JP2588181B2 - Magnetic and polymer particles - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This application is the same and assigned to the inventor, 1985.
It is a continuation-in-part application of application number 784,863, filed on October 4, 2008, the contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION The present invention relates to particles comprising a particular polymer in combination with a magnetic metal, compositions comprising the particles, methods of making and using the particles and compositions. More particularly, the invention relates to such particles where the polymer has a biochemical function.

Biologically active magnetic particles will find use in various formulation and diagnostic techniques. These include high gradient magnetic separation (HGMS), which uses a magnetic field to separate magnetic particles from a suspension. Biological materials of interest (eg cells, drugs)
If so, the material of interest can thereby be separated from other materials that are not associated with the magnetic particles.

Some properties are important when magnetic particles are effective in biological and diagnostic systems. First, the particles must have the required biological activity, affinity or reactivity, and will perform their function. Second, the particles must be suspendable in an aqueous medium for release into the biological or reaction system. It is also desirable that the particle suspension be stable (ie, not settle or agglomerate). Finally, a small particle size may be desired so that the suspension of magnetic particles can be sterile filtered in a conventional manner.

Many techniques have been proposed in the prior art for the preparation of magnetic particles or organic magnetic materials. U.S. Pat. No. 4,001,288 to Gable et al. Describes the preparation of magnetic organic-iron compounds with both water solubility and ferromagnetism. In the preparation shown by Gable et al., Ferrous iron in solution is treated with hydrogen peroxide and then treated with ammonium hydroxide to precipitate the iron oxide compound. The compound is then oxidized with peroxide, treated with a hydroxy-carboxylic acid, and reacted with an alkaline material to form a soluble material.

Gable et al. Also describe that proteins or "proteolytic products" are introduced into a ferrous solution and then reacted to precipitate iron oxide. The only material shown in the examples is the hydrolysis product of gelatin treated with hydrogen peroxide.

U.S. Pat. No. 4,452,773 to Molday describes "colloidal-sized" iron oxide particles coated with a polysaccharide (exemplified in the patent as dextran and dextran derivatives). Although the Mordi particles are made in a manner somewhat similar to those used in the particle preparation of the present invention, they appear to be substantially different from the particles of the present invention.

Czerlinski U.S. Patent No. 4,454,
No. 234 describes the preparation of coated magnetic particles that can be reversibly suspended in a solution (usually by suspension polymerization in a magnetic particle substrate)
It is shown. Suspension parameters exhibited by Kuzellinsky include the Curie temperature effect of his magnetic particles, which are used alternately to make the particles magnetic and non-magnetic. If the particles are magnetic, they tend to agglomerate, but can be resuspended when the particles are heated to a temperature above the Curie temperature of the magnetite contained therein.

Senyei et al. U.S. Pat. No. 4,230,685
The article describes the preparation of microspheres containing magnetite, albumin and protein A. The preparation shown by Seniay does not involve precipitating magnetite in the presence of these other components, but rather a coating of preformed magnetite particles.

Another patent that may be of interest is Robinson
on) et al., US Patent No. 4,152,210; Mosbac.
h) 4,335,094; Giaever 4,01
No. 8,886; No. 4,070,246 of Kennedy et al. Although all of these patents describe the preparation or use of magnetic biological particles, none of these are expected to be similar to those of the present invention.

Terms Active Halogen: Halogen atom bonded to a carbon atom bonded to an electron-withdrawing group or bonded to an adjacent carbon atom (eg; iodo-acetamide, iodo-acetate) Available coordination unit: not sterically hindered ( A coordination moiety suitable for coordinating metal atoms in a metal compound (eg, Fe ₃ O ₄ ), which can be freely accessed.

Biofunctional ligand: A molecule having a biological activity or having a specific affinity for a biological system that can bind to the ferromagnetic polymer particles of the invention and confer specific biological properties to these particles.

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

Denatured protein: a specific biological functional activity due to chemical or structural denaturation (eg, enzymatic, antigenic, antibody, etc.)
Lost protein. As used by Gable et al .: Protein residues in which some amide bond cleavages remain thereafter and have a molecular weight <10,000.

Extra-particle bonding: One functional group of a bifunctional compound and ferromagnetism-
One that is attached to a site on the polymer particle, while another functional group is attached to a site on a different molecule or molecule (generally a “ligand”).

Ferromagnetic: Permanent magnetic iron composition (having a true magnetic moment) Intraparticle bonding: Both functional groups of the bifunctional compound are bonded to different sites of the same magnetic-polymer particle.

Low ionic concentration: Aqueous solution with near neutral pH and a total cation concentration (usually from a buffer) of less than 40 mM.

Protein: Molecular weight 1 linked to amide (peptide) bond
Over 10,000 amino acid polymers.

Resuspension: The re-dispersion of a once-aggregated material (eg, by centrifugation, HGMS or flocculation) to obtain a stable suspension.

Sonication: Exposure to very strong ultrasound.

Stable suspension: A suspension that does not settle or otherwise agglomerate when allowed to stand at standard temperature and pressure for 2 days.

BRIEF DESCRIPTION OF THE INVENTION The present invention comprises the preparation of suspendable and resuspendable magnetic polymer particles and the particles made thereby. Such particles exhibit properties particularly useful in immunoassays, where the particles are made with a particular biofunctional ligand and then separated by high gradient magnetic separation techniques.

The method of the present invention comprises co-precipitating a metal ion as a magnetic compound in the presence of a polymer having an available coordination moiety (eg, [Fe (II) + Fe (III)] or [Fe (II) + Cr (I
II)] and reacting the polymer with a metal to precipitate and recover the magnetic-polymer particles. In addition, various biofunctional groups may be incorporated into the particles to obtain biofunctional reagents useful for use in immunoassays, cell capture, enzyme immobilization reactions, NMR imaging and other diagnostic and analytical techniques. .

DETAILED DESCRIPTION OF THE INVENTION According to one embodiment of the present invention, Fe (II) and Fe (II)
I) Solutions containing (typically Fecl ₂ and Fecl ₃ ) and polymers with available coordination moieties (eg proteins)
Is treated with a strong base such as ammonium hydroxide (NH ₄ OH) (titration and the like) to precipitate a magnetic iron oxide, such as magnetite (Fe ₃ O ₄ ), uniformly bound with the polymer. Precipitation is generally effected by rapid stirring and, optionally, sonication to produce resuspendable magnetic-polymer particles.

After precipitation, the particles are washed and then resuspended in a buffer at approximately neutral pH.

Another embodiment of the present invention involves using other metals besides iron in the coprecipitation reaction. In particular, Fe (III) can be replaced with any of a wide range of other transition metal ions. In some cases, iron can be completely replaced by a suitably selected transition metal ion. In many cases, the use of metals other than iron produces colored particles ranging from white to dark brown.

Magnetic-polymer particles made according to the present invention exhibit many beneficial properties. These particles are magnetic because they contain a form of a magnetic metal compound (eg, iron similar to the form of magnetite, or a similar compound). The particles are formulated so as to be resuspendable after aggregation and to form a relatively stable suspension that does not settle even after gently storing for several days. In addition, the particles made according to the present invention are relatively small (approximately 0.01-0.2 microns) and can therefore be sterile filtered. Finally, the particles made according to the present invention can be tailored to include certain biofunctional ligands useful in various analytical, diagnostic, and other biological / medical applications.

Following precipitation and resuspension of the magnetic-polymer particles, they may be treated with a bifunctional reagent to crosslink reactive sites present in the polymer. This cross-linking may be effective as an intra-particle cross-linking where the reactive sites bind to the same particle, or as a reactant of an extra-particle ligand that then cross-links to the polymer in a given particle. In the second case, a bifunctional reagent with a relatively short distance between the two functional groups is desirable to facilitate binding of the particulate polymer to the extra-particle species. Conversely, intraparticle crosslinking is facilitated by using a bifunctional reagent that is longer and sterically unhindered from the bends, so that two reactive sites on one particle can be linked by one bifunctional molecule.

Instead of using sonication during any of the precipitation or resuspension steps described above, other types of agitation (eg, mechanical agitation) can be performed.

Resuspension of the magnetic-polymer particles of the present invention is generally performed in a low ionic concentration buffer system (eg, a 40 mM phosphate system). The buffer system allows for resuspension of particles that do not resuspend in the nonionic solution. In addition to phosphate buffers, borate and sulfate systems can also be used.

The homogeneous association of the polymer and metal in the present invention
It is believed to be the result of coordination of the metals present during co-precipitation by coordination sites in the polymer. Certain coordination moieties are assumed to be more "available" than others based on both the strength of the coordination bonds that can be formed by one atom and the spatial barriers imposed by surrounding atoms. For example, it is known that oxygen atoms having a "free" electron pair complex iron more strongly than amine nitrogen atoms and, to a much greater extent, hydroxyl oxygen atoms. That is, polymers having oxy-acid functionality should provide better product particles than amine-substituted polymers. Similarly, a configuration that can be freely approached to near will obtain better performance than sites obstructed by either the approach path or the approach distance.

The above tendency is qualitatively observed in various experiments performed by the present inventors. The presence of "available coordination moieties" may be necessary for the production of the resuspendable magnetic-polymer particles of the present invention. For example, various polymers and their various copolymers, such as natural proteins, synthetic proteins, poly-amino acids, carboxoxy-poly-alkyl, alkoxy-poly-alkyl, amino-poly-alkyl, hydroxy-poly-alkyl, are all described herein. It is proven to produce the particles of the invention. In addition, other polymers such as sulfoxy-poly-alkyl, poly-acrylamine, poly-acrylic acid and substituted poly-alkylene make the particles of the present invention.

In selecting the transition metal to be used in the coprecipitation reaction,
Several criteria appear to be important. First, the final compound must have one or more unpaired electrons in its structure. Second, one of the metals must have a coordination site available for binding with the polymer. Third, the coprecipitation must be capable of forming a cubic or hexagonal (eg, cubic: spinel or inverse spinel) crystal structure. This last requirement appears to be the result of the need to be very tightly packed because the compound is magnetic.

Finally, the polymers useful in preparing the particles of the present invention may be "tailored" to include monomers that exhibit a particular biofunctional activity. The use of such polymers allows for the direct precipitation of biofunctional magnetic-polymer particles, which requires little processing to be useful in assays that rely on the specific biofunctional activity of the polymer. Or no need at all.

For certain applications, larger, less stable particles are useful. The particles of the present invention may be made to aggregate while still retaining both their biofunctionality and magnetism. Agglomeration of the particles can be achieved by treating the suspension with a predetermined amount of, for example, a barium chloride solution. This process may cause the particles to settle out of the suspension for a predetermined period of time, or perform further means,
Also, larger particles may be easily attacked by relatively small magnets.

The following examples illustrate various parameters of magnetic-polymer particle preparation and use: Example 1 General Preparation Procedure The starting aqueous solution for a representative (micro) preparation is as follows: 1 mg of the given protein / ml solution FeCl ₃ 500mg / ml FeCl ₂ 200mg / ml phosphate buffer (almost pH 7) 20mM ammonium hydroxide solution (NH ₄ OH) 7.5% or 15% To prepare 2.5ml protein solution and 20ml distilled water Start by diluting to FeCl ₂ 175μ and FeCl ₃ 140
Add μ. Finally, ammonium hydroxide solution about 200
Add μ.

It should be noted that the amount of base added is usually pre-calculated and takes into account the total amount of protein or polymer in the reaction mixture and the buffering capacity of that particular protein or polymer. The goal is to increase the pH to a degree sufficient to allow precipitation of the iron oxide, but to maintain substantial activity of the protein or polymer by not exceeding the pH that denatures the material.

These materials are all added to the reaction vessel with continuous stirring to promote a homogeneous reaction mixture. Black particles precipitate as soon as the base (NH ₄ OH) is added. The reaction mixture is centrifuged, typically at 3,000 rpm for 15 minutes, and the supernatant is discarded. The pellet is then broken up and the material is washed with 20 ml of phosphate buffer, typically in three washing cycles, with centrifugation. After the last wash, the pellet is broken up again and the material is resuspended in buffer under the influence of sonication. The final solution is a clear, pale yellow or brown solution that is a stable suspension of ferromagnetic-polymer particles.
Throughout the process, iron agglomerate residues are not always found.

Subsequent processing of the particles may proceed by any of the means described below, including intra-particle or extra-particle cross-linking with the biofunctional reagent.

Example 1A-Use of Metals Other than Iron As noted above, metals other than iron may be incorporated into the magnetic-polymer particles of the present invention. The following is a non-limiting table of ions that can be used to prepare the 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) Er (III) + Ru (III) Er (III) + Co (II) Mn (II) + Er (III) Cr (III) + Fe (II) In addition to the above table, the electromotive potential is Fe (II) To Fe (II
Fe (II) can also be used in combination with selected transition metals that are insufficient to oxidize to I). Of the metals listed in the table above, only V (III) is capable of oxidizing Fe (III) and is therefore unsuitable.

Example 2-Antibody-Bound Particles Two solutions (40 ml each) are rapidly mixed in an ultrasonic bath to facilitate mixing. Both contain 1.5 mg / ml bovine serum albumin (BSA). One is ammonium hydroxide (3
0% 8 ml, final concentration = 74 mM). The other contains 140 mg ferrous chloride and 280 mg ferric chloride (total iron concentration, after mixing
1 mg / ml). A black precipitate forms immediately. The mixture is neutralized by adding 6 ml of glacial acetic acid with stirring. Divide the sample into four tubes and centrifuge the precipitate (3000
(rpm, 15 min) and discard the small amount of iron remaining in the golden, slightly turbid supernatant. Resuspend the pellet in 20 ml of 20 mM phosphate buffer at neutral pH. Each of the four tubes containing 5 ml of particles is sonicated for 5 minutes in a Branson sonicator with a cup horn accessory.

The available amino groups on the particles react with succinimidyl-propiono-dithiopyridine (SPDP) to make these moieties for later binding to antibodies. This reaction (20 ml of particles + 5 mg of SPDP) proceeds for 1 hour in ice with stirring. The particles are then further stabilized by adding 25 mg of the amino-reactive crosslinking reagent ethylene-glycol-disuccinimide-ester (EGS). The mixture is reacted for a further hour with stirring. EGS cross-links with any of the remaining amino residues, which is very similar to the bond. The other remaining amino groups attach to one of the ends of the EGS molecule, while the others eventually hydrolyze to carboxyl groups.
This results in a net conversion of positively charged groups to negatively charged groups at the particle surface, which is believed to promote the stability of the colloidal suspension.

At the end of the reaction period, the preparation is transferred to a 50 ml test tube and the particles are "salted out" by adding 10 m of a 3 molar aqueous NaCl solution. After aggregation at room temperature for 10 minutes, the particles are centrifuged at 1500 rpm for 10 minutes. Discard the clear, colorless supernatant. After resuspending the pellet and centrifuging twice or more, add 20 ml of 20 mM phosphate buffer
Receive the final suspension in (sonication is not usually required at this point).

The particles are conjugated to a goat antiserum specific for horseradish peroxidase as a conveniently assayed antigen. Antiserum is activated by reaction with SPDP. 0.128 ml of antiserum containing 1.28 mg of total protein for 30 minutes at room temperature
Is reacted with 12.8 μg of SPDP and diluted to 0.512 ml of phosphate buffer. After 30 minutes, 3.1 mg of dithiothreitol (DTT) is added to convert SPDP to its free sulfhydryl form. The reacted Ab is separated in a small gel filtration column (desalting).

The thiolated antiserum and SPDP-activated particles are allowed to react by adding 6.4 ml of an antibody (0.64 mg of antiserum protein for 100% recovery) to give particles containing 14 mg of iron. The concentration in the reaction mixture is 1.0 mg / ml for antiserum proteins
And 2.2 mg / ml for particulate iron, for a total volume of 6.4 ml.
After 1 hour at 4 ° C., the particles are salted out and washed.

The pellet is resuspended in 3.2 ml phosphate buffer and sonicated for 1 minute.

Antigen binding activity of magnetite-bound anti-peroxidase was determined by incubating aliquots of particles with free horseradish peroxidase (HRP), salting out the particles, washing them, and resuspending the particles in a colorimetric assay for enzyme activity. To analyze. The antibody-bound particles captured as much enzyme more than 10 times as control particle preparations conjugated to similar antibodies specific for an irrelevant antigen.

Example 3 Particles Containing Radioactive Isotopes of Iodine Particles are made by the method described in Example 2, except that a small amount of radioiodinated ( ¹²⁵ I) BSA is added to the reaction as a tracer of the protein component and iron in the precipitation reaction And make four preparations with varying amounts of BSA as follows: A. 1.25 mg Fe / ml and 1.5 mg BSA / ml (normal concentration) B. 3.75 mg Fe / ml and 1.5 mg BSA / ml (high iron concentration C. 1.25 mg Fe / ml and 5.0 mg BSA / ml (high BSA concentration) D. 3.75 mg Fe / ml and 5.0 mg BSA / ml (high iron and BSA concentration) Immediately after precipitation, all washing steps are performed At no time, count the sample and quantify the radiolabel in the mixture. The particles are then centrifuged, washed, resuspended and counted. As a result, mixtures A and B
For full utilization of BSA (incorporation in magnetite pellets), only 60% contamination in high BSA sample (C), and increasing the amount of iron until almost the initial ratio of iron to protein (sample D) ) Was found to return to complete contamination. Assuming that the iron was completely converted to magnetite (Fe ₃ O ₄ ), the composition of the four preparations is as follows: A. 46% protein, 54% magnetite B. 22% protein, 78% magnetite C .63% protein, 37% magnetite D.49% protein, 51% magnetite Test the stability of protein-magnetite particles against BSA loss during sedimentation and resuspension by sonication. After resuspension, the particles are again "salted out" and the radioactive BSA remaining in the supernatant is counted. 40% average mixed into particles
(Standard deviation = 11%) counts were lost. Repeating this procedure reduced losses in the next wash (14
%, Standard deviation = 4%).

Example 5-Demonstration of a magnetic immunoassay using particles bound to antigen Particles were precipitated and SP as described in Example 2 above.
Use DP to bind to human IgM to make a magnetic antigen. Particle-I
gM magnetic antigen is alkaline phosphatase enzyme (Ab-Ap)
Binds to a commercially available antibody against human IgM bound. Ab-A
1: 500 dilution of p in 100 μl of phosphate containing 1% BSA
Incubate with about 250 μg of IgM-magnetite in. The amount of Ab-Ap bound to IgM-magnetite is
It is quantified by passing the incubation mixture through a small magnetic filter. The filter bed is then washed with excess buffer and buffered with the enzyme substrate.
After 15 minutes of incubation, the buffer is eluted and the amount of reaction product produced by the enzyme captured on the filter is determined by optical densitometry.

The above method forms the basis of a competitive immunoassay for human IgM. When a small amount of free IgM is added to the incubation mixture, the capture of the enzyme by the magnetic-antigen is specifically inhibited. A calibration curve is made by graphing the decrease in enzyme activity on the filter as a function of IgM in the incubation mixture. This allows the method to be used as a competitive immunoassay, allowing the determination of unknown quantities of IgM. The sensitivity is approximately 0.15 mg / ml (the concentration of IgM that reduces the specific capture of enzyme activity in the filter by 50%).

Example 6-Direct Preparation by Preparation of Particles Containing Antibodies Retaining Activity Seven precipitation reactions are performed using a mixture of BSA and goat antibody (goat anti-rabbit-IgG fraction of immunoglobulin). The total amount of antibody in each is either .75 or .375 mg / ml. Do not add BSA to this or .37
Add enough BSA to increase total protein to 5, .75, 1.5 or 3.0 mg / ml. FeCl ₂ 3.5 to each 2ml sample
mg and 7.0 mg of FeCl ₃ . The particles are precipitated by adding 20μ of 30% NH ₄ OH to pH 9.4. The formulation is then neutralized with acetic acid, spun in a centrifuge and washed. The pelleted particles are each resuspended by sonication for 2 minutes. One half of each preparation is treated with the bifunctional reagent EGS (0.31 mg / ml) for several hours. After EGS treatment, the particles are salted out with 1.5 M NaCl, washed and resuspended by sonication for 1 minute each.

Approximately 24 particles without EGS made of IgG (no BSA)
Settles out of suspension over time. This is 0.75
Both the mg / ml and 0.375 mg / ml preparations are true. However, both preparations that have undergone EGS treatment are half stable in suspension and can be used.

Twelve samples are tested for goat-anti-rabbit-Ig activity by hemagglutination. The microtiter plate is assembled to contain a series of recesses of goat red blood cells (SRBC) and a sub-agglutination concentrate of rabbit antibodies to SRBC. In each row, particles are added to each successive recess of the row at a concentration that is reduced by a factor of two. After several hours, the largest depression where aggregation can be seen is observed for each row. The more active particles aggregate at lower concentrations (higher recess numbers). The results for the 12 preparations are shown in the following table.

After storage at 4 ° C. for 2 weeks, hemagglutination was measured at 1.5 mg total protein /
Repeat for samples made in ml. Activity does not substantially change. The specificity of the binding is indicated by the fact that no hemagglutination is seen in the recesses of the control row where the anti-SRBC antibody has been excluded.

Conclusion: (i) Antigen binding activity is strongly affected by EGS. In particles made of the same total protein, it was found to be more active in preparations containing higher amounts of Ab.

(Ii) BS containing any but increasing amounts of Ab
In the particles containing A, it appears to be improved to 1.5 mg / ml total protein and above that point the activity is slightly reduced.

Example 7 Experiments are performed on magnetic particles containing ¹²⁵ I-labeled protein (HSA) to evaluate the stability of the particles relative to the protein composition.

Materials and Methods Particles were treated with ¹²⁵ I-labeled H
Prepare using SA. The three preparations are 0.05, 0.5 and
Made to contain a total protein concentration of HSA of 1.0 mg / ml. Each of these samples is prepared using the same amount of Fe. The relative proportions of the "hot" and "cold" proteins are adjusted such that the specific activity (4,000 cpm / μg) is the same in each case.

Results In each case, the amount of radiolabel present in the supernatant immediately after particle formation is assessed. The amount of radiolabel lost in subsequent washing steps is also measured. The final sonicated particle suspension is evaluated for radiolabel and the Fe content and protein / Fe ratio are measured accordingly. The results are summarized in Table IV.

Under the conditions used, the final Fe content is approximately 1-1.56 mg / m
l. In the case of preparations containing protein concentrations of 0.05 and 0.5 mg / ml, almost all of the labeled protein is incorporated into the initial precipitate. However, the protein is 1m
When used at a concentration of g / ml, a substantial proportion (approximately 30%) of the radiolabel remains in the supernatant. This indicates that particle formation is effective under these conditions for contaminating the protein to a protein concentration of 0.5 mg / ml. Higher protein concentrations result in a saturating effect, which seems to leave excess protein in solution.

The conclusion is that the protein Fe ratio is substantially equal to protein 0.
It rises from 05 to 0.5 mg / ml, but is further substantiated by the fact that 1.0 mg / ml shows no further increase.

The particles appear to be relatively stable with respect to protein content throughout the washing process (importantly, the particles have high HSA
Forming at a concentration loses more radioactive label during the subsequent particle washing step). ^It should also be noted that some of the ¹²⁵ I-label loss is measured by the loss of small amounts of unaffected protein / magnetite particles.

Experiments are also performed to evaluate the presence of free protein in these particle preparations by gel filtration. Preliminary experiments have shown that very little protein appears to be "particle-free" after sonication. Also, when the preparation is stored for up to 7 days, no more protein appears to "exude". Furthermore, the second sonication does not appear to increase the percentage of free protein.

According to the above technique, a polymer having a reactive group may be used to form a photoactive particle and then be combined with a specific biofunctional compound.

Example 8-Comparative Example In an attempt to repeat the Mordei preparation method and compare these methods to the present invention, two series of reaction mixtures are prepared. The first row of the mixture begins with dextran and iron concentrate as taught by Mordey and evaluates the effect of the dextran concentrate on the final particulate characteristics by sequentially decreasing the dextran concentration by a power of 10. A second series of reaction mixtures starts with dextran and iron concentrate according to the invention.
The dextran concentrate is increased sequentially by a power of 10. The preparation results for these series are summarized in Table III.

As is evident from the comparative data, preparations according to the procedure indicated by Moldy fail to obtain resuspendable particles. As the concentration of dextran decreases toward the range used in the present invention, the quality of the product particles decreases and the suspension of these particles becomes unstable. In contrast, as indicated by the present invention, dextran and iron concentrates produce marginally acceptable stable suspensions of weakly magnetic particles. Increasing the dextran concentration in the direction indicated by the morde, however, resulted in no precipitation and no particles were produced. This branch point in the conditions, as well as certain property differences in particle size, and the iron to polymer ratio, indicate that there is a significant difference between the process and product indicated by Moldy and that according to the present invention. Show.

From the above examples, the following conclusions are drawn by the invention: i) the particles of the invention are a) magnetic, b) stable in aqueous suspension, c) resuspendable, d. A) small (ie, filter-sterilizable); e) easily manufactured to contain specific biological functions; f) transparent in suspension.

ii) Particles may be made and subsequently conjugated to specific biofunctional ligands using conventional bifunctional reagents.

iii) Certain biofunctional ligands also act as polymers during co-precipitation or without significant loss of biological activity.

iv) The treatment of the particles with the bifunctional reagent does not significantly affect the particle size,
Above all, stability can be improved.

v) Upon binding to a particular biofunctional ligand, the particles can be used in magnetic immunoassays for the biological material of interest.

vi) Particles are NMR of adjacent protons in solution or tissue
Decrease relaxation time. This decrease is T ₂ (spin-spin)
The most notable is mitigation.

Although the present invention has been described in connection with specific examples, it will be understood by those skilled in the art that nonetheless other variations and parameters of the process conditions can be used without departing from the true spirit of the invention. Will. It is envisaged that the following claims should be constituted to include all such variations.

Statement of Industrial Utility The present invention comprises novel magnetic polymer particles and a method of making the same. These particles are useful in various biological / medical fields, including cell capture, and are used as control reagents in NMR imaging, immobilized enzyme reactants, immunoassays and other analytical and diagnostic techniques.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor D'Angelo, Lewis United States of America, New Jersey 08009, Berlin, Washburn Avenue 35 (72) Inventor of Liberty, Paul, A. United States of America, Pennsylvania 18966, Churchville, Green Drive 58 (56) References JP-A-55-143440 (JP, A) US Patent 4,169,804 (US, A) US Patent 4,247,406 (US, A) US Patent 4001,288 (US, A)

Claims (38)

(57) [Claims] A) preparing an aqueous solution of transition metal ions capable of undergoing a reaction to form a magnetic precipitate and a polymer having available coordination moieties in proportions suitable to produce a resuspendable product; 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. Preparation method. 2. The method of claim 1, further comprising the step of: d) resuspending said magnetic polymer particles in an aqueous solution. 3. The method of claim 2 wherein said aqueous solution has a low ionic concentration suitable for forming a stable suspension. 4. The method of claim 1, further comprising the step of: e) filtering the suspension through a filter having pores of at most 0.44 μm diameter. 5. The method of claim 1, further comprising the step of: f) reacting said magnetic polymer particles with a bifunctional compound specific for said polymer. 6. The method of claim 1, further comprising the step of: f) reacting said magnetic-polymer particles with a bifunctional compound suitable primarily for forming intraparticle bonds. 7. f) reacting said magnetic-polymer particles with a bifunctional compound suitable mainly for forming intraparticle bonds, wherein said bifunctional compound is arylnitrene, imide-
Ester, N-hydroxysuccinimide-ester, 2-diazo-3,3,3-trifluoropropionate, maleimide, pyridyl-disulfide, halonitrobenzene, isothiocyanate, halo-sulfonate,
The method of claim 1, further comprising having a terminal group selected from the group consisting of an active halogen and an active aldehyde. 8. The method of claim 1, further comprising the step of: g) reacting said magnetic-polymer particles with both a bifunctional compound and a biofunctional ligand. 9. The method of claim 1, further comprising the step of: g) reacting said magnetic-polymer particles with both an activator and a biofunctional ligand. 10. g) The magnetic-polymer particles are mainly a bifunctional compound suitable for forming an extraparticle bond, wherein the bifunctional compound is arylnitrene, imide-ester, N-hydroxy Succinimide-ester, 2-
Diazo-3,3,3-trifluoropropionate, maleimide, pyridyl-disulfide, halonitrobenzene,
Those having a terminal group selected from the group consisting of isothiocyanate, halo-sulfonate, active halogen and active aldehyde and antigens, antibodies, lectins, avidin, biotin, staphylococcal protein A (SPA), enzymes, serum proteins, Clq The method of claim 1, further comprising the step of reacting with a biofunctional ligand selected from the group consisting of: a complement protein; and a rheumatoid factor. G) activating the magnetic-polymer particles with an activator and antigen selected from the group consisting of water-soluble carbimide, glutamaldehyde, cyanogen halide, periodate, and tannic acid, an antibody, a lectin, Claims: further comprising reacting with a biofunctional ligand selected from the group consisting of avidin, biotin, staphylococcal protein A (SPA), enzymes, serum proteins, Clq, complement proteins, and rheumatoid factor. The method of claim 1. 12. The transition metal ions in the solution are Fe (II) and Fe (II).
The method according to claim 1, which is a mixture of (III). 13. The method of claim 1 wherein the weight ratio of metal to polymer in the solution is 100.
The method of claim 1, wherein the method is between 0: 1 and 1:10. 14. The method according to claim 1, wherein said reaction (step b) is performed under the influence of sonication. 15. The method according to claim 2, wherein said resuspension (step d) is performed under the influence of sonication. 16. The method according to claim 3, wherein said resuspension (step d) is performed under the influence of sonication. 17. The method according to claim 16, wherein a low concentration (<40 mM) of anions is added to the solution during resuspension (step d). 18. The method of claim 18, wherein said polymer is a synthetic protein, a natural protein, a poly-amino acid, a carboxy-poly-alkyl, an alkoxy-poly-alkyl, an amino-poly-alkyl, a hydroxy-poly-alkyl, a sulfoxy-poly-alkyl, a carboxy. -Poly-alkylene, alkoxy-poly-alkylene, amino-poly-alkylene,
The method of claim 1, wherein the method is selected from the group of hydroxy-poly-alkylenes, sulfoxy-poly-alkylenes, poly-silanes, poly-phosphines, and copolymers thereof. 19. The method according to claim 17, wherein said anion is selected from the group consisting of phosphate, borate and sulfate.
The method described in the section. 20. The method of claim 1 wherein said polymer contains an oxy-acid functionality having an available coordination moiety. 21. The method of claim 1, wherein said polymer is a protein having an available coordination moiety. 22) h) adding a salt to said suspension to increase its ionic concentration to a molar concentration suitable for causing agglomeration and precipitation of said magnetic-polymer particles; i) said agglomerated precipitated particles 4. The method of claim 3, further comprising: j) resuspending the magnetic-polymer particles in an aqueous solution of low ionic concentration suitable to form a stable suspension. 23. The method of claim 1, wherein said transition metal ion is selected from the group consisting of having at least one unpaired electron and forming a coprecipitate having a spinel or inverse spinel structure. . 24. The transition metal ions are of the following group: 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) 2. The method according to claim 1, comprising a pair of ions selected from Er (III) + Co (II) Mn (II) + Er (III) Cr (III) + Fe (II). 25. The transition metal ion comprising Fe (II) and Fe (II)
2. The method of claim 1 comprising a transition metal ion having an electromotive potential that is insufficient to oxidize (II) to Fe (III). 26) k) preparing an aqueous solution capable of forming a magnetic precipitate and a polymer having a coordinating moiety available by the reaction; 1) adding a strong base in excess of the stoichiometric amount; m) stirring the solution. N) precipitating the magnetic-polymer particles; o) recovering the magnetic-polymer particles; 27. The method according to claim 26, wherein the addition of the base (step 1) is carried out by titrating the solution with a base until the solution has a slightly basic pH. 28. The method of claim 26, wherein said polymer is a protein and the pH of said solution is kept lower than required to denature said protein. 29. A stable aqueous suspension of magnetic-polymer particles prepared by the method of claim 3. 30) a) preparing an aqueous solution of a transition metal ion capable of reacting to form a magnetic precipitate and a polymer having available coordination moieties in proportions suitable for making resuspendable particles; b) reacting the metal ions in the presence of the polymer to form a magnetic precipitate of magnetic polymer particles; c) recovering the magnetic-polymer particles; d) resuspending the magnetic-polymer particles in an aqueous solution. E) combining the magnetic-polymer particles with a bifunctional compound and a biofunctional ligand suitable for forming an extraparticle bond, wherein the biofunctional ligand is of the predetermined type; F) exposing the mixture containing the unknown amount of the predetermined type to a suspension of the magnetic polymer particles containing the biofunctional ligand, thereby causing the mixture to contain the biofunctional ligand. Binding the functional ligand to the predetermined type; g) passing the mixture through a magnetic filter having a magnetic field, the filter being suitable for retaining the magnetic-polymer particles; h) the magnetic field From the filter and elute the retained magnetic-polymer particles; i) the eluted magnetism by a preselected analytical method suitable to provide the desired data associated with the predetermined type. A predetermined type of analysis method comprising the step of analyzing the polymer particles. 31. Step d) resuspending the magnetic-polymer particles in an aqueous buffer solution having a low ionic concentration and filtering the suspension through a filter having pores of at most 0.44 μm in diameter. 31. The method of claim 30, comprising sterilizing the suspension. 32. The bifunctional compound as claimed in claim 1, wherein the arylfunctional compound is arylnitrene, imide-ester, N-hydroxysuccinimide-ester, 2-diazo-3,3,3-trifluoropropionate, maleimide, pyridyl-disulfide, halonitrobenzene, 31. The analytical method according to claim 30, having a terminal group selected from the group consisting of isothiocyanate, halo-sulfonate, active halogen and active aldehyde. 33. The biofunctional ligand selected from the group consisting of antigens, antibodies, lectins, avidin, biotin, staphylococcal protein A (SPA), enzymes, serum proteins, Clq, complement proteins, and rheumatoid factors. The analysis method according to claim 30. 34. The biofunctional ligands are antigens and their antibodies, haptens and their antibodies, hormones and their receptors, vitamins and their receptors, toxins and their receptors, drugs and their receptors, and enzymes. 31. The method of claim 30, wherein the method is selected to provide one half of a binding pair selected from the group of these cofactors. 35. The method according to claim 30, wherein the suspension is further treated with a flocculant to promote the flocculation of the magnetic-polymer particles.
Analysis method described in the item. 36. The method according to claim 35, wherein said coagulant is a salt of a Group II metal. 37. The analysis method according to claim 35, wherein the aggregated particles are filtered using a permanent magnet held by a hand. 38. The amount of agglutination caused by the aggregating agent is as follows:
Claims 1 to 3 minutes of suspension with a hand-held permanent magnet are sufficient to capture the agglomerated particles, but less than 1 hour are insufficient to precipitate the agglomerated particles from the suspension. Analysis method according to claim 35.