired data relative to the predetermined species.

The invention also relates to a method for producing magnetic polymer particles, in which:

(a) a first aqueous solution containing at least two species of transition metal ions capable of reacting with each other to form a magnetic precipitate and combining a polymer having biofunctional activity and accessible coordination sites in proportions that are based on the formation of a resuspendable precipitate product with biofunctional activity, with the proviso that that the polymer is not a polysaccharide or a derivative thereof;

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

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

The invention also relates to resuspendable magnetic polymer particles for use in the fields of analysis, biology, medical diagnosis and their use as contrast agents in NMR imaging.

In particular, the present invention encompasses a process for the production of suspendable and resuspendable magnetic polymer particles and of particles produced in this way. Such particles have useful properties, in particular in immunological test methods in which the particles are produced with a special biofunctional ligand and then deposited using magnetic steep gradient methods.

The process according to the invention includes both the coprecipitation of metal ions (eg FE (II) + Fe (III) or [Fe (II) + Cr (III)] in their function as magnetic compounds in the presence of a polymer with accessible coordination sites, as also the reaction of the polymer with the metal to form a precipitate and the recovery of the magnetic polymer particles. In addition, various types of biofunctional groups can be incorporated into the particles in order to obtain an effective biofunctional reagent which can be used in immunological test methods, in cell recognition and in reactive agents for enzyme immobilization, in NMR imaging and in other diagnostic, analytical and therapeutic techniques.

According to one aspect of the invention, a solution containing Fe (II) and Fe (III) (typically FeCl₂ and FeCl₃) and a polymer excluding a polysaccharide or a derivative thereof (e.g. a protein) with accessible coordination sites (by titration or otherwise) treated with a strong base such as ammonium hydroxide (NH 4 OH) to precipitate magnetic iron oxides such as magnetite (Fe 3 O 4) in a form that is intimately associated with that Polymer is combined. The precipitation is carried out in a characteristic manner to obtain resuspendable magnetic polymer particles with rapid stirring and possible shaking by means of ultrasound treatment.

The polymer is preferably selected from the group consisting of synthetic or natural proteins, polyamino acids, carboxypolyalkyl, alkoxypolyalkyl, aminopolyalkyl, hydroxypolyalkyl, sulfoxypolyalkyl compounds, carboxypolyalkylenes, alkoxypolyalkylenes, aminopolyalkylenes, hydroxypolyalkylenes, sulfoxypolyalkylenes, polyolsilanes, polyolsilanes, polyolsilanes, polyolsilanes, polyolsines thereof, polysilanes, polysilanes, polysilanes, polyolsines thereof, polysilanes, polysilanes, polysilanes, and polysilanes, polyolsines thereof, polysilanes, polysilanes, polysilanes, and polysilanes thereof, polysilanes, polysilanes, polysilanes, and polysilanes, their polyols, and polysilanes, More preferably, the polymer contains oxyacid functional groups with accessible coordination sites, most preferably this polymer is a protein.

After the precipitation, the particles are washed and then resuspended in a buffer solution at approximately neutral pH. The suspension can optionally be filtered through filters with a pore size of at most 0.44 µm in diameter.

Further embodiments of the present invention include the use of metals other than iron in the coprecipitation reaction. In particular, the Fe (III) can be replaced by any of the wide range of transition metal ions.

Further transition metal ions are preferably selected from a group consisting of those metal ions which form co-precipitates with at least one unpaired electron and a spinel or inverse spinel structure. In individual cases, the iron can be completely displaced by suitably selected transition metal ions. In many cases, the use of non-ferrous metals causes colored particles whose color ranges from white to dark brown.

The magnetic polymer particles made according to the invention show many useful properties. These particles are due to the inclusion of a magnetic metal compound (e.g. iron in a similar form to magnetite or a comparable compound). The particles can be formulated in such a way that they are resuspendable after aggregation and thus form relatively stable suspensions which do not settle even after several days of still standing. Furthermore, the particles made according to the invention can be very small (about 0.01-0.2 µm), which is why they can be filter sterilized. Finally, the particles made according to the invention can be tailored to include specific biofunctional ligands useful for a variety of analytical, diagnostic, and other bio-medical applications.

Following the precipitation and resuspension of the magnetic polymer particles, these can be treated with a bifunctional reagent for crosslinking reactive sites on the polymer. Preferably the bifunctional compound is specific to the polymer. This crosslinking can either be efficient with regard to intra-particulate crosslinking, in which the reactive sites are bound to the same particle, or it takes place as a reaction of an extra-particulate ligand, which is then crosslinked with the polymer on a given particle.

In the former case, the bifunctional compound has terminal groups, which consist of the group consisting of aryl nitrenes, imido esters, N-hydroxysuccinimide esters, 2-diazo-3,3,3-trifluoropropionate, maleimides, pyridyl disulfides, halogenated nitrobenzenes, isothiocyanates, halogen sulfonates, active halogens and active halogens Aldehydes are selected.

In the second case, a bifunctional reagent is desired which has a relatively short distance between its two functional groups in order to promote the link between the particle polymer and the extra-particulate species. In this case, the bifunctional compound has terminal groups, which consist of the group consisting of aryl nitrenes, imido esters, N-hydroxysuccinimide esters, 2-diazo-3,3,3-trifluoropropionate, maleimides, pyridyl disulfides, halogenated nitrobenzenes, isothiocyanates, halogen sulfonates, active halogens and active halogens Aldehydes are selected and a biofunctional ligand which is selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, Staphylococcus Protein A (SPA), enzymes, serum proteins, C1q, complement proteins and rheumatoid factor.

Conversely, the intra-particulate crosslinking is promoted by the use of a bifunctional, elongated reagent that is not sterically hindered in its binding, so that two reactive sites on a particle can be linked by a single bifunctional particle.

According to a further preferred embodiment according to the invention, the method described above includes the following as a further step:

that the magnetic polymer particles are reacted both with a bifunctional compound which is adapted to the formation of predominantly extra-particulate bonds, the bifunctional compound having terminal groups consisting of the group consisting of arylnitrenes, imido esters, N-hydroxysuccinimide esters, 2- Diazo-3,3,3-trifluoropropionate, maleimides, pyridyl disulfides, haiogeno nitrobenzenes, isothiocyanates, halogen sulfonates, active halogens and active aldehydes are selected as well as reacted with a biofunctional ligand which is selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, staphylococcus protein A (SPA), enzymes, serum proteins, C1q, complement proteins and rheumatoid factor ,

According to another preferred embodiment of the invention, the method described above includes the following as a further step:

that the magnetic polymer particles both with an activating agent selected from the group consisting of water-soluble carbodiimides, glutaraldehyde, cyanogen halides, periodates and tannic acid and with a biofunctional ligand selected from the group consisting of antigens, antibodies, lectins, avidin, Biotin, Staphylococcus Protein A (SPA), enzymes, serum proteins, C1q, complement proteins and rheumatoid factor react.

As an alternative to ultrasound treatment during the precipitation or resuspension steps set out above, another type of shaking (such as mechanical stirring) can also be used.

The resuspending of the magnetic polymer particles according to the invention is carried out in a characteristic manner in a buffer system with low ionic strength (eg 40 mM phosphate). The buffer system enables the resuspension of the particles, which are not resuspendable in nonionic solutions. In addition to the phosphate buffers, borate and sulfate systems can also be used.

According to a further preferred embodiment of the invention, the method described above includes the following as a further step:

that the magnetic polymer particles are selected both with an activating agent from the group consisting of water-soluble carbodiimides, glutaraldehyde, cyanohalides, periodates and tannic acid and with a biofunctional ligand which is selected from the group consisting of antigens, antibodies, lectins, avidin, biotin , Staphylococcus Protein A (SPA), enzymes, serum proteins, C1q, complement proteins and rheumatoid factor.

It can be assumed that the intimate union of polymer and metal according to the invention results from the coordination of the metal during coprecipitation as a result of the coordination sites on the polymer. The thesis is also argued that certain coordination points are "more accessible" than others due to the fact that both the strength of the coordination bond, which can be produced by the special atom, and the spatial disabilities caused by the atoms in the vicinity play a role play. For example, it is known that oxygen atoms that have a "free" iron complex electron pair have a stronger effect than nitrogen atoms in the amine and even more so than a hydroxyl oxygen atom. Thus, a polymer with oxyacid functional groups should give better product particles than an amine substituted polymer. Similarly, coordination centers that can be freely accessible at short intervals should produce better results than those that are disabled either in terms of the access route or in terms of distance from access.

The trends described above can be observed qualitatively in various experiments carried out by the inventors. The presence of "available coordination sites" appears necessary with regard to the production of resuspendable magnetic polymer particles according to the invention. For example, it could be shown that the present particles can be produced with various polymers such as natural or synthetic proteins, polyamino acids, carboxypolyalkyl, alkoxypolyalkyl, aminopolyalkyl, hydroxypolyalkyl compounds and various copolymers thereof. In addition, the particles according to the invention can also be produced with other polymers such as, for example, sulfoxypolyalkyl compounds, polyacrylamines, polyacrylic acid and substituted polyalkene compounds.

Various criteria appear to be important in the selection of the transition metals to be used in the coprecipitation reaction. First, the end product must have one or more unpaired electrons in its structure. Second, one of these metals must have a site available for polymer binding. Third, one of the metals must be able to have a cubic or hexagonally tightly packed crystalline structure (e.g. in terms of cubic spinel or inverse spinel structure). It is believed that the latter requirement stems from the need for an extremely tight packing density for a connection to be magnetic.

Finally, polymers useful for making the particles of the present invention can be "> must-tailor" in such a way that they include monomers which show a special biofunctional activity. The use of such a polymer permits the immediate precipitation of a biofunctional magnetic polymer particle, which either requires little post-treatment for the sensible use in determination methods which are based on the special biofunctional activity of the polymer.

Larger, less stable particles are useful in certain applications. The particles according to the invention can be produced in such a way that they agglomerate and at the same time retain both their biofunctional and their magnetic characteristics. Particle agglomeration can be accompanied by treating the suspension with a predetermined amount of, for example, a barium chloride solution. This treatment may be designed to cause the particles to separate from the suspension for a predetermined period of time to allow further procedural measures to be carried out, or to enable the larger particles to be easily attracted to relatively weak magnets.

The following examples serve to explain various parameters in the production and use of the magnetic polymer particles:

example 1

General preparative procedure:

The aqueous starting solutions for a typical (micro) preparation are as follows:

1 mg / ml solution of a given protein

500 mg / ml FeCl & sub3;

200 mg / ml FeCl & sub2;

20 mM phosphate buffer (approximately pH 7)

7.5% or 15% ammonium hydroxide solution (NH 4 OH)

Preparation begins by diluting 2.5 ml of protein solution with distilled water to a volume of 20 ml. 175 µl of FeCl 3 and 140 µl of FeCl 3 are added. Approx. 200 ul ammonium hydroxide solution are added to the slip.

It should not be neglected to point out that the amount of base to be added is usually calculated in advance in order to take into account the total amount of protein or polymer in the reaction mixture and the buffering capacity of that particular protein or polymer. The intent is to raise the pH sufficiently to enable precipitation of the iron oxides on the one hand and to maintain the crucial activity of the protein or polymer, on the other hand, provided that the pH at which this material denatures is not exceeded ,

All of these substances are added to a reaction vessel with constant stirring, so as to produce a homogeneous reaction mixture. Immediately after adding the base (NH 4 OH), black particles are precipitated. The reaction mixture is filtered off, specifically at 3000 rpm for 15 minutes, and then the supernatant is separated off. The aggregated residue is broken up and the material is washed in 20 ml of phosphate buffer, typically in 3 washing cycles with centrifugation. After the last washing, the aggregated residue is broken up again and the material is resuspended in the buffer under ultrasound treatment. The final solution is amber or brown and is in the form of a stable suspension of ferromagnetic polymer particles. No iron aggregates were found during the entire procedure.

The subsequent treatment of the particles can be carried out using process measures which are explained in detail below, including intra-particulate or extra-particulate crosslinking with biofunctional reaction partners.

Example 1A

Use of non-ferrous metals:

As discussed above, non-ferrous metals can also be incorporated into the magnetic polymer particles of the present invention. Below is a (non-exhaustive) table of those ions that can be used in the manufacture of magnetic polymer particles:

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)

He (III) + Ru (III)

He (III) + Co (II)

Mn (II) + Er (III)

Cr (III) + Fe (II)

In addition to the list above, Fe (II) can also be used in combination with a selected transition metal ion, the electromotive potential of which is insufficient for the oxidation of Fe (II) to Fe (III). Among the metals listed above, only V (III) is capable of oxidizing Fe (II) and is unsuitable for this.

Example 2

Particles coupled to antibodies:

Two solutions (40 ml each) are quickly combined in an ultrasonic bath for accelerated mixing. Both contained 1.5 mg / ml bovine serum albumin (BSA). One of them contained ammonium hydroxide (8 ml 30%, final concentration = 74 mM). The other contained 140 mg of Fe (II) chloride and 280 mg of Fe (III) chloride (total iron concentration 1 mg / ml after mixing). A black precipitate formed immediately afterwards. The mixture was neutralized by adding 6 ml of glacial acetic acid while stirring. The sample was divided into 4 tubes and the precipitate was washed by centrifugation (3000 rpm, 15 minutes); the small amount of iron remaining in the gold-colored and slightly cloudy supernatant was separated. The aggregated residue was resuspended in 20 ml of 20 mM phosphate buffer at neutral pH. All 4 tubes with their content of 5 ml particles were sonicated for 5 minutes in a Branson ultrasound device with a bowl horn connection.

The accessible amino groups on the particles were reacted with succinidylpropionodithiopyridine (SPDP) in order to prepare some of them for later coupling with the antibodies. This reaction (20 ml particles + 5 mg SPDP) was carried out for 1 hour in the cold and with stirring. 25 mg of the amino-reactive cross-linking reagent ethylene glycol disuccinimide ester (EGS) were then added to further stabilize the particles. The mixture was allowed to react with stirring for an additional hour. The EGS caused the crosslinking of any remaining amino residues that were present in sufficiently close proximity for the coupling. Otherwise, remaining amino groups would have been bound by one end of the EGS molecule while the others might have been hydrolyzed to a carboxyl group. This measure amounts to a smooth reversal from positively charged to negatively charged groups on the particle surface, a process that is believed to promote the stability of the colloidal suspension.

Towards the end of the reaction period, the preparation was placed in a 50 ml tube and then 10 ml of 3 molar NaCl solution in water was added to "salt out" the particles. After allowing 10 minutes to aggregate at room temperature, they were centrifuged at 1500 rpm for 10 minutes. The clear colorless supernatant was separated. The agglomerated residue was resuspended and centrifuged twice more before the final suspension was obtained in 20 ml phosphate buffer at a concentration of 20 mM (at this stage no ultrasound treatment was usually necessary).

The particles were coupled with horseradish-specific goat antiserum in the form of a conventional antigen for test procedures. Then the antiserum was activated by reaction with the SPDP. 0.128 ml of antiserum with a total content of 1.28 mg of protein was reacted with 12.8 micrograms of SPDP diluted in 0.512 ml of phosphate buffer for 30 minutes at room temperature. After 30 minutes, 3.1 mg of dithiothreitol (DTT)) was added to convert the SPDP to its free sulfhydryl form. The converted antibodies were separated (desalted) on a small gel filtration column.

Thiolated antiserum and SPDP-activated particles were reacted with each other by adding an amount of 6.4 ml containing the antibodies, which (0.64 mg antiserum protein with 100% recovery) to particles containing 14 mg iron. The concentration of antiserum protein and particulate iron in the reaction mixture was 1.0 mg / ml and 2.2 mg / ml, respectively, in a total volume of 6.4 ml. After one hour at a temperature of 4 ° C, the particles were salted out and washed. The aggregated residue was resuspended in 3.2 ml phosphate buffer under 1 minute ultrasound treatment.

The antigen binding activity of the magnetically bound antiperoxidase was determined by incubating an aliquot of particles with free horseradish peroxidase (HRP), the particles being salted out, washed and examined in resuspended form with regard to the enzyme activity in a coloring assay. The antibody-coupled particles took up more than ten times as much enzyme as the control particle preparation, which was coupled with a similar antibody specific for an irrelevant antigen.

Example 3

Particles containing iodine radioisotope:

Particles were prepared according to the method described in Example 2, except that a small amount of radioiodinated (125 J) BSA was added to the reaction as an indicator of the protein component and four preparations were made in which the amounts of iron and BSA in the precipitation reaction varied as follows:

A. 1.25 Fe / ml and 1.5 mg BSA / ml (usual concentrations

B. 3.75 mg Fe / ml u. 1.5 mg BSA / ml (higher iron concentration)

C. 1.25 mg Fe / ml u. 5.0 mg BSA / ml (higher BSA concentration)

D. 3.75 mg Fe / ml u. 5.0 mg BSA / ml (higher iron and BSA concentration)

Immediately after the precipitation, but before any washing process, a sample was measured for the quantitative determination of the radioactive label in the mixture. The particles were then centrifuged, washed, resuspended and counted. The results showed the complete consumption of the BSA (slip into the aggregated magnetite residue) for the mixtures A and 3, only a 60% inclusion in the high-percentage BSA sample (C) and a return to the complete slip when the iron content was increased and returning to the almost original iron to protein ratio (sample D). Assuming that the iron was completely converted to magnetite (Fe 3 O 4), the compositions of the 4 preparations were as follows:

A. 46% protein, 54% magnetite

B. 22% protein, 78% magnetite

C. 63% protein, 37% magnetite

D. 49% protein, 51% magnetite

The stability of the protein magnetite particles against the loss of BSA during sedimentation and resuspension was checked using ultrasound. After resuspension, the particles were again "salted out", and the radioactive BSA which remained in the supernatant was counted. An average of 40% of the count rate (st.dev. = 11%) that was incorporated into the particles was lost. When the procedure was repeated, the losses in the next wash were lower (14%, st.dev. = 4%).

Example 5

Demonstration of the magnetic immunoassay using particles coupled to antigen:

Particles were precipitated and coupled to human IgM using SDPD to form a magnetic antigen as described in Example 2 above. The magnetic IgM particle antigen had bound a commercial antibody against human IgM, to which the alkaline phosphatase enzyme (Ab-AP) was coupled. A 1: 500 dilution of Ab-AP was incubated with approximately 250 micrograms of IgM magnetite in 100 microliters of phosphate containing 1% BSA. The amount of Ab-Ap which was bound to the IgM magnetite was measured by passing the incubation mixture through a small magnetic filter. The filter bed was then washed with an excess of buffer and loaded with the buffer containing the enzyme substrate. After 15 minutes of incubation, the buffer was eluted and the amount of reaction product resulting from the enzyme, which was collected in the filter bed, was determined by measuring the optical density.

The above procedure formed the basis of a competitive immunoassay for human IgM. If free IgM was added to the incubation mixture, the uptake of the enzyme by the antigen magnetite was specifically inhibited. The decrease in the enzyme activity on the filter as a function of the IgM in the incubation mixture was plotted graphically to set up a calibration curve, which creates a possibility of using the method as an immunological assay for measuring unknown amounts of IgM. The sensitivity was approximately 0.15 mg / ml (the concentration of IgM, which resulted in a 50% reduction in the specific amount of enzyme activity captured in the filter).

Example 6

Direct production of antibody-containing particles that maintain their activity throughout the preparation:

Seven precipitation steps were carried out using mixtures of BSA and goat antibodies (IgG fraction of goat anti-rabbit immunoglobulins). The total antibody amount was either 0.75 or 0.375 mg / ml. Either zero BSA was added or enough BSA to bring the total protein to 0.375, 0.75, 1.5 or 3.0 mg / ml. 3.5 mg FeCl & sub2; and 7.0 mg FeCl & sub2; added. The particles were precipitated by adding 20 microliters of 30% NH4 OH to pH 9.4. The preparations were then neutralized with acetic acid, spun off in a centrifuge and washed. The pelleted particles were each resuspended under 2 minutes of ultrasound treatment. Half of each preparation was treated with the EGS bifunctional reagent (0.31 mg / ml) for several hours. After the EGS treatment, the particles were salted out with 1.5 M NaCl, washed and resuspended in each case under 1 minute of ultrasound treatment.

The particles produced with IgG (without BSA) and the particles not treated with BSA spontaneously settled out of the suspension within 24 hours. This was true for both the 0.75 mg / ml and 0.375 mg / ml preparations. In both cases, however, half of the preparations which had been subjected to the EGS treatment were stable and usable in suspension.

Twelve samples were checked for goat anti-rabbit Ig activity by hemagglutination. A series of round microtiter wells containing sheep erythrocytes (SRBC) and a sub-agglutinating concentration of rabbit antibodies to SRBC was set up. For each row, particles were added in decreasing concentration, which was graded twelve times in relation to all successive wells in the row. After several hours the round well was read with the maximum visible agglutination for each row. More active particles agglutinated at lower concentrations (hence a larger number of wells). A table of the results from 12 preparations tested is shown below. Table I Hemagglutination Results Goat Antirrabbit Particles Total Proetin on Precipitation

<S> = discontinued and therefore not tested for hemagglutination.

Note: Fractions of round wells refer to a pattern in which the last round well did not clearly show agglutination (e.g. 9.5 = 9 wells with agglutinations and the tenth well with partial agglutination).

After storage at 4 ° C for 2 weeks, hemagglutination was repeated with the samples made with 1.5 mg / ml total protein. The activity was essentially unchanged. Binding specificity was demonstrated by the fact that no hemagglutination was observed in the control series of wells in which the anti-SRBC antibody was omitted.

Conclusions

(i) The antigen binding activity was hardly affected by EGS. For particles which were produced with the same total protein content, a higher activity was also found in preparations with a higher Ab content.

(ii) Improvements up to a total protein of 1.5 mg / ml and a slight decrease in activity above that point were observed on particles containing either amount of Ab but increasing amounts of BSA.

Example 7

Experiments were carried out on magnetic particles containing 125 J-labeled protein (HSA) to determine particle stability in terms of protein composition. Material and methodology:

Particles were made using 125 J-labeled HSA which had been iodinated using JCl. Three preparations with a total protein concentration of 0.05, 0.5 and 1.0 mg HSA were prepared. Each of these samples was produced using the same amount of Fe. The relative ratio of "hot" to "cold" protein was adjusted so that the specific activity (4000 cpm / pg) was the same in each case.

Results

In each case, the amount of radiolabel present in the supernatant was determined immediately after particle formation. The loss of radiolabel in the subsequent wash was also determined. The final ultrasound treated suspension was tested for radiolabelling and iron content to determine the protein / iron ratio. The results are summarized in Table IV.

Under the conditions used, the final iron content was approximately 1-1.56 mg / ml. Almost all of the labeled protein was incorporated into the initial precipitate in the preparations that had a protein concentration of 0.05 and 5.0 mg / ml. However, if protein was used at a concentration of 1 mg / ml, a substantial proportion (approximately 30%) of the radioactively labeled material remained in the supernatant. This was an indication that under these conditions the formation of the particles was efficient in terms of protein incorporation up to protein concentrations of 0.5 mg / ml. Higher protein concentrations appeared to result in a saturation effect, with excess protein remaining in the supernatant. This conclusion was further supported by the fact that the protein / iron ratio was increased significantly as the protein content increased from 0.05 to 0.5 mg / ml, but no further increase was observed at a concentration of 1.0 mg / ml was.

The particles appeared relatively stable in terms of protein content throughout the wash. (Significantly, the particles formed at high HSA concentration lost more radioactive material during the subsequent washing of the particles). It should also not be neglected to point out that part of the loss of 125 J-label is due to the loss of small but intact protein / magnetite particles.

Experiments were also carried out in which the presence of free protein in these particle preparations was determined by gel filtration. Previous experiments have indicated that only a very small amount of protein appeared to be "free of particles" after the ultrasound treatment. In addition, no other protein appeared when "leaching out", ie when the preparations were kept for 7 days. Furthermore, a second ultrasound treatment did not appear to increase the level of free protein. Table II Protein concentration Original 125 J-HSA first supernatant precipitated particles first wash. second wash third wash Ultraschallbeh. Particle suspension iron content protein: iron ratio * Obtained by difference

Using the above technique, a polymer with reactive groups can be used to form a photoactivatable particle, which can then be coupled to specific biofunctional compounds.

Example 8

Comparative example

In an attempt to repeat the Molday preparative measures and compare them with the present inventive procedures, two series of reaction mixtures were prepared. The first series of blends started with Molday dextran and iron concentrations, with dextran concentrations decreasing by the order of 10 to determine the effects of dextran concentrations on the final particle characteristics. The second series of reaction mixtures was started with dextran and iron concentrations according to the invention, the dextran concentrations increasing in the order of 10. The results of these preparations are summarized in Table 111. Table III Procedure Results Fe (III) Fe (II) Dextran According to Bas. Addition after heating. According to * Sonic Molday not mag. Rod. Susp. Rod. magn. Suspens. Magn. Przp. no change not resus pend. bar burst. color solution Note: kV = no trial = cannot be used with the procedure used * = after ultrasound treatment

As can be seen from the comparison values, the preparations are not able to deliver resuspendable particles according to the procedures mediated by Molday. As soon as the dextran concentrations are reduced to the range which is used according to the invention, the product quality of the particles is reduced and the suspension of these particles becomes unstable. In contrast, the dextran and iron concentrations according to the invention in the limit range give acceptable, stable suspensions of weakly magnetic particles. However, as soon as the dextran concentrations are increased in the direction of the values ​​taught by Molday, the precipitation fails and no particles are formed. This divergence of the conditions as well as certain qualitative differences in the ratio of the particle size and iron to the polymer are an indication of the extreme differences between the method and product according to Molday and those according to the invention.

Using the above examples, the inventors drew the following conclusions:

i) The particles according to the present invention are

a) magnetic

b) stable in aqueous suspension

c) resuspendable

d) small (and therefore filter sterilizable)

e) easy to manufacture with the inclusion of a spec. bilog. activity

f) clear in suspension.

ii) Particles can be made and then bound to specific biofunctional ligands using conventional bifunctional reagents.

iii) Specific biofunctional ligands can also act as the polymer during coprecipitation without significant loss of biological effectiveness.

iv) Treatment of the particles with bifunctional reagents can improve overall stability, both that of the particles and that of the suspensions, without unduly affecting the particle size.

v) The particles can be used in the case of coupling to specific biofunctional ligands in magnetic immunoassays of interesting biological substances.

vi) The particles decrease the NMR relaxation time of neighboring protons in solutions or tissues. This reduction is most pronounced in terms of T 2 (spin-spin) relaxation.

Nevertheless, while the invention has been described with reference to the specific examples, it will be apparent to those skilled in the art that variations in process conditions and parameters may be employed. The following claims are to be interpreted to include such modifications.

The invention encompasses new magnetic polymer particles and methods of making them. These particles are in a variety of biological and medical fields including cell recognition, when used as a contrast medium for NMR imaging, immobilizing enzyme reactions, as an immunoassay and other analytical and diagnostic techniques.