WO2012137202A1 - Composition comprenant un indicateur de fer attaché à une microparticule et utilisations de celle-ci pour quantifier du fer non lié à la transferrine (ntbi) et du fer labile cellulaire (lci) - Google Patents

Composition comprenant un indicateur de fer attaché à une microparticule et utilisations de celle-ci pour quantifier du fer non lié à la transferrine (ntbi) et du fer labile cellulaire (lci) Download PDF

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WO2012137202A1
WO2012137202A1 PCT/IL2012/050119 IL2012050119W WO2012137202A1 WO 2012137202 A1 WO2012137202 A1 WO 2012137202A1 IL 2012050119 W IL2012050119 W IL 2012050119W WO 2012137202 A1 WO2012137202 A1 WO 2012137202A1
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iron
composition
matter
levels
indicator
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PCT/IL2012/050119
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English (en)
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Ioav Zvi Cabantchik
William Breuer
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2012137202A1 publication Critical patent/WO2012137202A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood

Definitions

  • the present invention in some embodiments thereof, relates to a composition comprising a iron indicator attached to a microparticle and uses of same for quantifying labile iron in biological fluids, often identified with non-transferrin bound iron (NTBI) (which is considered non-labile).
  • NTBI non-transferrin bound iron
  • Systemic iron overload is a pathological condition characterized by persistently high levels of iron in plasma and interstitial fluids. In that condition, the levels of iron in the circulation exceed by > 70% the binding capacity of transferrin generating chemical forms identified as non-transferrin bound iron (NTBI).
  • NTBI non-transferrin bound iron
  • plasma NTBI might be considered a marker of systemic iron overload per se, more importantly, it might serve as indicator of impending organ iron accumulation and ensuing damage. This is because some forms of NTBI present in plasma over extended periods of time can infiltrate cells and cause tissue iron to attain levels that override cellular antioxidant capacities and thereby affect vital functions, leading to oxidative cell damage and death.
  • Clinical management of systemic iron overload requires sensitive and simple diagnostic tests that reflect the level of patients' overall iron burden, but more importantly the component of iron directly implicated in cell toxicity, i.e. labile iron.
  • the most commonly used indicators for assessing systemic iron overload have thus far been serum ferritin levels (in some cases in conjunction with transferrin saturation) and T2* MRI (and/or biopsies when possible) for assessing end organ accumulation . These indicators are mostly associated with iron accumulated over extended periods of time, with serum ferritin reflecting iron accumulated primarily in the liver and spleen.
  • Plasma NTBI may be conceived as an early indicator of systemic iron overload and some of its components as indicators of chelation efficacy.
  • NTBI is detected in plasma
  • NTBI appears in various chemical forms, depending on the origin and degree of iron overload, the history of blood transfusions, bone-marrow transplantation and treatment (chelation or phlebotomy or chemotherapy). At present it is not clear which of the NTBI components is of pathophysiological relevance as marker of systemic iron overload and/or as a source of tissue iron overload and ensuing toxicity.
  • NTBI tissue iron overload
  • the chemical nature of the permeating substrate namely, the NTBI forms in plasma/interstitial fluids in the various pathological conditions and the various "opportunistic" membrane routes through which the NTBI forms can gain cellular access.
  • those routes differ among cells types, both qualitatively and quantitatively, their relative contribution to tissue iron overload will vary accordingly. This is best exemplified in the differential loading of parenchymal versus Kupfer cells in hemochromatosis versus thalassemia major or the ostensible lack of cardiac iron loading in thalassemia intermedia versus thalassemia major, despite the high levels of plasma NTBI.
  • Plasma NTBI is comprised of both organic and inorganic complexes, some non- specifically associated with plasma proteins.
  • the relevant plasma NTBI permeating components are either: a. the small and "free" ligand-metal, in which case ingress might require metal reduction by membrane associated reductases followed by translocation or diffusion of the free-iron via putative transporters or channels (that can handle only free or hydrated divalent ions) or b. the iron complex, in which case more complex machineries that can handle iron-ligands should be implicated.
  • the major permeating species are the protein-bound iron complexes
  • their tissue ingress would be limited to bulk endocytotic routes (mostly adsorptive endocytosis) followed by intracellular metal release and translocation from vesicles to cytosol.
  • a serial or parallel combination of the above mechanisms, bulk and ionic transport can possibly account for the handling of all NTBI forms by different tissues in the different forms of iron overload.
  • the relative contribution of the operative ingress routes of NTBI into cells might differ in different tissues, depending on the available repertoire of transporters/channels vis a vis the NTBI forms present at a particular site. Determination of NTBI.
  • NTBI appears in plasma in multiple forms, some bound to small organic ligands which, in turn, might be free (filterable) or adsorbed to proteins, some cryptic or occluded to chelators and partially accessible to cells. These forms might change in the same patient depending on the time of sampling vis a vis its patho-physiological status and current treatment regimen. Because of technical difficulties in revealing the apparently cryptic NTBI in plasma/ serum (protein-bound complexes that are not easily accessible/chelatable), most of the published NTBI assays rely on strategies that attempt to "differentially" extract NTBI from macromolecular plasma/sera components and render the extract filterable and/or detectable by analytical chelating agents.
  • NTBI extracted from plasma/serum is not derived from highly saturated TBI (plasma TBI at full saturation ranges 40-50 ⁇ and NTBI is generally less than 1/10 th that value).
  • analysis is preceded by storage of frozen sera (or heparinized plasma) under conditions that, ideally, should preserve both the TBI and the NTBI components and particularly their possible deterioration or inter-conversion (during storage or analysis).
  • LPI labile plasma iron
  • DCI directly chelatable iron
  • NTBI labile cell iron
  • NTBI assays were designed to reveal all forms of non TBI, a goal attained generally by adding strong mobilizing agents that can extract all NTBI forms, the easily accessible and the "cryptic" ones, followed by filtration and chemical analysis of the extracted material (by standard colorimetric assays, ICP-MS or in combination with HPLC ).
  • LPI and DCI assays were designed to reveal labile forms of iron in fluids while avoiding potential complications associated with very high concentrations of iron- mobilizing agents.
  • the advantage of DCI and LPI assays is their ability to provide a measure for clinical efficacy of chelation/ phlebotomy in reducing/eliminating NTBFs labile/ chelatable component.
  • the assays are based on reading of fluorescence by high throughput devices, designed to be non laborious and suitable for all bio fluids.
  • a potential limitation of the existing LPI and DCI assays is incomplete detection of some NTBI complexes with low redox-activity and/or limited accessibility of the iron-detector agent to some chenical forms bound/adsorbed to proteins.
  • composition-of-matter comprising an iron indicator attached to a microparticle, wherein the iron indicator comprises an iron binding moiety and a signal generating moiety, wherein an intensity of the signal generated by the signal generating moiety is related to an amount of the iron bound be the iron binding moiety.
  • the iron binding moiety and the signal generating moiety are covalently attached forming a single molecule.
  • the iron binding moiety and the signal generating moiety are distinct molecules.
  • the iron binding moiety comprises deferoxamine (DFO).
  • the iron indicator comprises dihydrorhodamine 123 (DHR) or carboxydihydrofluorescin (CDCF).
  • the iron indicator is a cell permeant iron indicator.
  • the iron indicator comprises calcein.
  • the iron indicator is selected from the group consisting of calcein, deferiprone, deferasirox and salicyl-aldehyde hydrazone (SIH).
  • the iron binding moiety comprises a modified apo-transferrin.
  • the iron indicator is fluorescein-apo-transferrin.
  • the signal generating moiety is a fluorophore.
  • the fluorophore is selected from the group consisting of Fluorescein, Rhodamine, nitrobenzfurazan, fluorogenic ⁇ - galactosidase and a green fluorescent protein.
  • the iron binding moiety is selected from the group consisting of apo-transferrin, lactoferrin, ovotransferrin, desferoxamine, phenanthroline, ferritin, porphyrin, EDTA and DPTA.
  • the iron indicator comprises an enzyme
  • the enzyme is an aconitase enzyme.
  • the microparticle has a weight similar to, or lower than a mammalian cell.
  • the microparticle is selected from the group consisting of a polystyrene bead, a silica bead or an agarose bead.
  • the iron indicator is conjugated directly to the microparticle.
  • the iron indicator is conjugated indirectly to the microparticle.
  • the indirect conjugation is via an adhesive protein.
  • the adhesive protein comprises a histone or an albumin.
  • the indirect conjugation is via an extension arm.
  • the extension arm is composed of a dendrimer and a multifunctional bridge.
  • a method of quantifying free iron levels in a sample comprising:
  • the free iron comprises non- transferrin bound iron.
  • the free iron comprises directly chelatable iron.
  • the free iron comprises labile plasma iron.
  • the iron indicator comprises fluorescein and defemoxamme.
  • the fluorescein and deferoxamine are covalently attached to form fluorescinated defemoxamme (Fl-DFO).
  • the iron indicator comprises dihydrorhodamine 123 (DHR)
  • said iron indicator comprises rhodamine(R) and deferrioxamine.
  • said rhodamine and deferoxamine are covalently attached to form R-deferrioxamine (R-DFO).
  • the method further comprising:
  • step (d) detecting and quantifying the signal of step (C), the signal being the background signal of the chelation reaction.
  • the iron binding moiety in excess is identical to the iron binding moiety of the iron indicator.
  • a method of calibrating free iron levels in a fluid sample comprising:
  • the method further comprising contacting the plurality of samples with an iron chelator so as to release the iron binding moiety following step (b); and detecting and quantifying a signal' generated following contact with the iron chelator, and wherein the calibration curve depicts the change in the signal vs the signal against the predetermined distinct free iron concentration values.
  • a method of quantifying labile cellular iron in a biological sample comprising:
  • step (b) contacting loaded cells of step (a) with a cell permeant metal chelator so as to remove the iron from the iron binding moiety; (c) recording a signal generated in (a) and in (b), wherein the mean change in signal intensity is indicative of a level of labile cell iron;
  • the iron indicator comprises calcein.
  • a biological fluid comprising:
  • the reducing agent is ascorbic acid, dithionite, dithiothreitol and mercaptoacetic acid.
  • the detector molecule is selected from the group consisting of dihydrorhodamine, dihydrorhodamine, carboxy- dihydrofluorescein and dihydroresorufin.
  • the redox active iron reaction products are reactive oxygen species.
  • a method of determining a presence, absence or risk of a disorder associated with abnormal levels of free iron in a biological fluid or cells of a subject comprising:
  • the medicament comprises an iron chelation therapy.
  • the medicament comprises iron chelation therapy, and whereas a reduction in the levels following the therapy is indicative of efficacious treatment.
  • the medicament is selected from the group consisting of oral iron supplementation, folic acid, vitamin B-12, erythropoietin, blood transfusion and hyperbaric oxygen, and whereas an increase in the levels following the therapy is indicative of efficacious treatment.
  • kits for determining a presence of a disorder associated with abnormal levels of free iron in a biological sample of a subject comprising the composition of matter.
  • the kit further comprising at least one of a mobilizing agent and a compound devoid of iron and being capable of binding endogenous apo-Transferrin
  • the compound comprises gallium or cobalt.
  • the mobilizing agent is selected from the group consisting of sodium-oxalate, nitrilotriaacetate, ascorbate and salicylate.
  • FIGs. 1A-B are schematic illustrations showing fluorescence metal sensors on beads (FMSB) as probes for labile iron.
  • FMSB containing fluorescein-DFO (FDFO) or calcein green (CALG) undergo fluorescence quenching when reacting with labile iron and dequenching when treated with a strong chelator.
  • Plasma or other fluids containing labile Fe will react accordingly, with or without the addition of a mild metal mobilizer (e.g., nitrilotriacetic acid ⁇ ImM or deferiprone ⁇ 30 ⁇ ).
  • a mild metal mobilizer e.g., nitrilotriacetic acid ⁇ ImM or deferiprone ⁇ 30 ⁇ .
  • the sample fluorescence is read by a flow cytometer ( Figure IB) after 60 minutes incubation at room temperature; a parallel (for CALG also sequential) assay is carried out with beads in the presence of excess (0.1 mM) deferoxamine (DFO) which demonstrably prevents (for CALG also removes) Fe from binding to the respective FMS; in the last step, the iron is quantitated using a calibration curve.
  • DFO deferoxamine
  • FIG. 2 is a schematic illustration of the construction of the FMSB and some of its chemical properties.
  • FIGs. 4A-B are schemes showing FMSB as probes for labile iron. Measurement of plasma NTBI as directly chelatable iron (DCI) by flow cytometry (FC).
  • Figure 4 A Fluorescein+DFO coated beads DenB(F+D) used for quantitative assessment of DCI by flow cytometry.
  • Figure 4B Rhodamine+DFO coated beads DenB(R+D) used for quantitative assessment of DCI by flow cytometry.
  • FIGs. 5A-C are schematic illustrations showing measurement of LCI in blood cells.
  • Figure 5A - is an image of a CALG bead generated according to the teachings of the present invention.
  • Figure 5B - In a first step, blood or other cells are loaded with calcein-green -acetomethoxy-ester (CALG-AM) for 10' at room temperature (that leads to the intracellular generation of free CALG and binding of LCI). Cells are washed with buffered saline and in the next step the sample is analyzed by flow cytometry before and after addition of a permeant chelator. Addition of the chelator causes a rise in fluorescence AF which is proportional to LCI.
  • CALG-AM calcein-green -acetomethoxy-ester
  • FIG. 5C - demonstrates the conversion of fluorescence intensity shift AF into concentration of LCI using CALGB.
  • a specific carrier molecule labeled with the fluorescence metal sensor (FMS) CALG is attached to polystyrene beads of 3-6 ⁇ mean diameter. These beads are used to simulate CALG loaded cells for flow cytometry.
  • FIG. 6 shows a schematic illustration of the method of measuring DCI and LCI according to the teachings of some embodiments of the invention.
  • the present invention in some embodiments thereof, relates to a composition comprising a iron indicator attached to a microparticle and uses of same for quantifying non-transferrin bound iron (NTBI) and as reference for quantifying LCI in circulating blood and other cells.
  • NTBI non-transferrin bound iron
  • Non-transferrin bound iron is a marker of systemic iron overload and an indicator of impending organ damage.
  • the present inventor has previously devised compositions and assays for measuring free iron levels using indicator molecules composed of an iron binding moiety and a signal generating moiety wherein an intensity of the signal generated by the signal generating moiety is stochiometrically related to the amount of the iron bound by the iron binding moiety.
  • the present inventor has generated diverse assay conditions which make use of microparticles coated with the above-mentioned indicator molecules for recruiting chelatable iron from biofluids while concentrating the chelatable iron (by binding with high affinity) from diluted samples, overcoming media impurities, the major obstacle in presently available assays.
  • the beads are read selectively in a flow cytometer (or equivalent instrument) with changes in signal reflecting changes in chelatable iron.
  • the sensitivity of the method is 10-20 fold higher than the presently available ones and has a wide range of applications beyond plasma and serum, especially to urine and cerebrospinal fluid to which there is a growing interest and increasing demand.
  • the microparticle (also termed herein "bead”) technology is of a special advantage in the following three modalities:
  • the bead microspheres are constructed of derivatized polystyrene or silica (by derivatization it is meant a bead covalently bound to dendrimers (PAMAM generation 1- 3) onto which: a. fiuoresceinated -DFO is coupled covalently (via the free carboxyl of fluorescein, while the DFO hydroxamates are protected via prior metal chelation) or b.
  • RITC rhodamine isothiocyante
  • FITC fluorescein-isothiocyanate
  • DFO-ITC DFO-ITC
  • ITC isothiocyanate
  • CALG is coupled (via the free carboxyl of fluorescein, while the CALG carboxyl-metal binding groups are protected via prior metal chelation) .
  • CALGB only the DFO recoverable fluorescence is taken as a measure of DCI.
  • the same approach of using a mobilizing agent to extract all chelatable forms of iron can be used for extracting labile iron form intact cells (like red or white blood cells) with the permeable chelator deferiprone as a mobilizer (30-100 micromolar for 1- 3 h ) and assessing the extracted iron in the medium (after cell separation) by DCI in a fluorescence plate reader or DCIB [F+D)B or (R+D)B] in a flow cytometer.
  • This procedure makes use of the high iron affinity of desferoxamine, which enables ready transfer of deferiprone-bound iron to the detector moiety used in DCI or DCIB.
  • LPI in fluids LPIB
  • the beads or microspheres are constructed as described using either DHR (dihydrorhodamine) or dichloro-dihydro fluorescein, coupled to protein and the protein coupled to the beads.
  • DHR dihydrorhodamine
  • dichloro-dihydro fluorescein coupled to protein and the protein coupled to the beads.
  • a possible variation on this technique would involve incubation of the protein-bound probe with the bio-fluid followed by attachment of the probe to beads.
  • An example could be soluble avidin-conjugated probe, which is incubated with the bio-fluid and then quantitatively trapped on biotinylated beads after completion of the reaction.
  • LPI on beads LPIB is detected as a time dependent rise in fluorescence prompted by ascorbate and blocked by deferoxamine. The rise in fluorescence after a predetermined period of time (20 minutes at room temperature) is proportional to the level of labile iron.
  • LCI LCI in cells by flow cytometry
  • the labile cell iron content is indicative of cumulative events of exposure to LPI. Those are also manifested in circulating blood cells as a raise in LPI.
  • LPI is determined in cells with the probe calcein CAL (green or blue, CALG and CALB, respectively) which is loaded into cells as its acetomethoxy-precursor CAL-AM.
  • the intracellular CAL probe that is formed in cells from CAL-AM interacts with labile iron and undergoes quenching commensurate with the LCI levels. This is depicted for attached cells based on the outlined protocol. .
  • the key question is how to convert the fluorescence changes elicited by chelator AF into actual LCI concentrations.
  • CALG coated beads titrated with given amounts of labile iron are made and measured them the flow cytometer before and after addition of a strong chelator that can displace the CALG- bound iron (Figure 5C) .
  • the titration of the beads with iron is depicted in Figure 5C.
  • the lower left panel depicts the fluorescence distributions after addition of Fe and the upper those after addition of chelator (100 ⁇ of deferiprone, another cell permeant iron chelator).
  • a plot of the weighted reciprocal change in fluorescence vs Fe was linear for a wide range of Fe concentrations, allowing conversion of shifts in fluorescence of CAL-laden cells elicited by chelators into concentrations.
  • Figure 6 provides a general description of quantifying DCI and LCI using by flow cytometry, as applied to CALG.
  • Labile iron binds to fluorescent bead and quenches its fluorescence (F) by an amount AF.
  • the changes in fluorescence (AF) are stoichiometric, namely 1 : 1 with Fe binding and the latter proportional to the the # of atoms of labile iron in solution.
  • AF should be prevented/recovered in the presence of a strong but specific Fe chelator (e.g. excess DFO for measurements of directly chelatable iron (DCI) in fluids and for calibration of measurements of labile cell iron (LCI) with CALG using CALG-beads.
  • a strong but specific Fe chelator e.g. excess DFO for measurements of directly chelatable iron (DCI) in fluids and for calibration of measurements of labile cell iron (LCI) with CALG using CALG-beads.
  • composition-of- matter comprising an iron indicator coating a microparticle, wherein the iron indicator comprises an iron binding moiety and a signal generating moiety, wherein an intensity of the signal generated by the signal generating moiety is related to an amount of the iron bound by the iron binding moiety.
  • the iron binding moiety and the signal generating moiety might be contiguous in the same molecule (i.e., covalently attached to form a single molecule) or distinct molecules bound on separate, but proximal sites of a polymer or a multimer such as a dendrimer (see Figure 3).
  • iron binding moiety refers to an iron chelator, which binds to or combines with iron ions, including all synthetic and natural organic compounds known to bind iron, and any molecule of biological origin, or by-product or modified product of a molecule of biological origin, such as proteins, sugars or carbohydrates, lipids and nucleic acids, and any combination thereof, that may bind iron ions.
  • iron binding proteins include but are not limited to transferrin, apo- transferrin, lactoferrin, ovotransferrin, p97-melanotransferrin, ferritin, Ferric uptake repressor (FUR) proteins, calcineurin, acid phosphatase, ferredoxin. .
  • the iron binding moiety is transferrin (Tf), since iron binding to Tf or apo-Tf is significantly faster than to other known iron binding molecules, which enhances the probability that free iron (including NTBI) will bind the indicator molecule rather than to endogenous apo-Tf.
  • Tf transferrin
  • Chemical moieties which are suitable for use as the iron binding moiety of this aspect of the present invention include iron chelators.
  • an iron chelator refers to a molecule comprising nonmetal atoms, two or more of which atoms are capable of linking or binding with a iron ion to form a heterocyclic ring including the metal ion.
  • iron chelators include but are not limited to desferoxamine (DFO), phenanthroline, ethylene diamine tetra-acetic acid (EDTA), diethylene triamine- pentaacetic acid (DTPA), N,N'-bis[2-hydroxybenzoyl]ethylene diamine-N,N'-diacetic acid (HBED) and the like.
  • DFO desferoxamine
  • EDTA ethylene diamine tetra-acetic acid
  • DTPA diethylene triamine- pentaacetic acid
  • HBED N,N'-bis[2-hydroxybenzoyl]ethylene diamine-N,N'-diacetic acid
  • Other examples of iron chelators and related compounds are provided in U.S. Pat. NOs. 4,840,958, 5,480,894 4,585,780, 5,925,318 and in Hider (1996) Acta Heamatologica 95:6-12.
  • the signal generating moiety of the indicator molecule is selected such that the intensity of signal generated therefrom is related to the amount of the iron which is bound to the iron binding moiety.
  • the intensity of the signal is stoichiometrically related to the iron bound by the iron binding moiety.
  • ionic iron is its inherent ability to affect the fiuorescence properties of fluorophores when in atomic or molecular contact, usually resulting in the quenching of the fluorescence signal [Lakowicz, J.R. (1983) Principles of fluorescence spectroscopy, Plenum Press, New York, pp.266 ff].
  • the signal generating moiety is a fluorophore, which can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light.
  • a fluorophore as the signal generating moiety allows the generation of a direct correlation between changes in fluorescence and NTBI concentration.
  • the fluorophores are selected fluorescent in most channels of the flow-cytometer.
  • fluorophores suitable for use as the signal generating moiety of the present invention along with approximate absorption (Abs) and fluorescence emission (Em) is provided in Table 1 below.
  • the listed fluorophores are available from Molecular Probes (www.molecularprobes.com).
  • a fluorophore can be a protein belonging to the green fluorescent protein family including but not limited to the green fluorescent protein, the yellow fluorescent protein, the cyan fluorescent protein and the red fluorescent protein as well as their enhanced derivatives.
  • the signal generating moiety of the indicator molecule can be an enzyme which when in the presence of a suitable substrate generates chromogenic products.
  • enzymes include but are not limited to alkaline phosphatase, ⁇ - galactosidase, ⁇ -D-glucoronidase and the like.
  • a naturally occurring molecule such as an enzyme can comprise the indicator molecule of the present invention, wherein following iron binding a measurable conformational and/or a functional alteration is effected.
  • the aconitase enzyme is activated following iron binding [Klausner, R.D. et al. . (1993) Cell 72:19-28].
  • the indicator molecule is DFO e.g., fluoresceinated deferoxamine (Fl-DFO).
  • the indicator molecule is 5-4,6-dichlorotriazinyl aminofluorescein (DCTF)-apo-transferrin i.e., Fl-aTf.
  • DCTF 5-4,6-dichlorotriazinyl aminofluorescein
  • Fl-aTf 5-4,6-dichlorotriazinyl aminofluorescein
  • the iron indicator is a cell permeant iron indicator. This is beneficial for measuring cellular iron.
  • the iron indicators is selected from the group consisting of calcein, deferiprone, deferasirox (Exjade) and salicyl-aldehyde hydrazone (SIH).
  • the iron indicator is calcein (calcein- AM).
  • the indicator molecules of the present invention can be synthesized using well known chemical synthesis procedures. Detailed protocols describing how to use reactive fluorophores are available in wwwdotmolecularprobesdotcom.
  • amine-reactive fluorophores i.e., including a reactive group such as dichlorotriazinyl, isothiocyanate, succinimidyl ester, sulfonyl chloride and the like
  • amine-reactive fluorophores can be used to modify proteins, peptides and various synthetic molecules with primary amines (see wwwdotprobesdotcom/media/pis/mpOO 143.pdf).
  • unconjugated fluorophore is removed, usually by gel filtration, dialysis, HPLC or a combination of these techniques.
  • the presence of free fluorophore, particularly if it remains chemically reactive, can greatly complicate subsequent analysis with the indicator of the present invention.
  • the indicator molecules of the invention are characterized by a high fluorescence yield yet retain the critical parameters of the unlabeled iron binding moiety (i.e., iron binding).
  • the indicator molecule of the present invention can be a chimeric protein including a protein fluorophore (e.g., GFP) linked to an iron binding protein.
  • a protein fluorophore e.g., GFP
  • Such a chimeric protein can be produced via well known recombinant techniques.
  • indicator molecules can also be used according to this aspect of the present invention.
  • calceins are commercially available from Molecular Probes Inc, FL-DFO, synthesized by reacting FITC with desferoxamine (available from Evrogen, Moscow, Russian Republic) and the respective isothiocyanate derivatives of DFO (DFO-ITC) and DTPA ( DTPA -ITC) are obtainable from Macrocyclics Inc (Dallas, TX, USA), RITC and derivaives are available from Fluka (Sigma-aldrich Co St louis MO).
  • the molecule comprises DFO.
  • the iron indicator comprises dihydrorhodamine 123 (DHR) or carboxydihydro fluorescein (CDCF).
  • the iron indicator comprises calcein.
  • the molecule comprises a modified apo- transferrin.
  • the molecule comprises fluorescein-apo- transferrin.
  • the fluorophore is selected from the group consisting of Fluorescein, Rhodamine, nitrobenzfurazan, fluorogenic ⁇ -galactosidase and a green fluorescent protein.
  • the indicator molecule coats a microparticle.
  • microparticles or “beads” refers to particles having a mean diameter 0.1 to 100 ⁇ (e.g., 5-10 ⁇ ). Microparticles have a much larger surface-to-volume ratio than at the macroscale, rendering the compositions of the invention highly advantageous in terms of affinity to free iron.
  • the microparticles comprise microspheres.
  • the microparticle should preferably be made of such material and be of such size as to stay suspended, with minimal agitation if necessary, in solution or suspension (i.e., once the sample is added). It should preferably not settle any faster than cells of interest in the sample, as further described herein below.
  • the material from which the microparticles are made should be such as to avoid clumping or aggregation, i.e., the formation of doublets, triplets, quadruplets and other multiplets. This can be minimized by using mild detergents such as Triton X-100 (0.05 %).
  • Microparticles may be used in accordance with the present teachings include, but are not limited to fixed human red blood cells, coumarin beads, liposomes, cell nuclei and microorganisms.
  • particularly advantageous examples of microparticles that may be used in the invention include microbeads, such as agarose beads, polyacrylamide beads, polystyrene beads and silica gel beads.
  • microparticles for use in the methods and compositions described here include plastic microbeads. While plastic microbeads are usually solid, they may also be hollow inside and could be vesicles and other microcarriers. They do not have to be perfect spheres in order to function in the methods described here. Plastic materials such as polystyrene, polyacrylamide and other latex materials may be employed for fabricating the beads, but other plastic materials such as polyvinyl chloride, polypropylene and the like may also be used. According to a specific embodiment, the microparticles are polystyrene beads, silica beads or agarose beads.
  • the microparticle has a molecular size of 4- 10 ⁇ similar to, or lower than most mammalian cells.
  • the indicator molecules coat the microparticles.
  • the coating is homogeneous. Attachment or conjugation of the indicator molecule to the microparticle is effected such that the functionality of the indicator molecule is unharmed (that is, without compromising neither the signal intensity nor the binding to the metal binding moiety).
  • microparticle to which the iron indicator is bound may be composed of a polymeric microparticle bead onto which the iron indicator is covalently attached.
  • the binding may be direct or via a spacer layer bound to the polymer.
  • the iron indicator on the microparticle should remain quantitatively accessible to iron for quenching and, where applicable, for the iron binding moiety provided in excess for dequenching (important for elucidating the background signal);
  • a spacer (linear or branched-as in polyethylene glycols, dendrimers or globular proteins) should be introduced between the bead and the iron indicator.
  • the iron indicator is conjugated directly to the microparticle.
  • the iron indicator is conjugated indirectly to the microparticle.
  • the indirect conjugation is via an adhesive protein.
  • the adhesive protein comprises a histone or an albumin, (serum albumin, ovalbumin, lactalbumin).
  • the iron indicator is conjugated indirectly to the microparticle via an extension arm.
  • the extension arm is composed of a dendrimer or a multifunctional bridge.
  • Figure 2 describes specific configurations (non-limiting) of conjugating the iron indicator to the microparticle.
  • a specific coat labeled with the iron indicator is covalently coupled to the surface of (COOH or NH2-derivatized) polystyrene (or silica) 4-10 ⁇ mean diameter bead.
  • the iron indicator consists of probes like calcein green (CALG) or fluorescein + deferoxamine (F+DFO), which are bound covalently to the coated beads (either separately or as F-DFO conjugate)
  • the iron indicator comprise rhodamine (R)+ deferoxamine (R+DFO), which are bound covalently to the coated beads (either separately or as R-DFO conjugate).
  • Polymer bead Polystyrene, silica or the like (4-10- ⁇ diameter) with terminal primary amino or carboxyl groups.
  • PAMAM generation 1-3 a. dendrimer (PAMAM generation 1-3) or polyethylene glycol (linear or branched) coupled covalently to pre-activated microparticle, or
  • multifunctional bridge lysine or orthogonally-protected diaminobutyric acid derivative with hindered Dde variant ivDde.
  • bead-dendrimer via N-(a and ⁇ protected) -COOH
  • COOH- not involved in metal binding the -COOH groups involved in metal binding are protected during coupling with excess Co
  • F or R and DFO are covalently attached to dendrimer NH2 sites (via their -NCS or ITC- derivatives FITC or RITC and DFO-ITC) or
  • compositions of the present invention can be widely used to directly and sensitively detect free iron levels in biological fluids and inside cells, the latter also termed "labile cellular iron (LCI)".
  • LCI labile cellular iron
  • normal cellular iron levels are between about 0.2-1.5 ⁇ .
  • abnormal cellular levels are typically below about 0.2 ⁇ or above about 1.5 ⁇ .
  • free iron levels refers to non-transferrin bound iron
  • NTBI NTBI
  • NTBI refers to directly chelatable iron (DCI) which is accessible to exogenous iron chelators; mobilizer-dependent chelatable iron (MDCI) which is accessible to exogenous iron chelators upon addition of mobilizing agents; and labile plasma iron (LPI) including redox active iron, which redox activity is eliminated upon addition of exogenous iron chelators.
  • DCI directly chelatable iron
  • MDCI mobilizer-dependent chelatable iron
  • LPI labile plasma iron
  • a method of quantifying free iron levels in a sample comprising:
  • the free iron comprises non-transferrin bound iron.
  • the free iron comprises directly chelatable iron.
  • the free iron comprises labile plasma iron.
  • the iron indicator comprises fluoresceinated deferoxamine (Fl-DFO).
  • the iron indicator comprises calcein green
  • the iron indicator comprises dihydrorhodamine 123 (DHR)
  • biological sample refers to a biological fluid such as blood, serum, plasma, lymph, bile fluid, urine, saliva, sputum, synovial fluid, semen, tears, cerebrospinal fluid, bronchioalveolar large fluid, ascites fluid, pus and the like.
  • the sample may be free of cells or comprise cells. Methods of quantifying intracellular free iron levels are further described herein below.
  • a sample of the biological fluid is contacted with the composition-of- matter of the present invention.
  • Contacting is effected under conditions suitable for binding of the iron-binding moiety of the indicator molecule to free iron.
  • fluorescence detection sensitivity is oftentimes compromised by background signals, which may originate from endogenous sample constituents or from unbound or nonspecifically indicator molecules (i.e., background fluorescence).
  • background fluorescence i.e., unbound or nonspecifically indicator molecules
  • parallel samples are preferably incubated with excess unlabeled chelator molecule, which scavenges all iron in the sample.
  • the method further comprises:
  • step (d) detecting and quantifying the signal of step (C), the signal being the background signal of the chelation reaction.
  • the iron binding moiety in excess is identical to the labeled iron binding moiety (i.e., of the iron indicator).
  • the background signal is typically deduced from the signals obtained in all the samples tested.
  • the signal is thus detected and quantified to determine the levels of free iron in the biological fluid of the subject.
  • signal detection may be effected by any suitable instrumentation such as a flow cytometer e.g., FACS.
  • the intensity of signal produced may be analyzed manually or using a computer program. Any abnormality in the levels of free iron in the sample is indicative of the presence of a disorder.
  • free iron quantification is preferably effected alongside a look-up table or a calibration curve so as to enable accurate iron determination. Methods of calibrating iron levels are further described herein below.
  • Embodiments of the method of this aspect of the present invention include the use of iron mobilizing agents and removal of endogenous apo-transferrin from the sample, to accurately determine the levels of free iron in the sample.
  • the fluid sample obtained from the subject is preferably treated with a mobilizing agent, described hereinabove, prior to contacting with the compositions of the present invention.
  • mobilizing agents include but are not limited to sodium oxalate, nitrilotriaacetate, ascorbate and salicylate.
  • mobilizing reagent employed must balance the ability to detect maximal free iron levels, without contributing to iron release from iron- containing transferrin in a given sample.
  • 10 mM sodium oxalate is used as the mobilizing reagent according to this aspect of the present invention.
  • mobilizing agents or other combinations thereof can be used providing that they cause quenching of an indicator molecule of the present invention which is not quenched in the absence of a mobilizing reagent while causing no quenching of samples which contain no free iron such as normal serum samples or human transferrin saturated with various concentrations of iron.
  • Another embodiment of the method of this aspect of the present invention includes the exclusion of endogenous apo-transferrin and/or iron free transferrin from the sample prior to free iron determination.
  • Apo-transferrin is universally found in human sera, except in cases of extreme iron- overload where the transferrin is 100 % iron-saturated. Therefore the detection of free iron may be rendered more difficult once the sample contains nearly normal levels of apo-Transferrin.
  • the use of Fl-aTF as a probe equalizes the probability that the mobilized iron will bind to the indicator molecule or to endogenous apo-Transferrin in the sample.
  • Exclusion of endogenous apo-transferrin can be effected by incubating (i.e., pre- clearing) the sample with anti-apo-transferrin antibodies, such as solid phase coupled anti-transferrin antibodies available from Pharmacia, Uppsala and Bio-Rad Laboratories, Hercules, CA. Additionally or alternatively anionic beads such as MacroPrep® High S support beads available from Bio-Rad Laboratories, Hercules, CA can be used to exclude apo-transferrin from the sample.
  • anti-apo-transferrin antibodies such as solid phase coupled anti-transferrin antibodies available from Pharmacia, Uppsala and Bio-Rad Laboratories, Hercules, CA.
  • anionic beads such as MacroPrep® High S support beads available from Bio-Rad Laboratories, Hercules, CA can be used to exclude apo-transferrin from the sample.
  • exclusion of apo-transferrin is effected by co-incubating the sample with an apo-transferrin binding metal other than iron such as Gallium and Cobalt.
  • an apo-transferrin binding metal other than iron such as Gallium and Cobalt.
  • These metals mimic iron and bind to the indicator molecule of the present invention, preventing their reaction with iron [Breuer and Cabantchik Analytical Biochemistry 299, 194-202 (2001)].
  • a preferably used indicator molecule is Fl-aTf, described hereinabove, which is not affected by Gallium due to a biochemical mechanism which is yet to be determined.
  • This apparent insensitivity to Gallium gives the Fl-aTf indicator an iron-binding advantage over the endogenous Apo- transferrin, overcoming most of its interference.
  • NTBI also refers to labile plasma iron (LPI) including redox active iron, which redox activity is eliminated upon addition of exogenous iron chelators.
  • LPI labile plasma iron
  • Redox active iron i.e., ferrous iron
  • Ferric iron i.e., Fe(III)
  • ferrous iron i.e., Fe(II)
  • ROS oxygen species
  • Free radical toxicity is produced primarily by the hydroxy radical ( ⁇ ). Most of the ⁇ generated in vivo comes from iron-dependent reduction of H 2 0 2 [Halliwel (1986) Archi. Biochem. Biophys. 46:501-14].
  • ROS redox active iron and its reaction products
  • a method of quantifying redox active iron in a biological fluid comprising:
  • a sample of the biological fluid is contacted with a reducing agent.
  • the reducing agent is selected capable of reducing free iron from ferric to ferrous form.
  • Suitable reducing agents according to this aspect of the present invention include, but are not limited to, ascorbic acid, dithionite, mercaptoacetic acid, dithiothreitol.
  • a physiological concentration of ascorbic acid is used according to this aspect of the present invention.
  • Reduced iron is capable of reacting with any oxygen species dissolved in the sample to generate redox active iron reaction products (e.g., ROS).
  • redox active iron reaction products e.g., ROS
  • Reducer-treated sample is then contacted with a detector molecule, which can be measurably activated upon interaction with the newly generated redox active iron reaction product.
  • the detection molecule is selected such that its activation is related to the amount of the redox active iron reaction products. Specific examples include, but are not limited to, dihydrorhodamine, carboxy-dichloro-dihydrofluorescein, dihydroethidium and dihydroresorufin.
  • the detector molecule is a molecule which undergoes a spectrophotometric/fluorescence change following interaction with the redox active iron reaction products, such as ROS.
  • redox active iron reaction products such as ROS. Examples include but are not limited to dihydrorhodamine-123 (DHR), which converts to fluorescent rhodamine, dichloro- dihydrofluorescein, which converts to dichlorofluorescein, dihydroresorufm which converts to resorufm and the like.
  • Quantification of detector molecule activation is effected according to the methodology described herein above.
  • Labile cellular iron is also an important pathological measure.
  • labile plasma iron refers to a component that is redox active and chelatable in native plasma or serum or plasma diluted in salt solutions or serum following mobilization with a mild extracting agent.
  • the present invention further provides for a method of quantifying labile cellular iron in a biological sample, the method comprising:
  • step (b) contacting loaded cells of step (a) with a cell permeant metal chelator so as to remove the iron from the iron binding moiety;
  • Cells in which cellular iron is recorded include but are not limited to, red blood cells, white blood cells and cells taken from biopsies.
  • the iron indicator is first contacted with the reaction buffer and only then the diluted sample is added. Rationale: The initial incubation is meant to bind all the contaminating iron in solutions prior to addition of the biological test-solution.
  • the microparticles are added to a 240 microliter solution of buffered saline (+/- 50 micromolar DFO) and preincubated for 0.5-1 hr at room temperature, with frequent mixing.
  • Figures 5A-B provide specific and non-limiting assay conditions for measuring LCI (Labile cell iron).
  • the iron indicator is added as a non-fluorescent precursor that permeates the cells and releases a fluorescent indicator that binds labile cell iron (LCI) and undergoes quenching.
  • a strong permeant chelator identical or different from that in the iron indicator removes the iron from the metal indicator causing cell fluorescence F to rise, so that:
  • Measuring fluorescence on a flow cytometer before and after addition of chelator allows determination of AF.
  • the method of calibrating further comprises contacting the plurality of samples with an iron chelator so as to release said iron binding moiety following step (b); and detecting and quantifying a signal' generated following contact with said iron chelator, and wherein the calibration curve depicts said change in the signal versus the signal' against the predetermined distinct free iron concentration values.
  • compositions of the present invention can be included in a diagnostic or therapeutic kit.
  • indicator sets including one or more of the following components described hereinabove (e.g., the composition comprising indicator molecules in solution or attached to microparticles along with a mobilizing agent, a non-labeled iron chelator, an apo-transferrin binding metal other than iron, an anti-apo transferrin antibody or anionic beads), can be packaged in a one or more containers with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
  • compositions of the present invention can be widely used to directly and sensitively detect NTBI which persists in sera of patients, even with low transferrin saturation.
  • diagnosis refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome associated with abnormal levels of free iron in a biological fluid or cells), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery, determining a risk for the pathology and screening of a subject for the pathology.
  • a pathology e.g., a disease, disorder, condition or syndrome associated with abnormal levels of free iron in a biological fluid or cells
  • disorders and conditions which are associated with abnormal levels of free iron include, but are not limited to, hemolytic diseases hemoglobinopathies, thalassemia, thalassemia major, anemia, sickle cell anemia, aplastic anemia, megaloblastic anemia, myelodyplasia, diseases which require repeated transfusions, diseases which require dialysis, hereditary hemachromatosis, cancer, heart diseases, Myelo Dysplasia Syndrome (MDS), iron poisoning and rheumatoid arthritis.
  • hemolytic diseases hemoglobinopathies thalassemia, thalassemia major, anemia, sickle cell anemia, aplastic anemia, megaloblastic anemia, myelodyplasia
  • diseases which require repeated transfusions diseases which require dialysis, hereditary hemachromatosis, cancer, heart diseases, Myelo Dysplasia Syndrome (MDS), iron poisoning and rheumatoid arthritis.
  • the present teachings further provide for a method of treating a subject having a disorder associated with abnormal levels of free iron in a biological fluid or cells, the method comprising:
  • the medicament comprises an iron chelation therapy.
  • the iron chelation therapy comprises Deferoxamine (Desferal, DFO), Deferiprone (Ferriprox, LI), Exjade (Deferasirox), Deferasirox, FB50701 (Ferrokin Bioesciences) or a combination of same e.g., Desferal and Deferiprone.
  • the present teachings further contemplate a method of determining efficacy of treatment of a disorder associated with abnormal levels of free iron in a biological fluid or cells, the method comprising;
  • the medicament comprises iron chelation therapy, and whereas a reduction in the levels following the therapy is indicative of efficacious treatment.
  • a reduction in the levels following the therapy is indicative of efficacious treatment.
  • normal LPI/plasma NTBI levels are below about
  • the medicament is selected from the group consisting of oral iron supplementation, folic acid, vitamin B-12, erythropoietin, blood transfusion and hyperbaric oxygen, and whereas an increase in said levels following said therapy is indicative of efficacious treatment.
  • Preferred individual subjects according to the present invention are mammals such as canines, felines, ovines, porcines, equines, bovines, humans and the like.
  • Quantification of free iron levels is effected as described hereinabove.
  • Determining iron levels originating from a biological sample of a patient is preferably effected by comparison to a normal sample, which sample is characterized by normal levels of free iron (i.e., no detectable free iron). If free iron levels exceed normal values, the subject is informed of the diagnosis.
  • the availability of an accurate and non-invasive free iron quantification method is also useful in disease management.
  • the diagnostic method of the present invention can aid a medical professional diagnosing of a patient even with low levels of free iron and instructing the patient on the type of diet to maintain in terms of iron content and iron availability for adsorption, which can result in iron overload.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Step 1 Covalent attachment of calcein (CALG) to ovalbumin:
  • a complex of CALG with Cobalt (CA:Co) is generated by mixing 2 mM CALG A (Sigma, Molecular Probes) with 4 mM CoCl 2 in a solution containing 50 mM MES, 100 mM NaCl, pH 6.5.
  • Ovalbumin (OVA) in powder form is added and allowed to dissolve, giving a final concentration of 0.1 mM.
  • dry EDC is added in 2 separate portions at 20 min intervals to give a final concentration of 25 mM. Total reaction time is 40 min.
  • reaction mixture is then passed through a Sephadex G-25 column pre-equilibrated with PBS and the conjugate OVA-CALG-Co is eluted in the void volume. This results in a 2-fold dilution of the input OVA, giving a final OVA concentration of 0.05 mM.
  • concentration of CALG-Co in the OVA-CALG-Co conjugate is determined by absorbance at 490 nm using a calibration curve of free CA-Co.
  • the ratio OVA:CALG is typically 1 :2.
  • Step 3 Binding of OVA-CALG-Co to dendrimer polystyrene beads (Den- B).
  • the Den-B beads (washed once in DDW and packed by centrifugation are supplemented with a volume of OVA-CALG-Co solution (0.05 mM OVA) equivalent to 2 volumes of the original bead suspension and allowed to passively adsorb to the beads by mixing for 10 min.
  • OVA-CA-Co is added (10% w/v, MW 14,215; 64 amino groups) to a final concentration of 0.07 mM and the mixture is vigorously mixed for 10 min followed by bath sonication..
  • the bis-suberimidate crosslinker BS3 To the mixture of beads+ OVA-CA-Co is then added the bis-suberimidate crosslinker BS3 to 1 mM and the mixture is vigorously mixed for 20 min at room temperature. BS3 is then neutralized by addition of L-lysine to a final concentration of 50 mM and further incubation for 20 min. The mixture is then diluted with 4 volumes of PBS containing 0.05 % Triton-X- 100 and washed once in PBS/ Triton-X 0.05%>. The beads are then re-suspended in PBS/ Triton-X 0.05%/ EDTA 2 mM/ DTP A 2 mM and mixed for 20 min, to remove the Cobalt.
  • the beads are then washed 3 times with PBS/ Triton-X 0.05%>, resuspended in the same buffer at ⁇ 2.5 % w/v.
  • the beads are sonicated in a bath sonicator to break up clumps and passed through a 30 ⁇ filter mesh to remove aggregates. At this stage the beads are ready for use.
  • the beads examined by epifluorescence microscopy are depicted in the figure.
  • the beads are added to a final concentration of -0.025% w/v to 1 ml aliquots of HBS containing Fe:NTA (ratio 1 :2) at Fe concentrations ranging from 0 to 10 ⁇ .
  • AFm represents the maximal attainable change in fluorescence by a given preparation of beads.
  • Den-B Beads are reacted with fluorescein isothiocyanate FITC (0.1 mM in 50 mM NaHC0 3 (pH 8.5-9.0) buffer for 2 h at room temperature, washed in the same buffer and subsequently reacted with deferrioxamine isothiocyanate (DFO-ITC, from Macrocyclics Inc, Dallas TX) 0.2 mM for 2 h at room temperature, washed with 0.05 % Triton x-100 in PBS, PBs, sonicated (bath sonicator).
  • fluorescein isothiocyanate FITC 0.1 mM in 50 mM NaHC0 3 (pH 8.5-9.0) buffer for 2 h at room temperature
  • DFO-ITC deferrioxamine isothiocyanate
  • Den-B (F+D) beads were used to determine the concentration of standard labile iron in solution in the absence and presence of 50 ⁇ DFO and the fluorescence intensity was read in a flow cytometer and the difference between ⁇ DFO AF was determined and used for constructing the calibration curve shown in Figure 4A.
  • Den B Coupling of Den (R+D) to beads to yield Den B (R+D).
  • 0.1ml of amino-coated polystyrene beads (Spherotech; 6.2um diam) are added to eppendorf tubes and washed twice distilled water ( by centrifugation at 10K rpm x 1 min).
  • a 100 ⁇ of Den (R+D) are added together with 50 ul Ovalbumin (Sigma 45mg/ml in DDW), incubate for 5 min, and centrifuged 10K rpm 10 sec, and the supernatant is aspirated. These steps are repeated twice more (we referred to these steps as coating of the beads with dendrimer with the aid of ovalbumin as additional crosslinking bridge)) .
  • the suspension (150 ⁇ 1) is transferred to 2 ml Eppendorf and subjected to crosslinking (while vortexing) by addition of 17 ⁇ of BS3(Bis(sulfosuccinimidyl) suberate; Pierce- Thermo Scientific, cat# 21580; prepared .Fresh 10 mM in DDW).
  • the reaction of crosslinking is carried out on rotatory mixer for lhr rm temp in the dark. 1 ml PBS+0.05% Triton-X(PBS-T) are added together with 60 ⁇ of Lysine 0.5M in DDW pH 7.3 (to stop the reaction and neutralize extra reagent) for 20 min room temp dark.
  • Figure 4B shows fluorescence intensity dispersity FL2 (560->610 nm) of Rhodamine+DFO coated beads DenB(R+D) analyzed by flow cytometry. Titration of DenB(R+D) with the indicated concentrations of Fe as analyzed by flow cytometry (tested 1 month apart). The relative change in fluorescence AF produced by the indicated concentration of Fe is plotted in the inset (semilog scale). The figure further shows linear correlation in the comparison of NTBI values of 10 thalassemia patients obtained by the plate reader method of DCI (based on Fl-DFO) versus those obtained by the flow cytometric method based on DenB(R+D).
  • Ovalbumin-F-DFO (ovaFD) is formed by reacting: 2.8 mg of ovalbumin with 20-fold excess of EDC [l-ethyl-3-(3-dimethylaminopropyl) carbodiimide)], N- hydroxysuccinimide sulfonic acid in 3 fold molar excess) and 1 mM FDFO:Fe at pH 6.5 for 2 h, followed by gel filtration. Unreacted components and Fe were removed by dialysis against 10 mM acetic acid; 1 mM EDTA; 150 mM NaCl, pH 4.5 followed by dialysis against saline. Aminopropyl-containing polystyrene beads (diameter 6.2 um) are coated with Fluorescein-Desferrioxamine by the same procedure as described above for CALGB,
  • Figure 1A- Labile iron (Fe) binds to a fluorescent bead that carries a fluorescent metal sensor (FMS) and quenches its fluorescence (F) by an amount AF.
  • the change in fluorescence (AF) is stoichiometric, namely 1 : 1 with Fe binding and the latter proportional to the number of atoms of labile iron in a given solution.
  • the relevant AF is that which is affected (blocked) by the presence of a strong/ specific Fe chelator:
  • DCI directly chelatable iron
  • DFO chelator deferoxamine
  • LCI labile cell iron
  • SIH salicyl-aldehyde hydrazone
  • Figure IB - Labile iron (Fe) binds to a fluorescent bead that carries a fluorescent metal sensor (FMS) and quenches its fluorescence (F) by an amount AF.
  • the change in fluorescence (AF) is stoichiometric, namely 1 : 1 with Fe binding and the latter proportional to the number of atoms of labile iron in a given solution.
  • the relevant AF is that which is affected (blocked ) by the presence of a strong/ specific Fe chelator:
  • DCI directly chelatable iron
  • DFO chelator deferoxamine
  • LCI labile cell iron
  • SIH salicyl-aldehyde hydrazone

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Abstract

L'invention concerne une composition de matière. Cette composition de matière comprend un indicateur de fer attaché à une microparticule, l'indicateur de fer comprenant une fraction de liaison au fer et une fraction générant un signal, une intensité du signal généré par la fraction générant un signal étant corrélée à une quantité du fer lié par la fraction de liaison au fer. L'invention concerne également des utilisations cliniques de cette composition.
PCT/IL2012/050119 2011-04-05 2012-04-03 Composition comprenant un indicateur de fer attaché à une microparticule et utilisations de celle-ci pour quantifier du fer non lié à la transferrine (ntbi) et du fer labile cellulaire (lci) WO2012137202A1 (fr)

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CN106796244A (zh) * 2014-03-28 2017-05-31 瑟乐斯佩克株式会社 用以判断子宫内膜异位性卵巢囊肿致癌可能性之资料取得方法及其诊断装置
CN113640266A (zh) * 2021-08-11 2021-11-12 郑州大学 一种用于细胞中铁蛋白储存和释放铁的检测方法
WO2023028658A1 (fr) * 2021-09-01 2023-03-09 Telix International Pty Ltd Procédé de préparation

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