WO2000043199A1

WO2000043199A1 - Magnetically responsive gel particle

Info

Publication number: WO2000043199A1
Application number: PCT/US2000/001311
Authority: WO
Inventors: Joseph F. Lawlor; Gordon C. Siek; Joseph D. Musto
Original assignee: Reference Diagnostics, Inc.
Priority date: 1999-01-20
Filing date: 2000-01-19
Publication date: 2000-07-27
Also published as: JP2002535634A; EP1152888A4; AU2731700A; EP1152888A1

Abstract

Gel particles, methods of preparing gel particles, and methods of using gel particles are disclosed. The gel particles contain a magnetically responsive particle, and optionally a scavenger.

Description

MAGNETICALLY RESPONSIVE GEL PARTICLE Cross-Reference to Related Patent Application This application claims priority under 35 U.S.C. section 119(e)(1) to U.S. Patent Application Serial No. 60/116,633, filed January 20, 1999, and entitled "Magnetically Responsive Particle".

Background of the Invention The invention relates to gel particles.

In humans the metabolic requirements for iron can be determined by the rate of absorption which is matched by a normally fixed rate of loss. A state of iron deficiency can occur if dietary intake of iron is insufficient, if iron absoφtion by mucosal membranes is impaired, or if excessive bleeding occurs. Severe cases may lead to iron deficiency anemia. Conversely, genetic defects in the absoφtion of iron can result in an overload of iron which can lead to chronic liver disease and premature death. Iron is stored as a component of the protein ferritin and as part of insoluble hemosiderin. Iron is transported from the absoφtion and storage sites to the sites of use via specific iron-binding proteins — most notably transferrin, which is usually one-third saturated with iron.

While serum ferritin concentrations are a useful index of storage iron, they are not as informative in clinical practice when iron deficiency is often complicated by concurrent inflammatory or chronic disorders. In these situations, as well as for the assessment of patients with iron overload, the best discrimination is obtained from combined measurements of serum iron concentration and the total iron-binding capacity (TIBC), which is a measure of both unsaturated and saturated transferrin — expressed in terms of iron concentration. From these two measurements one is able to calculate the percent transferrin saturation, a sensitive index for iron overload. The measurement of TIBC involves first adding excess iron, usually as ferric ion, to the serum specimen and allowing the iron to bind to (e.g., saturate) all the available sites on the iron-binding proteins. Then the free (unbound) iron that remains is removed, by absoφtion or adsoφtion, and the total iron which remains bound is measured. Each step of this process causes some dilution of the serum, but it has been shown that a threefold dilution in the determination of TIBC is advantageous, since this brings the iron-saturated transferrin into the same concentration range as the original serum iron concentration. This allows better control of the instrument and chemistry parameters.

The typical absorants/adsorbants that have been used to remove excess ferric ion, without stripping transferrin-bound iron, include magnesium carbonate, amberlite resin, and activated alumina. These all usually require centrifugation, which yields a supernate that contains the protein-bound iron for analysis. N popular modification uses dry alumina columns for adsoφtion. In this format, the diluted serum, containing excess iron, is poured onto the column and the eluate is collected for TIBC analysis. Summary of the Invention Accordingly, in one aspect, the invention features a gel particle that includes a scavenger, a magnetically responsive particle (MRP) and a gel. The scavenger can be, for example, a cell, a receptor, charcoal, silica or alumina. The scavenger and the MRP are disposed within the gel.

A scavenger is a particle that can have an analyte (e.g., DNN, a polynucleotide, a protein, a metalloprotein, a metal ion, or a nucleoside acid, such as cyclic AMP, cyclic GMP or their intracellular messengers) attach thereto. Examples of scavengers include a cell, a receptor, charcoal, silica and alumina. N gel particle is a particle that includes a gel having a scavenger and an MRP disposed within the gel (e.g., the scavenger and the MRP are encapsulated in the gel, which is a shape-retaining gel).

Within the gel particles, the scavenger may or may not be attached (e.g., chemically bound) to the MRP. Examples of gels include polyacrylamide, agarose and alginate. Gels can be gelled or cross-linked.

Gel particles can be formed by adding a scavenger and an MRP to a solution of liquefied gel (e.g., sodium alginate) to form a suspension, and then expanding the suspension through an air jet. Gel particles can also be formed using emulsification, dripping and Rayleigh jet. Methods of preparing a gel particle containing a cell are disclosed in, for example, U.S. Patents No. 4,701,326; 5,567,451; and 5,846,530.

In another aspect, the invention features a gel particle that includes a scavenger, an MRP and a gel. The scavenger and the MRP are disposed within the gel. The gel should have a porosity that allows one or more desired analytes through while not allowing undesired analytes through. The particular undesired analytes will vary depending upon the desired analyte(s). Examples of desired analytes include DNA, a protein, a metalloprotein or a metal ion. Examples of undesired analytes include cellular debris, cell walls, proteins or antibodies. In some embodiments, a desired analyte can be a metal ion (e.g., iron ion), and an undesired analyte can be the desired analyte bound to a protein (e.g., a metal ion, such as an iron ion, bound to a protein, such as transferrin). In certain embodiments, a desired analyte is iron ion, and an undesired analyte is iron-bound transferrin. The porosity of gel can be, for example, about 1-1000 nanometers (mn) (e.g., about 20-200 mn, or about 200-500 nm).

In other aspects, the invention features a sample (e.g., from a patient) interacting with one of these gel particles.

In a further aspect, the invention features a method of removing an analyte from a sample. The analyte can be, for example, a protein, DNA, a metal ion, a metalloprotein, a steroid, or a metal-containing analyte. The method includes contacting the sample with a gel particle that a magnetically responsive particle. The method also includes removing the analyte from the sample.

Methods, reagents, and reaction mixtures of the invention provide advantageous methods of determining TIBC. TIBC methods of the invention can (a) eliminate centrifugation, (b) be rapid, (c) use small amounts of serum (e.g,. less than about 500 uL), and/or (d) can be adapted to on-line automation or semi-automation.

Nil publications, patent applications, and patents cited herein are incoφorated by reference. Other features and advantages of the invention will be apparent from the following description of the figures, detailed description, and from the claims.

Brief Description of the Figures Fig. 1 is a graph of a regression analysis of a correlation study; and Fig. 2 is a graph of a regression analysis of a linearity study.

Detailed Description Sample

The preferred sample is serum, but plasma can also be used. Suitable sample sizes can be less than 1,000 microliters (e.g., less than about 500 microliters, such as about 200 microliters or 100 microliters). They can be 10-500 microliters (e.g., 5-10 microliters, or 50-75 microliters). The sample can be from a patient (e.g., a human patient) or from a culture (e.g., tissue culture or fermentation process. Sequestration

Embodiments of the invention can allow, for example, the determination of TIBC in a sample, without a centrifugation step.

The free iron in a sample can be sequestered by contacting the sample with a gel particle containing a scavenger (e.g., alumina) and an MRP. The gel particle can serve to separate the sample into a TIBC phase and a phase which includes sequestered Fe. The partition into phases can be effected without centrifugation and/or without removing either phase from the container which holds the sample. Scavengers

Scavengers should be capable of binding an analyte of interest. Scavengers include cell receptors, ion absorbers (e.g., alumina, silica or charcoal), metal complexing agents (e.g., crown ether, and dendrimers) and ion exchange resins. Other scavengers include polyacrylic acid, deferoxamine and other iron-binding iminocarboxylic acids (e.g., EDTN, EGTA, ΝTA and/or HEDTN), and other iron- binding carboxylic acids (e.g., tartaric acid, malonic acid, succinic acid, citric acid and/or tricarballic acid). For embodiments in which the analyte is iron, the scavenger does not need to be specific for iron as long as it absorbs Fe^"1-1" and/or Fe^4"^. In certain embodiments, the scavenger can be neutral grade alumina, such as commercially available from Scientific Adsorbents, Inc. (Atlanta, GA), J.T. Baker (Phillipsburg, NJ), or Sigma Chemical Co. (St. Louis, MO). Magnetically Responsive Particles (MRP's) MRP's are particles which respond to a magnetic field. In embodiments in which iron is the analyte, MRP's which include an iron binding ligand can be included in the gel particle. The free iron passes through the gel, and is bound to the MRP's. A magnetic field is applied to the sample to partition the gel particles (e.g., by bringing them to the bottom of the sample container) and to form a phase which is free of MRP's, the TIBC phase. The iron in the TIBC phase (e.g., supernatant phase) is then measured.

An MRP can be made by the entrapment or precipitation of iron, nickel or cobalt salts in the particle rendering the particle paramagnetic. The particles should not release or leach free iron. A suitable example is a particle made from styrene co- polymerized with acrylic acid in the presence of an iron, nickel or cobalt salt which are encapsulated with an additional co-polymerized surface layer. Similar particles are available commercially as ESTAPOR^® supeφaramagnetic particles. Another suitable example is a particle is polyacrolein polymerized with precipitated metal salts (e.g., iron salts, such as magnetite), and then ground to size. Such particles are commercially available from, for example, Cortex Biochem, Inc., under the trademark MagAcrolein . Other suitable particles are commercially available from Polysciences, Inc. (Warrington, PN, trademark PolyMag^® and Bangs Laboratories (Fishers, IN).

Other suitable particles are those which are the product of the polymerization of monomers such as methacrylate, divinylbenzene, acrylamide, acrylic acid, or polyethylene in various combinations and proportions. Polymerization should be performed in the presence of iron, nickel or cobalt salts and an additional polymerization step should be used to encapsulate the particles.

Other MRP's include particles of cellulose, agarose (e.g., type IN agarose, commercially available from Sigma Chemical Co., located in St. Louis, MO, or commercially available from FMC Bioproducts, located in Rockland, ME), or other polysaccharides with an iron, nickel or cobalt salt precipitated within the particle. These particles can be washed in strong acid to remove surface iron, nickel or cobalt which could leach from the particle. An example of such a particle is sold by Cortex under the name MAGCELL^®.

Iron, nickel or cobalt can also be covalently attached to a particle. Iron chelating polymers can be used as iron binding ligands in methods of the invention. U.S. Patents No. 5,487,888 and 3,899,472 describe a variety of chelating polymers. These polymers can be used to encapsulate magnetic particles for use in methods described herein. In certain embodiments, the particle is preferably less than about 1 ,000 microns in diameter, and more preferably less than about 10 microns, or less than about 1 micron in diameter. In some embodiments, the particle is about 1 micron in diameter. Measurement of Iron

Iron in a sample can be measured (e.g., with a colorometric method). For example, the sample can be reacted with a dye (e.g., ferene or ferrozine, or other pyridyl or phenanthroline dyes which form colored complexes with iron).

Determination of TIBC

The following method can be used to measure TIBC.

(1) Mix a solution containing Fe at a concentration sufficient to saturate the iron binding proteins in the solution with serum. Under these conditions the added iron binds to the serum iron binding proteins to "saturate" any available binding sites;

(2) incubate for five minutes;

(3) add a slurry of gel particles;

(4) incubate for five minutes;

(5) put a magnet into contact with the sample tube, and the iron which did not attach to the serum proteins (which is sequestered by the gel particles) is pulled from suspension/solution; and

(6) measure the supernatant Fe to determine the TIBC of the sample. The method need not be performed in any particular sequence of steps.

For example, the Fe solution can be added to the sample (e.g., serum) first, and allowed time to saturate all the available sites. After this step, the gel particles can be added to remove excess iron, the sample can be incubated, a magnetic field can be applied, and the measurement of supernatant iron can be made. In a preferred embodiment, the gel particles and high Fe concentration are mixed, and used as a single reagent. The sample (e.g., serum) is added to this reagent and allowed time for the iron to transfer to the available sites on the serum proteins, a magnetic field is applied, and a measurement of supernatant iron is made. In this embodiment, the affinity of iron for the sites in the sample (e.g., serum protein sites) is much greater than the affinity of iron for the solid-phase ligand.

In another embodiment, the gel particles and the sample (e.g., serum) are first combined, then the high Fe concentration solution is added (wherein the serum proteins and the gel particles compete for the excess iron sample), allowed to incubate, a magnetic field applied, and a measurement of supernatant iron made. The following examples are illustrative and not be construed as limiting. Examples In the following examples, the alumina was neutral grade alumina purchased from Scientific Adsorbents, Inc. (Atlanta, GN), the agarose was type IN purchased from Sigma Chemical Co. (St. Louis, MO), and the paramagnetic particles were MagAcrolein^® polyacrolein paramagnetic particles purchased from Cortex Biochem, Inc. (San Diego, CA). Correlation Study

Experiments were conducted to obtain a comparative evaluation between the magnetic separation TIBC methodology and the alumina column separation TIBC methodology. 103 patient samples were taken, spanning from 107 to 570 ug/dL TIBC per patient.

The magnetic separation TIBC methodology used an iron saturating reagent that contained 24 mg/L ferric chloride (i.e., 800ug/dL Fe ^) in 1 milliMolar (mM) citric acid solution (Reference Diagnostics product number 4021) and a magnetic separation reagent that contained 300 mg/mL magnetically responsive alumina particles in 20mm MOPS buffer, pH 7.5 (Reference Diagnostics product number 4022).

The alumina column separation TIBC methodology used an iron saturating solution that contained not less than 500 ug/dL in 1mm citric acid solution and disposable columns that were prefilled with 300mg of alumina (J&S Medical Associates, Inc., product numbers ICM 117 and ICM 127).

Serum iron reagents and a calibrator were used with ferene chromogen at 40 niM (DMA, Inc. product number 1580-300).

All iron measurements we performed on a Roche COBAS Mira Plus chemistry analyzer using the DMA serum iron reagent.

A regression analysis of the results was performed for an objective correlation. The results of the regression analysis are shown in Fig. 1 (y = 1.02x - 5.7; with a correlation coefficient of 0.99).

Results

Precision Study

A study was conducted to test the precision (both within run and between runs) of the magnetic TIBC method. Precision was evaluated with quality control materials at two TIBC concentrations. All iron measurements were performed on the Roche COBAS Mira Plus chemistry analyzer using DMA iron reagents.

With run precision was determined by performing 25 magnetic separations for each control and measuring the iron content of each supernate (all in the same run). For an average level of 314 ug/dL, the standard deviation was 6.1 ug/dL and the coefficient of variation (c.v.) was 1.9%. For an average level of 158 ug/dL, the standard deviation was 4.5 ug/dL and the c.v. was 2.8%.

Between run precision was determined by performing a magnetic separation for each control 25 different times over about two weeks, and each time the iron content of the two supernatant solutions were measured in a different run. For an average level of 302 ug/dL, the standard deviation was 13.2 ug/dL and the c.v. was 4.4%. For an average level of 155 ug/dL, the standard deviation was 10.3 ug/dL and the c.v. was 6.6%. Linearity Study

A study was conducted to determine whether a linear response exists across the expected range of TIBC measurements.

Two serum specimens, at the high and low ends of the typical patient distribution range, were each measured using the magnetic TIBC method. They

were then combined in different proportions to produce four additional samples. The proportions (high/low) were: 80/20; 60/40; 40/60; and 20/80. The samples were also measured using the magnetic alumina TIBC method and compared to the expected TIBC concentrations. The results are shown in Fig. 2 (y = 1.01x + 7.4; with a correlation coefficient of 0.999).

Interference Study

An interference study was conducted to determine whether certain substances interfered with the magnetic TIBC measurements.

Bilirubin, hemoglobin, and ascorbic acid were added to samples in various amounts to determine whether these substances caused significant interference. Triglyceride interference was determined by diluting a high triglyceride specimen (Tg=1466 mg/dL, TIBC=340 ug/dL) with a specimen that had much lower triglyceride and TIBC concentrations (Tg=72 mg/dL, TIBC=94 ug/dL). The two specimens were mixed in varying proportions (80/20; 40/60; 60/40; and 20/80) to yield four additional samples with intermediate triglyceride and TIBC concentrations. Each sample was tested with the magnetic TIBC method and the results were compared to calculated (expected) TIBC values.

These tests showed that the magnetic TIBC method is not interfered by: bilirubin at a level of up to at least 35 mg/dL; ascorbic acid at a level of up to at least 50 mg/dL; hemoglobin at a level of up to at least 200 mg/dL; and triglyceride at a level of up to at least 1450 mg/dL.

Other embodiments are in the claims.

What is claimed is:

Claims

1. A gel particle, comprising: a scavenger selected from the group consisting of alumina and silica; a magnetically responsive particle; and a gel, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

2. The gel particle of claim 1, wherein the scavenger is alumina.

3. The gel particle of claim 1 , wherein the scavenger is silica.

4. The gel particle of claim 1 , wherein the gel has a porosity of from about 10 nanometer to about 1000 nanometers.

5. The gel particle of claim 1 , wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

6. A gel particle, comprising: a scavenger; a magnetically responsive particle; and a gel having a porosity of from about 10 nanometers to about 1000 nanometers, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

7. The gel particle of claim 6, wherein the gel has a porosity of from about 20 nanometers to about 200 nanometers.

8. The gel particle of claim 6, wherein the gel has a porosity of from about 200 nanometers to about 500 nanometers.

9. The gel particle of claim 6, wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

10. A composition, comprising: a sample; and a gel particle, comprising: a scavenger selected from the group consisting of alumina and silica; a magnetically responsive particle; and a gel, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

11. The composition of claim 10, wherein the scavenger is alumina.

12. The composition of claim 10, wherein the scavenger is silica.

13. The composition of claim 10, wherein the gel has a porosity of from about 10 nanometer to about 1000 nanometers.

14. The composition of claim 10, wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

15. A composition, comprising: a gel particle, comprising: a sample from a patient; a scavenger; a magnetically responsive particle; and a gel having a porosity of from about 10 nanometers to about 1000 nanometers, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

16. The composition of claim 15, wherein the gel has a porosity of from about 20 nanometers to about 200 nanometers.

17. The composition of claim 15 , wherein the gel has a porosity of from about 200 nanometers to about 500 nanometers.

18. The composition of claim 15, wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

19. A method of removing an analyte from a sample, comprising: contacting the sample including the analyte with a gel particle comprising a magnetically responsive particle; and removing the analyte from the sample.

20. The method of claim 19, wherein the analyte comprises a metal- containing analyte.

21. The method of claim 20, wherein the metal-containing analyte comprises a metal ion or a metalloprotein.

22. The method of claim 20, wherein the metal-containing analyte comprises an iron ion.

23. The method of claim 19, wherein the analyte is removed from the sample by exposing the sample to a magnetic field.

24. The method of claim 19, wherein the analyte comprises DNA, a polynucleotide or a protein.

25. The method of claim 19, wherein the gel particle comprises: a scavenger selected from the group consisting of alumina and silica; a magnetically responsive particle; and a gel, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

26. The method of claim 25, wherein the scavenger is alumina.

27. The method of claim 25, wherein the scavenger is silica.

28. The method of claim 25, wherein the gel has a porosity of from about

10 nanometer to about 1000 nanometers.

29. The method of claim 25, wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

30. The method of claim 19, wherein the gel particle, comprises: a scavenger; a magnetically responsive particle; and a gel having a porosity of from about 10 nanometers to about 1000 nanometers, wherein the scavenger and the magnetically responsive particle are disposed within the gel.

31. The method of claim 30, wherein the gel has a porosity of from about

20 nanometers to about 200 nanometers.

32. The method of claim 30, wherein the gel has a porosity of from about 200 nanometers to about 500 nanometers.

33. The method of claim 30, wherein the gel comprises a material selected from the group consisting of polyacrylamide, agarose and alginate.

34. The gel particle of claim 1, wherein the scavenger is not attached to the magnetically responsive particle.

35. The gel particle of claims 1, wherein the scavenger is not chemically bound to the magnetically responsive particle.

36. The composition of claim 10, wherein the scavenger is not attached to the magnetically responsive particle.

37. The composition of claim 10, wherein the scavenger is not chemically bound to the magnetically responsive particle.

38. The method of claim 25, wherein the scavenger is not attached to the magnetically responsive particle.

39. The method of claim 25, wherein the scavenger is not chemically bound to the magnetically responsive particle.

40. The gel particle of claim 1, wherein the gel has a porosity sufficient to allow a metal ion to pass therethrough without allowing the metal ion bound to a protein to pass therethrough.

41. The gel particle of claim 10, wherein the gel has a porosity sufficient to allow a metal ion to pass therethrough without allowing the metal ion bound to a protein to pass therethrough.

42. The method of claim 25, wherein the gel has a porosity sufficient to allow a metal ion to pass therethrough without allowing the metal ion bound to a protein to pass therethrough.

43. The method of claim 19, wherein the analyte comprises an nucleoside.

44. The method of claim 43, wherein the nucleoside is selected from the group consisting of c AMP and cGMP.