JP2009106936A - Separation efficiency enhanced by controlled flocculation of magnetic nanoparticles - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To facilitate the visualization of a biological portion isolated by a method for isolating a target biological portion from a biological sample with the use of colloidal magnetic particles. <P>SOLUTION: Flocculation of magnetic nanoparticles on the surface of an isolated target biological portion is controlled. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the field of bioaffinity separation and diagnostic testing of biological samples. More particularly, the present invention is used in magnetic separation assays and enrichment procedures to control endogenous magnetic particle aggregation factors that would interfere with visualization of isolated portions if uncontrolled. Compositions and methods are provided. Methods for constructing and synthesizing reversible aggregation factors are also provided, and the resulting composition facilitates the recovery of rare biological material while facilitating the observation of such isolated material.

In order to more fully describe the state of the art relating to this invention, several publications are referenced in this application by parenthesized numbers. The disclosure of each of these publications is deemed to be included herein by reference.
Many laboratory and clinical procedures employ biospecific affinity reactions. Usually, such reactions are used in diagnostic tests of biological samples or for the separation of a wide range of target substances, in particular biological parts such as cells, viruses, proteins, nucleic acids and the like. Various methods are available for the analysis or separation of the target substance, and are based on complex formation between the substance of interest and another substance that specifically binds to the target substance. Separation of the complex from unbound material can be accomplished gravitationally, for example by sedimentation of finely divided particles or beads linked to the target material, or alternatively by centrifugation. If desired, such particles or beads can be made magnetic to facilitate the binding / free separation step. Magnetic particles are well known in the art for their use in immunity and other biospecific affinity reactions. For example, U.S. Pat. No. 4,554,088 and ["Immunoassays for Clinical Chemistry", 1983, pp. 147-062, edited by Hunter et al., Churchill Livingstone, Edinburgh]. In general, any material that facilitates magnetic or gravity separation can be employed for this purpose. However, over the last two decades, magnetic materials that are excellent for performing such separations have led to their use in many applications.

  In general, magnetic particles fall into two broad categories. The first category includes particles that are permanently magnetizable, ie ferromagnetic. The second category includes particles that exhibit bulk magnetic behavior only when subjected to a magnetic field. The latter is called magnetically responsive particles. Sometimes, a substance exhibiting magnetic response behavior is described as a superparamagnetic substance. However, materials that exhibit bulk ferromagnetic properties, such as magnetic iron oxide, can be characterized as superparamagnetic only when crystallized to a diameter of about 30 nm or less. In contrast, larger crystalline ferromagnetic materials retain their permanent magnetic properties after exposure to a magnetic field and then tend to aggregate due to strong particle-particle interactions.

  Magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7 to 1.5 microns), and colloidal or nanoparticles (<200 nm). The latter, also called ferrofluid or ferrofluid-like, has many of the characteristics of typical ferrofluids [Liberti et al., Pp. 777-790, edited by E. Pelizzetti, “Fine Particles Science and Technology”, 1996, Kluwer Acad. Publishers, Netherlands].

  Small magnetic particles are very useful for analysis involving biospecific affinity reactions. This is because they are conveniently coated with a biofunctional polymer (eg, protein), giving a very high surface area and providing appropriate reaction kinetics. Magnetic particles in the range of 0.7 to 1.5 microns are, for example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 773; 4,554,088; and 4,659,678. Some of these particles are disclosed to be useful solid phase supports for immunological reagents.

In addition to the above small magnetic particles, there is a classification of large magnetic particles in the size range of approximately 1.5-50 micron, which also have superparamagnetic behavior. Typical of such materials are those invented by Ugelstad [US Pat. No. 4,654,267] and manufactured by Dynal (Oslo, Norway). The Eugelstadt method relates to the synthesis of polymer particles that are caused to swell and have magnetite crystals incorporated into the swollen particles. Other materials in the same size range are prepared by synthesizing particles in the presence of dispersed magnetic crystals. This results in confinement of magnetite crystals in the polymer matrix, thus making the resulting material magnetic. In both cases, the resulting particles have superparamagnetic behavior, as evidenced by their ability to disperse easily when the magnetic field is removed. Unlike magnetic colloids or nanoparticles, these materials, like small magnetic particles, are easily separated by simple laboratory magnets depending on the amount of magnetic material per particle. Thus, the separation is performed with a gradient from as low as a few hundred gauss / cm to about 1.5 kilogauss / cm. On the other hand, colloidal magnetic particles (less than approximately 200 nm) require substantially higher magnetic gradients due to their diffusion energy, small magnetic mass per particle and Stokes friction. Owen et al U.S. Pat. No. 4,795,698 relates to polymer-coated colloidal superparamagnetic particles. Such particles are produced by precipitation of magnetic species in the presence of biofunctional polymers. The structure of the resulting particles, referred to herein as single-shot particles, is a micro-aggregation in which one or more ferromagnetic crystals having a diameter of 5-10 nm are incorporated into a polymer body having a diameter on the order of 50 nm. It has been found to be a mass. The resulting particles show a clear tendency to be maintained in an aqueous suspension for an observation period of several months. Molday U.S. Pat. No. 4,452,773 describes materials similar in nature to those described by Owen et al. From Fe ^{+ 2} / Fe ⁺³ to base in the presence of very high concentrations of dextran. Produced by forming magnetite and other iron oxides upon addition. The material so produced has colloidal properties and has proven very useful for cell separation. This technology is commercialized by Miltenyi Biotec, Bergisch Grandbach, Germany.

  Another method for producing superparamagnetic colloidal particles is described in US Pat. No. 5,597,531. In contrast to the particles described in the Owen et al. Patent, these latter particles are produced on preformed superparamagnetic crystals dispersed, for example, by sonic energy into quasi-stable crystal clusters in the size range of about 25-120 nm. It is manufactured by directly coating a biofunctional polymer on the surface. The resulting particles are referred to herein as direct coated (DC) particles, but exhibit a significantly greater magnetic moment than Owen et al. Or Morday nanoparticles of the same overall size, greater than about 6 k Gauss / cm. It can be separated efficiently in a magnetic gradient.

  Magnetic separation techniques are known in which a magnetic field is applied to a fluid medium to separate a ferromagnetic material from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to be maintained in suspension, coupled with their relatively weak magnetic responsiveness, separates such particles from the fluid medium in which they are suspended. Requires the use of high gradient magnetic separation (HGMS) technology. In an HGMS system, the magnetic field gradient, ie the spatial derivative, has a stronger influence on the behavior of suspended particles than is exerted by the strength of the magnetic field at a given point. High gradient magnetic separation is useful for separating a wide variety of magnetically labeled biological materials, including eukaryotic cells, prokaryotic cells, viruses, nucleic acids, proteins, and hydrocarbons. In known methods, antibodies, antibody fragments, specific binding proteins (eg, protein A, streptavidin), lectins, etc. are at least on a substance capable of specifically recognizing and binding a receptor. The biological material was separable by HGMS, provided that one characterization factor was present. HGMS systems can be divided into two broad categories. The one category includes magnetic separation systems that employ a magnetic circuit located entirely external to the separation chamber or vessel. Examples of such external separators (or open field gradient separators) are described in US Pat. No. 5,186,827. In some of the embodiments described in this' 827 patent, the indispensable magnetic field gradient is such that permanent magnets are placed around a non-magnetic container and something like a pole of a magnet is placed opposite the field. Generate by The degree of magnetic field gradient in the test medium obtained with such a system is limited by the strength of the magnets and the spacing between the magnets. Therefore, the gradient obtained by the external gradient system is finite. In co-pending application 60 / 098,021, a means for maximizing radial gradient and a method for maximizing separation efficiency with a novel vessel design is disclosed.

  Another type of HGMS separator is (1) enhancing the applied magnetic field; and (2) a ferromagnetic collection structure (ferromagnetic) disposed in the test medium to create a magnetic field gradient in the test medium. collection structure). Previously disclosed internal HGMS systems include fine steel wool or gauze packed inside a column placed adjacent to a magnet. The applied magnetic field is concentrated in the vicinity of the steel wire so that the suspended magnetic particles are attracted and attached to the wire surface. The gradient produced on such wires is inversely proportional to the wire diameter, while the magnetic “reach” decreases with diameter. Therefore, very high gradients can be generated.

  One major drawback of the internal gradient system is that steel wool, gauze material, steel microbeads, etc. are trapped by non-magnetic components of the test medium due to capillarity in the vicinity of or between the intersecting wires. is there. Although various coating procedures have been applied to such internal gradient columns [US Pat. Nos. 5,693,539; 4,375,407], the large surface area of such systems is still due to absorption. This creates a recovery problem. Therefore, an internal gradient system is undesirable, especially when the collection of very low frequency capture moieties is the ultimate goal of separation. Furthermore, these systems are difficult to automate and costly.

  On the other hand, the HGMS approach using external gradients for cell separation offers a number of conveniences. First, simple laboratory tubes such as test tubes and centrifuge tubes or vacutainers used for blood collection may be employed. A quadrupole / hexapole device [US Pat. No. 5,186,827] or [US Pat. No. 5,466,574] if the external gradient is such that the separated cells can be efficiently further stratified. As in the case of the opposing dipole arrangement described in, the cells can be easily washed or subsequently manipulated. Furthermore, the recovery of cells from a tube or similar container is a simple and efficient process. This is especially true when compared to recovery from high gradient columns. Such separation vessels also provide another feature that is the ability to reduce the volume of the original sample. For example, if a particular human blood cell subset (eg, magnetically labeled CD34 + cells) is isolated from blood that has been diluted 20% with buffer to reduce viscosity, a 15 ml conical tube can be separated on a suitable quadrupole magnetic device. It can be used as a vessel.

  After appropriate washing and / or separation and resuspension to remove unbound cells, CD34 + cells can be resuspended very efficiently in a volume of 200 μl. This can be done, for example, starting with 12 ml of solution (blood, ferrofluid and dilution buffer) in a 15 ml conical tube, separating, discarding the “supernatant” and subsequent washing “supernatant”, then This can be achieved by resuspending the collected cells in 3 ml of a suitable cell buffer. A second separation is then performed, which (when necessary to carry out the labeling / staining reaction) can include further separation / washing steps, and the last isolated cells can easily be brought to a final volume of 200 μl. Resuspend. By reducing the volume in this continuous manner and using a vortex mixer for resuspension, the cells attached to the tube above the resuspension volume are collected in a reduced volume. When taken care of in a properly treated vessel and performed rapidly, cell recovery is very efficient, ranging between 70-90%.

  The efficiency with which magnetic separation can be performed and the recovery and purity of magnetically labeled cells will depend on many factors. These are the number of cells to be separated, the receptor density of the cells, the magnetic load per cell, the non-specific binding (NSB) of the magnetic substance, the technology used, the nature of the vessel, the nature of the vessel surface, the viscosity of the medium And points such as the magnetic separator used. This is usually the case if the level of non-specific binding of the system is substantially constant, and the target population reduces purity. As an example, a 0.2% NSB system recovers 80% of the population, which is 0.25% in the raw mixture and has a purity of 50%. On the other hand, if the initial population is 1.0%, the purity will be 80%. The fact is not clear, but the smaller the target cell population, the more difficult it is to magnetically label and recover. Furthermore, labeling and recovery are obviously dependent on the nature of the magnetic particles used. For example, when cells are incubated with large magnetic particles such as dynal beads, the beads tend to be too large to diffuse and are labeled by collisions caused by mixing the system. Thus, if cells are present in the population at 1 cell / ml blood or less frequently, the probability of labeling target cells, such as tumor cells in very early cancers, is the magnetic particles added to the system. It will be related to the number and length of time to mix. Mixing cells with such particles for a significant amount of time is detrimental and it is necessary to increase the particle concentration as much as possible. However, there is a limit to the amount of magnetic particles that can be added to the system, including systems that contain rare cells mixed with other blood cells and rare cells that are mixed with large amounts of magnetic particles by separation. In which case the ability to count and investigate the cells of interest is not significantly improved.

  There is another drawback to using large quantities of magnetic particles to isolate cells with a rare frequency (1-50 cells / ml blood). Despite the fact that large magnetic particles allow the use of external gradients with a very simple design and relatively low magnetic gradients, large particles tend to surround the cells in a cage-like fashion That makes it difficult to analyze. Therefore, the particles should be released before analysis, and particles that often cause other problems should be released.

  Theoretically, colloidal magnetic particles can be used with high gradient magnetic separation to separate a cell subset of interest from a mixed population of eukaryotic cells, particularly when the subset of interest is only a small fraction of the total population. If it only contains, it is the method chosen. With proper magnetic loading, sufficient force is exerted on the cells, facilitating its isolation even in moderately diluted whole blood grade viscosity media. As noted above, colloidal magnetic materials of less than about 200 nanometers perform Brownian motion and significantly promote their ability to magnetically label by colliding with rare cells. This is illustrated in US Pat. No. 5,541,072, where the results of a very efficient tumor cell removal experiment are described, employing 100 nm magnetic particles (ferrofluid). It is important to note, however, that colloidal materials below the size range generally do not interfere with cell observation. Cells so recovered can be examined by flow cytometry with minimal forward scatter effects or by microscopic observations employing visual or fluorescent techniques. Because of their diffusive properties, such materials readily “discover” and magnetically label rare biological moieties such as tumor cells in the blood, as opposed to large magnetic particles.

  However, for the reasons described above, there are obvious problems that arise with the use of ferrofluid-like materials for cell separation in an external field gradient system, which is the selected device design. Direct monoclonal antibody conjugates of Owen et al. Materials or Morday nanoparticles, such as those manufactured by Miltenyi Biotech, such as the quadrupole or hexapole magnetic device described in US Pat. No. 5,186,827, It does not have enough magnetic moment for use in cell separation employing the best available external magnetic gradient device. They are even less effective when used for separation in moderately diluted whole blood. More promising results are obtained using materials similar to those described in US Pat. No. 5,698,271, which are substantially more magnetic. In model spiking experiments, SKBR3 cells (breast tumor lineage) have a high EpCAM (epithelial cell-adhesion molecule) determinant density, even at very low spiking densities (1-5 cells / ml blood). Efficiently separated from whole blood with a direct conjugate of anti-EpCAM MAb ferrofluid. PC3 cells (prostate tumor lineage), on the other hand, have a low antigen density and can only be separated with significantly lower efficiency. It is plausible that it is the result of inadequate magnetic loading on these low density receptor cells.

  From the above discussion, for separations involving rare events or for cells with very low density receptors, the beneficial properties of both colloidal magnetic materials and large magnetic particles (eg, diffusion-based labeling and large magnetism, respectively) It would be advantageous to provide a magnetic separation system that combines moments). It can be envisaged that the separation process starts with magnetic colloids or nanoparticles, which, due to their Brownian motion, will quickly find and label rare cells or cells with very low density receptors. Let's go. Once that labeling is achieved, it is desirable to convert the magnetic moment of the nanoparticle to a value equivalent to that of a large magnetic particle. As such, it could be separated in a gradient field, such as a single external field gradient separator, where the magnetically labeled portion is used for larger particles. In the case of very low density receptor cells, even in high gradient external field separators are not fully recovered and the use of such principles clearly increases the separation efficiency. It is also highly desirable to be able to revert the magnetic moment of the labeled portion back to its original colloidal magnetic label density in applications where cells are used after analysis or for certain biological purposes. This approach will allow separation from excessive magnetic material and facilitate subsequent analysis or use.

  Liberty et al., US Pat. No. 5,466,574, describes a system having some of the above-mentioned characteristics relating to the “loading” of magnetic material onto cells. When cells are first labeled with a specific monoclonal antibody (with or without biotinylation) and subsequently magnetically labeled with goat anti-mouse ferrofluid or streptavidin-ferrofluid (respectively), the separation is performed in the presence of excess monoclonal antibody. Was promoted. The unique ability of ferrofluids to create this “no wash” facilitating procedure is due to the immunochemical cross-linking of free ferrofluid in solution to ferrofluid-bound target cells. This time, the ferrofluid that binds to the monoclonal antibody on the cell binds to the free ferrofluid in solution via the free monoclonal antibody. This results in an immunochemical cluster of monoclonal antibody / ferrofluid “grown” from the monoclonal antibody labeled cell determinant (referred to as chaining). Thus, magnetic colloids are “artificially” loaded onto cells, making them more magnetic and easier to separate. It has been found that the phenomenon follows the rules of immunochemistry and that a high excess of monoclonal antibody results in decreased chaining (monoclonal excess zone) and loss of separation efficiency. Similarly, high levels of ferrofluid also reduce chaining (ferrofluid excess zone). Chaining has been found useful in removing unwanted cells, such as tumor cells, in bone marrow or peripheral blood “transplantation”. In this way, very high levels of magnetic material (visually brown edges around the cells when viewed under a microscope) are loaded onto the labeled cells in a high gradient field of only 8-12 kGauss / cm gradient. Very efficient separation can occur. On the other hand, cells labeled with “monomer” ferrofluids were found to be separated with lower efficiency in the same gradient.

  A number of problems are encountered in attempting to use chaining to separate rare cells from whole blood. First, spiked cells are actually recovered efficiently, but they are densely coated (chained) with ferrofluid and their ability to analyze them is significantly reduced. This approach is therefore not ideal for applications where positively selected cells are to be observed by microscopy or flow cytometry. Furthermore, chaining appears to promote non-specific binding. In short, it is extremely difficult to design a chain-based assay in which the level of chaining simultaneously causes separation enhancement, unshielded observation of isolated cells, and an acceptable level of non-specific binding. The chain reaction is difficult to control because it requires immunochemical stoichiometry. For example, most (> 99%) added monoclonal antibody (or tagging ligand) is always free in solution, regardless of the affinity of the antibody. Therefore, in general, the amount of ferrofluid required to achieve an immunochemical equivalent (where the best separation occurs due to chaining) is particularly observed when selected cells are observed and / or further studied. If done, it results in more chaining than desired. Chaining can be reduced by simultaneously reducing the labeling of the monoclonal antibody and the addition of ferrofluid, but this comes at the expense of separation efficiency. Another disadvantage of using chaining to facilitate separation is the inability to reverse chain in some practical ways. If the chain can be reversed and the concomitant increase in non-specific binding is reduced, the phenomenon will provide a viable approach that allows the desired “loading” of the magnetic material. Another disadvantage of this method is that it requires a two-step reaction. That is, the reaction with the target primary monoclonal antibody in the first step is followed by the magnetic fluid specific for the primary monoclonal antibody in the second step. This approach does not work for assays that conjugate primary antibodies directly to ferrofluids.

  US Pat. No. 5,108,933 to Liberty et al. Discloses weak magnetic colloidal materials such as those described by Owen et al. Or Morday in immunoassays employing external field magnetic separators. Such materials are described therein as agglomerated and resuspendable colloidal magnetic materials, which are external magnetic field systems such as those commercially available at the time (Ciba Corning (Ciba Corning), Walpole, Mass .; Serono Diagnostics, Norwell, Mass.) Remain substantially unperturbed. In contrast, as described above, materials made by the method disclosed in the '531 patent are substantially more magnetic and separate at their separators. In the '933 patent, a means for converting colloids into coagulum is disclosed, making them separable in the separator described above. Thus, such substances could be used to perform the binding / free separation step of the immunoassay. There is no mention in 933 about the necessity or method of reversing the agglomeration reaction.

  In light of previous or recent discoveries of naturally occurring ferrofluid aggregation factors, the present invention provides compositions that control the aggregation of ferrofluids by endogenous factors during the isolation and immunochemical characterization of rare target biological parts and Recognized the need for a method. Such compositions and methods can be advantageously used to facilitate analysis and observation of biological parts so isolated. Furthermore, the present invention allows the use of materials that are substantially less magnetic as well as the possibility of using lower magnetic gradients. In the case of a fixed gradient, the present invention provides for capture or isolation of moieties that would otherwise have insufficient magnetic label to capture.

  In accordance with the present invention, methods, compositions and kits are provided for controlling the aggregation of ferromagnetic nanoparticles by endogenous aggregation factors. Magnetic fluid agglomeration often creates problems during subsequent observation of isolated targets. The method of the present invention facilitates visualization of isolated biological parts by allowing the observer to control the level of aggregation. In one embodiment of the invention, a method for inhibiting aggregation of magnetic nanoparticles on the surface of an isolated target moiety is provided. This method is characterized by obtaining a biological specimen suspected of containing the target biological part. Next, an immunomagnetic suspension is prepared by mixing the specimen with colloidal magnetic particles linked to a biospecific ligand having affinity for one characterization factor of the target biological part. Thereafter, this immunomagnetic suspension is subjected to a magnetic field to obtain a target biological fraction enriched fraction. If desired, the fraction is then examined to determine the characteristics of the target biological part so isolated. Inhibition of ferrofluid aggregation facilitates subsequent cell analysis, as ferrofluid aggregates on the cell surface are excluded. The absence of such aggregates is important for several types of analysis including, for example, flow cytometry and immunofluorescence microscopy.

  The reagents provided herein sufficiently inhibit or eliminate endogenous aggregation factors. This factor exclusion or inhibition step can be performed before or simultaneously with the addition of ferrofluid to the biological specimen for separation and enrichment.

  In order to further characterize the target biological part isolated using the method of the present invention, the method optionally comprises adding at least one additional characterization factor on the target biological part to the target biological part-enriched fraction. And adding at least one biospecific reagent that recognizes and efficiently labels. The labeled target biological moiety is then separated in a magnetic field to remove unbound biospecific reagents. A non-cell elimination agent is added to the separated biological part to allow for the elimination of non-nuclear components present in the sample. After purification of the target biological portion, it is ready for analysis using a variety of different analysis platforms. Target biological parts include, but are not limited to, tumor cells, virus-infected cells, maternal circulating fetal cells, viral particles, bacterial cells, leukocytes, cardiomyocytes, epithelial cells, endothelial cells, proteins, hormones, DNA and RNA. The target biological part is analyzed by a technique selected from the group consisting of multi-parameter flow cytometry, immunofluorescence microscopy, laser scanning cytometry, bright field basic image analysis, capillary volume measurement, manual cell analysis and automated cell analysis Can do. Aggregation inhibitors suitable for use in the methods of the invention include, but are not limited to, reducing agents, animal serum proteins, immune complexes, hydrocarbons, chelating agents, an unconjugated ferrofluid, And diaminobutane. When the endogenous aggregation factor is of the IgM class and is reactive to ferrofluid, preferred aggregation inhibitors are mercaptoethane sulfonate [MES], mercaptopropane sulfonate [MPS] and dithiothreitol [DTT]. It is a reducing agent. In particularly preferred embodiments, the biospecific ligand is a monoclonal antibody having affinity for epithelial cell adhesion molecules.

  In an alternative preferred embodiment of the present invention, a method is provided for isolating a target biological moiety from a biological sample by controlling the aggregation of magnetic nanoparticles. This method obtains a biological specimen suspected of containing the target biological moiety and makes the biological specimen a reagent effective to inactivate any endogenous aggregating factor present. With touching. An immunomagnetic suspension comprising a mixture of the specimen and colloidal magnetic particles linked to a biospecific ligand having binding affinity for at least one characterization factor present on the target biological portion Prepared and the magnetic particles are further linked to a first exogenous aggregation promoting factor comprising one member of a specific binding pair. A second multivalent exogenous aggregation promoting factor is then added to the immunomagnetic suspension to increase the aggregation of the particles, the second aggregation promoting factor being the other member of the specific binding pair Which reversibly binds to the magnetically labeled target biological moiety. The sample is then subjected to a magnetic field to obtain a target biological part-enriched fraction. This preferred embodiment takes advantage of the fact that aggregating ferrofluid on the target moiety in a controlled and reversible manner results in substantially improved isolation efficiency.

  In a further embodiment, the above method further comprises adding at least one biospecific reagent that recognizes and labels at least one additional characterization factor on the target biological moiety. The target biological moiety so labeled is then separated in a magnetic field to remove unbound biospecific reagents. A non-cell elimination agent is added to the separated target biological part to allow for the elimination of the non-nucleated part present in the sample. The target biological part is then purified and further analyzed. In order to reverse the aggregation mediated by exogenous aggregation factors, the specific binding pair can be added in excess to the purified biological part to reduce aggregation of ferrofluid on the cell, for example Facilitates observation of cells with a microscope. Suitable specific binding pairs for this purpose include but are not limited to biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-hydrocarbon, protein A-antibody Fc , Avidin-biotin, biotin analog-streptavidin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. Preferably, the biospecific ligand is a monoclonal antibody having affinity for epithelial cell adhesion molecules. Exemplary biospecific reagents include monoclonal antibodies, polyclonal antibodies, detectably labeled antibodies, antibody fragments, and single chain antibodies. The isolated target biological part is selected from the group consisting of multi-parameter flow cytometry, immunofluorescence microscopy, laser scanning cytometry, bright field basic image analysis, capillary volume measurement, manual cell analysis and automated cell analysis Can be analyzed.

  In accordance with the present invention, controlling the aggregation of ferrofluids in a sample has some of the aforementioned unexpected benefits, for example, increasing the efficiency of separation of certain parts. The addition of exogenous aggregation promoting factors increases the magnetic load and consequently reduces the amount of ferrofluid required to isolate the target portion while increasing the separation efficiency. Increased magnetic loading also allows for a decrease in incubation time, facilitating isolation of the target biological part in the presence of a sub-optimal magnetic field.

  In further embodiments of the invention, kits are provided that facilitate the performance of the methods described herein. A kit for isolating a target biological moiety comprises (i) a coated magnetic nanoparticle comprising a magnetic core material, a protein-based coating material, and an antibody that specifically binds to the first characterization factor of the target biological moiety; Here, the antibody is directly or indirectly linked to the base coating material. (Ii) at least one antibody having binding specificity for a second characterization factor of the target biological moiety; (iii) an endogenous aggregation inhibitor; and (iv) to exclude anucleated cells from the analysis. Non-cell elimination agent.

  The present invention also provides kits for improving the isolation efficiency of certain biological moieties that would be required to isolate low antigen density tumor cells from a biological sample. This kit achieves such enhancement using controlled reversible aggregation of magnetic nanoparticles. Such a kit is specific to (i) a reagent effective to inactivate endogenous aggregation factors; (ii) a magnetic core material, a protein-based coating material, and a first characterization factor of the target biological moiety. Coated magnetic nanoparticles comprising an antibody that binds electrically, wherein the antibody is directly or indirectly linked to the base coating material, and the magnetic particles are further linked to a first exogenous aggregation promoting factor. The factor comprises one member of a specific binding pair. (Iii) at least one antibody having binding specificity for a second characterization factor of the tumor cell; (iv) a second exogenous aggregation promoting factor, wherein the second aggregation factor is the Contains the other member of the specific binding pair. And (v) a non-cell elimination agent to exclude anuclear sample components other than the tumor cells from the analysis. If desired, the kit can include a reagent that reverses the exogenous aggregation factor. Specific binding pairs useful for such kits include, but are not limited to, biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-hydrocarbon, protein A-antibody Fc And avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. Reagents effective to inactivate endogenous aggregation factors include reducing agents, animal serum proteins, immune complexes, hydrocarbons, chelators, unconjugated ferrofluids, and diaminobutane.

  The methods, compositions and kits of the present invention control the aggregation of magnetic nanoparticles, thus facilitating the isolation, visualization, and characterization of rare biological material or cells from biological specimens.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1H depict a series depicting a microscopic observation of a blood donor sample taken with a high level of endogenous aggregation factor compared to one with a low level of endogenous aggregation factor. It is a micrograph. Breast cancer cells are spiked into whole blood, selected using EPCAM colloidal magnetic particles, and stained in suspension. 1A, transmitted light only, low aggregation; FIG. 1B, transmitted light only, high aggregation; FIG. 1C, cells stained with Hoechst nuclear staining, low aggregation; FIG. 1D, cells stained with Hoechst nuclear staining, high aggregation; FIG. 1E, cells stained with the epithelial cell marker Cytokeratin Alexa 488 (Alexa 488), low aggregation; FIG. 1F, cells stained with the epithelial cell marker Cytokeratin Alexa 488 (Alexa 488), high aggregation; FIG. , Cells stained with tumor cell marker erb2 conjugated to phycoerythrin, low aggregation; FIG. 1H, cells stained with tumor cell marker erb2 conjugated to phycoerythrin, high aggregation.

DETAILED DESCRIPTION OF THE INVENTION The term “target biological part” as used herein refers to a wide range of biological or medical objects. Fine particle analytes of biological origin including, for example, hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemicals, nucleic acid molecules (eg, RNA and / or DNA) and biological particles such as cells, viruses, bacteria, etc. including. In preferred embodiments of the invention, rare cells such as maternally circulating fetal cells or circulating cancer cells are efficiently produced from non-target cells and / or other biological parts using the methods, compositions and kits of the invention. Can be isolated. The term “biological specimen” includes, but is not limited to, cell-containing body fluids, peripheral blood, tissue homogenates, nipple aspiration and other rare cell sources that can be obtained from a human subject. A typical tissue homogenate could be obtained from symptomatic lymph nodes of breast cancer patients. The term “determinant” when used in reference to any of the target biological moieties is specifically bound by a biospecific ligand or biospecific reagent and participates in selective binding to a specific binding substance, which It refers to that part of the target biological part to be loaded and its presence is necessary for selective binding to occur. In basic terms, a determinant is a molecular contact region on a target biological part that is recognized by a receptor in a specific binding pair reaction. As used herein, the term “specific binding pair” refers to antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-hydrocarbon, nucleic acid (RNA or DNA) hydrolysis sequence, Fc Receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin and virus-receptor interactions. Various other determinant-specific binding agent combinations are contemplated for use in the practice of the methods of the invention, and such will be apparent to those skilled in the art. The term “antibody” as used herein includes immunoglobulins, monoclonal or polyclonal antibodies, immunoreactive immunoglobulin fragments, and single chain antibodies. Peptides, oligonucleotides or combinations thereof are also contemplated for use in the present invention, and they specifically recognize determinants with similar specificity as conventionally generated antibodies. The term “detectable label” is used herein to detect the presence of a target biological moiety in the test sample, either directly or indirectly, whose detection or measurement by physical or chemical means. Any substance that indicates Representative examples of useful detectable labels include, but are not limited to: molecules or ions that can be detected directly or indirectly based on the following: light absorption, fluorescence, reflectance, light scattering, phosphorescence or luminescent properties; Molecules or ions detectable by radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Among the group of molecules that can be indirectly detected based on light absorption or fluorescence are, for example, suitable substrates that convert from non-light-absorbing to light-absorbing molecules or from non-fluorescent to fluorescent molecules, for example. Various enzymes that produce Nucleic acid dyes or other reporter molecules are sometimes referred to herein as non-cell eliminators and can identify both target biological parts and certain non-target biological parts such as intact nucleated cells. Is added to the sample to eliminate any remaining anucleated cells or other potential inhibitory sample components prior to analysis by flow cytometry, microscopy, or other analytical platform. enable. Non-cell eliminators can react with DNA, RNA, proteins or lipids, and the amount of signal obtained is typical of what is obtained for cells, or the resulting image is a cell and nuclear membrane, Clarify typical features of cells such as the nucleus and mitochondria.

The term “optimal” as used herein to describe ferrofluid concentration, magnetic field strength, or incubation time refers to the conditions used for a standard unmodified assay, separation, isolation or enrichment.
The term “sub-optimal” as used herein to describe ferrofluid concentration, magnetic field strength, or incubation time refers to conditions used for assay, separation, isolation or enrichment It produces inferior results compared to those obtained under conditions.

As used herein, the term “ferrofluid” refers to suspended magnetic nanoparticles. The terms ferrofluid and magnetic nanoparticles are used interchangeably herein.
Endogenous ferrofluid aggregation factor is present in a sample isolated from the test subject. Exogenous aggregation factors are those listed herein that are added and / or reversed as desired by the investigator.

  Preferred magnetic particles for use in the present invention behave as a colloid and are superparamagnetic particles. A colloid is characterized by its size, i.e. smaller than 200 nm, i.e. of a size that does not inhibit the analysis. Superparamagnetic particles become magnetic only when they are subjected to a magnetic field gradient and do not become permanent permanently. The colloidal superparamagnetic particles are not separated from the aqueous solution for an extended period of time and are not allowed to settle. These particles consist of either a single crystal of iron oxide or an agglomerate of such a crystal surrounded by either physically adsorbed or covalently bound molecules. Colloidal magnetic particles having the above properties can be prepared as described in US Pat. Nos. 4,795,698; 5,512,332 and 5,597,531. Monoclonal antibodies that recognize a specific subset of cells and are conjugated to magnetic particles are preferred for use in the present invention.

  In the course of research on rare cell isolation, a factor that was found in the blood of certain patients and caused aggregation of magnetic nanoparticles was discovered. Furthermore, this accelerated aggregation effect appears to be easily reversible. Thus, rare cells with low receptor density are moderately diluted more efficiently and more efficiently than those that specifically bind to the characterization factor of the rare cell of interest by “loading” more ferrofluid. Isolated from whole blood in an external field quadrupole or hexapole separator. When the isolated cells are examined by microscopic observation, ferrofluid clusters are present in the cell membrane. The reversal of the promoting effect disaggregates the cluster and facilitates microscopic analysis of the cells. This intrinsic promoting effect is present at various levels in about 90% of apparently normal donor blood samples. By manipulating the assay conditions, the resulting ferrofluid cluster can be extinguished without damaging the cells of interest. Thus, the present invention provides a method to eliminate the effects of endogenous ferrofluid aggregation factors and a means to build agents that can control and reverse ferrofluid aggregation, if desired. This allows for effective and effective isolation of rare cells and other biological parts such as viruses and bacteria, and subsequent analysis.

  Endogenous ferrofluid aggregates found in blood have the following characteristics, as described in US Pat. No. 5,597,531: (1) it is present in plasma or serum; (2) It is sensitive to millimolar concentrations of dithiothreitol; and (3) it reacts with the “naked” crystalline region above the direct coated ferrofluid. Control experiments revealed that the factor was not ferritin, transferrin, fibrinogen, Clq, human anti-mouse or anti-BSA antibody. The aggregate is not of the IgG subclass. This is because IgG-lost plasma also causes aggregation of the ferrofluid. Ferrofluid aggregation appears to correlate with the concentration of ferrofluid or serum. Aggregation dependence on the concentration of either component is similar to that observed in the sedimentation curve, and based on the above observations, the material was assumed to be IgM. This was finally proved by removing the IgM from the plasma sample by immunoaffinity purification. The resulting IgM depleted plasma did not cause ferrofluid aggregation.

  A control experiment in which the same plasma sample was absorbed throughout the BSA affinity medium showed ferrofluid aggregation. Further studies demonstrated that absorption of large amounts of serum containing ferrofluid with the aggregation factor only removed a small amount of total IgM and that purified human IgM produced ferrofluid aggregation. These observations and identification studies of the ability to cause aggregation with absorbed aggregation factors and absorption / desorption materials lead to the conclusion that the aggregation factor in these experiments is indeed highly specific IgM. Based on its inability to inhibit aggregation by any component used to form the ferrofluid, except for magnetite crystals that are incompletely coated with protein or magnetite crystals that are partially coated with soap, It is believed that the epitope recognized by the IgM is present on the magnetite crystalline surface. Although the role of this specific IgM is not known, it exists at various levels in a substantial portion of the human population. This antibody may play a role in iron metabolism.

  Methods for manipulating endogenous promoters are described herein, which allows for patient-to-patient comparison of cells isolated in a meaningful way. Since endogenous aggregation promoters vary in the patient population, it is difficult to create a standard procedure to isolate rare circulating cells that are tumor cells, fetal cells in maternal blood or virus-infected cells. Recent discoveries described in the co-pending application (09 / 248,388) demonstrated that the size of circulating cells is directly related to the systemic tumor tissue volume and disease stage of breast cancer patients. Similarly, systemic viral tissue mass has been shown to be substantial in the prognosis of HIV infection. The need for accurate quantification of such parts has become even more important. The methods described herein are conveniently performed with a test kit that can be advantageously used in a clinical setting.

With the discovery of the presence of this ferrofluid agglutination factor (FFAF) in donor sera, careful studies have used monoclonal antibody-conjugated ferrofluids to significantly increase the recovery of spiked low density tumor cells. Confirmed to improve. In these studies, cells spiked with FFAF-containing blood at a level of 1 cell / 2 ml are collected as regularly with an efficiency of 70% or more. In contrast, in donor blood in the absence of FFAF, recovery efficiency drops to approximately 15-25%. From these experiments, the following observations were made:
1. Low density receptor cells are often isolated with lower efficiency than high density receptors;
2. Separation efficiency varies considerably for low density receptor cells, and this variation depends on the blood donor;
3. After ferrofluid / magnetic isolation from different donor blood, ferrofluid aggregates about 90% of the patient sample when any cell type (high or low density characterization factor cells) is examined microscopically;
4). Recovered tumor cells have varying degrees of visual ferrofluid aggregation on their surface.
Moreover, since the aggregated material is sensitive to DTT, the recovered cells can be easily visualized by microscopic observation on morphological characteristics by reducing the aggregation of ferrofluid before observation.

  Comparing the phenomenon of FFAF and chaining (see US Pat. No. 5,466,574), which facilitates cell recovery, can lead to some interesting conclusions. As in many individuals, excellent recovery of low density receptor spiked cells (eg, 1-5 cells / ml blood) is obtained when FFAF is present in blood at excessively low concentrations. The quality of the recovered cells is very suitable for morphological investigations or further manipulations, as assessed by the level of magnetic material on their surface. When FFAF is at a high concentration, recovery is excellent, but the quality of the recovered cells is unacceptable because clots on the cells interfere with their observation. Therefore, unlike monoclonal antibody-ferrofluid “chains”, which are limited by the components of chaining, ie concentration, there are levels of FFAF that can lead to very effective cell separation, thus studying efficiently. An isolated population of cells obtained is provided. In certain embodiments of the invention, endogenous FFAF in the sample is initially inactivated so that aggregation can be controlled by adding the compositions described herein by investigators. In this way, aggregation can be promoted or inhibited as desired.

  FFAF and any similar material should be evaluated in assays that isolate rare cells. Such factors should be controlled to develop a test that works reliably for all patients. For example, many individuals have antibodies to components such as anti-rodent antibodies or hydrocarbons, which could be found on the surface of some magnetic nanoparticles. Other potential aggregates that are normally present in the blood include Clq, rheumatoid factor, and clotting proteins. Such reactions are either controlled to keep them constant from specimen to specimen or completely eliminated. In the case of anti-rodent antibodies, this is achieved by adding rodent proteins to the system so as to inhibit their aggregation effects. This addition is quite different from adding such components to the immunoassay system. In the latter case, rodent antibodies generally facilitate isolation, but they tend to increase false positives because the captured antibody is linked to the labeled antibody in a sandwich reaction. In contrast, additional antibodies used in the methods of the present invention facilitate the recovery of low density receptor cells. Thus, in some cases, aggregation factors can artificially increase false positives, and in other cases, facilitate the isolation of the target biological moiety.

  In addition to competitively inhibiting such factors, they can be disabled or absorbed from solution, or the determinants on the magnetic colloid to which such factors bind are removed. Can do. Specific chelating agents that inhibit various activation systems, including mouse or other animal serum proteins, immune complexes, hydrocarbons, complement, and interactions with antibodies that react with C1q, such as diaminobutane FFAF activity can be inhibited by preparing special buffers containing optimal amounts of additives, such as compounds that inhibit oxidatively. When FFAF is IgM, the reducing agent effectively disables FFAF without affecting the ligand used to label the cells. Thus, such factors can be selectively disabled chemically or enzymatically. Absorption with a suitable material, eg, unconjugated ferrofluid, removes the aggregation factor in the sample by another route.

  It would also be possible to keep the amount of aggregator constant from patient to patient so that they always have the same level of facilitator. This can be achieved in various ways. For example, all endogenous factors can be reduced to the same level in all patient samples. However, this seems to be a difficult and unrealistic solution. Alternatively, a two-step process can be used. As a first step, all endogenous FFAF is disabled without affecting the specific ligand binding of the ferrofluid to its target or subsequent cellular analysis. The second step involves a controlled agglutination reaction. The above two-step process can be performed using colloidal magnetic material conjugated to two ligands. One ligand, such as a monoclonal antibody, will be directed to cell surface determinants and will efficiently label the cells. The second ligand does not react with any blood component, but has binding affinity for the multivalent component that would be added after binding to the first ligand. Thus, additional magnetic colloids bind to colloids already bound to cells, thereby facilitating their magnetic loading just as FFAF does. Similarly, it is preferable if the reaction of the second ligand and its multivalent component are reversible. By selecting the correct components of the second reaction and their concentrations, it becomes possible to add the ferrofluid and the aggregation factor simultaneously.

  Thus, FFAFs are provided that facilitate control of ferrofluid aggregation, thereby facilitating the recovery of rarely produced biological moieties. The ideal aggregation factor mediates the reversible aggregation effect. Reversal of aggregation eliminates magnetic nanoparticle clusters and facilitates visualization of isolated cells. The factors of the present invention operate in a manner similar to the ideal magnetic particle conditions described above, i.e. by converting colloidal nanoparticles bound to the target into large particles with the ability to easily reverse the process. To do. The identification and investigation of endogenous factors that are present in the blood of most normal donors and facilitate the isolation efficiency of low density receptor rare cells are described herein.

  Preferred FFAFs contain specific multivalent substances that recognize determinants on ferrofluid magnetic particles, thereby cross-linking the particles. This factor may be a reagent that is naturally produced in plasma or added externally. Several types of exogenous reagents are suitable for this purpose, including but not limited to IgG, dimeric IgG, IgM, streptavidin, avidin, protein A, protein G, dimeric or tetrameric poly-A or Includes poly-T, or specific oligonucleotide sequences. The second ligand can be introduced onto the ferrofluid and can be recognized by FFAF. Several types of selectables such as haptens, biotin, biotin analogs (iminobiotin, desthiobiotin), sheep IgG, goat IgG, rat IgG, poly-A or poly-T, or oligonucleotides. There are two ligands. The FFAF-secondary ligand interaction can be either reversible or irreversible, with reversible interactions being preferred. Used to reverse FFAF-secondary ligand interactions such as reducing agents, excess hapten, excess hapten analog, excess second reagent analog, salt concentration change, pH change or temperature change. There are several reagents that can be done.

  When the assay or separation is performed under optimal conditions, the percent recovery of the target cells is maximal. However, it is clear that the conditions can be modified when exogenous aggregation is induced. For high density surface receptor target cells, the amount of ferrofluid required for maximum recovery is fairly high without exogenous aggregation, and each target is magnetically responsive by saturating all available binding sites Is guaranteed. One benefit of the present invention is that less total ferrofluid is required for separation because it is not necessary to saturate all of the available binding sites. This is due to the ability to form crosslinks of magnetic particles mediated by exogenous aggregation factors and increase the amount of magnetism per binding site. The use of many single magnetic particles per cell makes the cells magnetically responsive, i.e., instead of one particle binding to one cell surface antigen, it binds multiple particle aggregates to a single antigen. And will maintain the same magnetic force as optimally captured cells. Each particle now has the ability to bind to a cell surface antigen or another particle. In other words, despite using a small total ferrofluid, each target cell has the same magnetic responsiveness and the resulting separation efficiency is comparable to that obtained under optimal conditions. Optimally, 10 μg of ferrofluid is used per ml of sample. This concentration can be reduced by a factor of 10 according to the invention.

  In addition to reducing the amount of ferrofluid required, the magnetic force as well as the incubation time can be reduced. Since the aggregate of the present invention is larger than the individual magnetic particles in non-aggregated separation, the aggregate can be moved even with a lower magnetic field strength. Exogenous aggregation proceeds to produce primary and reversible large magnetic particles from small magnetic particles. Indeed, the benefits of large particles include the ability to use weak magnetic fields for separation and can be applied to the present invention. Typically used quadrupole magnetic separators maintain a magnetic field gradient of 6.3 k Gauss / cm at the vessel surface. Magnetic arrays such as dipoles have approximately half the magnetic field strength at the vessel surface and can be used to carry out the method of the present invention.

  In non-aggregated systems, longer incubation times are required to increase the number of magnetic particles per cell for effective separation. In this system, one magnetic particle binds to one cell surface antigen. However, in an exogenous-induced aggregation system, the induced aggregate can achieve the same number of particles per cell by generating multiple particles per antigen. This makes it possible to reduce the incubation time. This is because not all available binding sites need to bind to the magnetic particles. By adding this second binding pair member, the particles can now bind to other particles instead of only binding to free cell surface antigens. Thus, as explained above, the binding site need not be saturated to have the same magnetic responsiveness as optimal conditions, which allows for a reduction in incubation time. The minimum time for magnetic incubation is 30 minutes. With the present invention, these times can be reduced by a factor of three. However, it is not intended to simultaneously reduce both the ferrofluid concentration and the incubation time. Variations of these steps are used independently of each other to maintain maximum recovery of the target biological part.

The second ligand recognized by the exogenous ferrofluid aggregation factor is coupled to ferrofluid in addition to the monoclonal antibody by standard coupling chemistry. The second ligand may be a small molecule such as a hapten or biotin analog or a large molecule such as an antibody or specific protein or a polymer such as a polypeptide or polyoligonucleotide. A biotin analog such as desthiobiotin is preferred for conjugation to magnetic particles as the second ligand of the present invention. This is because it shows a lower affinity (Ka = 10 ⁶ M ^{−1 for} streptavidin, compared to natural biotin (Ka = 10 ¹⁵ M ⁻¹ )). The interaction between streptavidin and desthiobiotin can be easily disrupted by the addition of excess biotin. The combination of desthiobiotin and avidin has been used to remove magnetic particles or insoluble phases from the target material (PCT / US94 / 10124 and US Pat. No. 5,332,679). In the present invention, the combination is used only for agglomerating and deaggregating the magnetic particles, and not for removing the magnetic particles from the target substance.

  The reaction vessel used in the present invention may be either glass or plastic, but a plastic tube is preferred. The bottom of the tube may be round or conical. Different lengths or diameters of tubes can be used to process different volumes of samples. For example, in some cases, a 50 ml conical tube can be used to process 20 ml or more of blood. In one embodiment of the invention, a 12 × 75 mm polystyrene tube or a 15 ml conical tube is used. The reaction vessel used during the incubation with magnetic particles and the vessel used during magnetic separation need not be the same. Two different types of containers can be used, one for incubation and another for magnetic separation. However, it is preferred to use only one container in both cases. The magnetic separation vessel may be a tube or a flow-through chamber or some other device.

  The test medium used to practice the present invention may be any liquid or solution containing the target substance, preferably blood. The test sample in the reaction vessel can be incubated with a ferrofluid conjugated to an antibody specific for the target substance and a second ligand specific for FFAF. The exogenous FFAF is added to the test sample simultaneously with the magnetic fluid or after the magnetic fluid is bound to the target substance. If desired, a reagent that inhibits or disables naturally occurring aggregation factors can be added simultaneously with or prior to the addition of the ferrofluid. After an optimal incubation time, the magnetically labeled target is separated from the remaining test media in a magnetic separator. The magnetic separator and separation time are selected based on the test medium and the reaction vessel. Preferably, a high gradient magnetic separator is used, such as that described in US Pat. No. 5,186,827. After aspirating the uncollected liquid, the collected cells are suspended in an isotonic buffer or permeabilization solution to increase the permeability of the cells for intracellular staining. The magnetically labeled cells are magnetically re-separated to remove the permeabilizing reagent. The collected cells are resuspended in a small amount of cell buffer for staining with labeling material. The buffer volume will be 100-300 μl. If desired, the cell buffer can contain a deaggregation reagent such as biotin as described above. The final concentration of biotin may be 1-5 mM. Incubation time for antibody staining or for deaggregation of ferrofluid with deaggregation reagent may be 10-60 minutes, preferably 15 minutes. After optimal staining with antibodies or deaggregation of ferrofluid, excess reagent can be removed by another magnetic separation. After aspirating the uncollected liquid, the collected cells are resuspended in a small amount of isotonic buffer. The amount of this buffer may be 100-500 μl. The ferrofluid labeled cells can be further processed or analyzed by flow cytometry or microscopy.

  Although only magnetic particles conjugated to antibodies have been described above, other types of conjugated magnetic particles are contemplated for use in the present invention. Magnetic particles conjugated to proteins other than antibodies can be used. For example, streptavidin-conjugated magnetic particles that bind to target cells labeled with an antibody-biotin conjugate can be used. After labeling the target cells, excess antibody-biotin can be removed by a washing step using centrifugation. The target cells labeled with antibody-biotin are then incubated with streptavidin ferrofluid for magnetic labeling of the cells. Desthiobiotin conjugated to any polymer or protein (aggregation factor) is added to the test medium to aggregate the ferrofluid. Aggregation factors can be added simultaneously with the magnetic particles or after the magnetic particles have bound to the target cells. The number of desthiobiotin per polymer or protein should be greater than 1 for the aggregate ferrofluid. Preferably, desthiobiotin conjugated to bovine serum albumin (BSA) can be used. The number of desthiobiotin relative to BSA will be 2-10. Such desthiobiotin / protein conjugates can be synthesized as described below.

Although the present invention has been described herein with reference primarily to tumor cell selection from blood, the present invention is not limited to tumor cell selection. Other cell types present in blood, leukophoresis or bone marrow can be selected, such as CD34, CD4 and fetal cells. The antigenic determinants on those cells will be low to high. More generally, the present invention is directed to the isolation of any cell that requires magnetic enhancement for its effective isolation.
The following examples are given to illustrate various embodiments of the present invention. These examples are in no way intended to limit the scope of the invention.

Example I
The following data illustrates the effect of the FFAFs of the invention on the recovery of low and high density receptor tumor cells spiked into a blood sample. A representative FFAF has been identified as a specific IgM present in most donor blood samples. Reducing agents such as dithiothreitol (DTT) and mercaptoethane sulfonate (MES) that cleave disulfide bonds inhibit ferrofluid aggregation in blood by converting pentameric IgM to its monomeric form. did. DTT is not a preferred reagent for use in the present invention. This is because high concentrations alter cell morphology and are toxic to target cells and leukocytes.

  This example describes the effect of MES on ferrofluid aggregation and tumor cell recovery of both high and low antigen density tumor cells from spiked blood. The protocol used for this study is: Blood (2 ml) is placed in a 12 × 75 mm polystyrene tube and 1 ml of Imunicon dilution-wash buffer is added to dilute the blood. Next, 100 μl of cell buffer (isotonic 7 mM phosphate with 1% BSA and 50 mM EDTA, pH 7.4) containing approximately 1000 SKBR3 or PC3 cells was added. Increase the volume (without exceeding 150 μl) and add MEA to the blood sample to obtain different concentrations of reducing agent. After mixing, EpCAM MAb (GA73.3; 50 μl) conjugated ferrofluid magnetic particles are added to the sample. The final concentration of magnetic particles was 5 μg / ml. The blood sample is mixed well and incubated for 15 minutes at room temperature. After incubation, the tube containing the blood sample was placed in a quadrupole magnetic separator. Magnetic separation was performed for 10 minutes. The supernatant was aspirated and the tube was removed from the magnetic apparatus. Magnetically collected cells were resuspended in 1 ml of dilution-wash buffer and then re-separated for 5 minutes in a quadrupole magnetic separator. After discarding the supernatant and removing from the quadrupole device, the target cells were resuspended in 150 μl of diluted wash buffer. A portion (5 μl) of this sample was spotted on a microscope slide. Next, a photograph of the collected cells was taken using a microscope equipped with a digital camera.

  The remaining samples were subjected to flow cytometry analysis to assess tumor cell recovery using the following procedure. Phycoerythrin (PE) -conjugated MAb (5 μl) specific for tumor cells (Neu24.7) and 5 μl peridinin-chlorophyll protein (PerCP) -conjugated CD45 monoclonal antibody were added to the sample. It was then incubated for 15 minutes. After incubation, 1 ml of dilution-wash buffer was added and magnetic separation was performed to remove excess stained antibody. Magnetically collected cells were resuspended in 500 μl of dilution-wash buffer. Nucleic acid dye (10 μl) and 5 μl of 3 mM size fluorescent beads (5000) were added to this sample. The samples were then analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using FL1 as the sensitivity limit. The fraction of fluorescent beads obtained with a flow cytometer is used to determine the amount of sample analyzed by flow cytometry, thus facilitating the calculation of spiked tumor cell recovery.

  When viewed under a microscope, in the absence of MES, the final sample showed free ferrofluid aggregates and ferrofluid aggregates on tumor cells. Increasing the MES concentration decreased ferrofluid aggregates and no aggregates were observed at higher concentrations of MES. These visual results were then compared to the tumor cell recovery measured by flow cytometry. Although the addition of MES had little effect on the recovery of SKBR3 cells (high antigen density), microscopic observation revealed that it reduced ferrofluid aggregation in solution and on the cell surface. In contrast, MES had a significant effect on PC3 cell (low antigen density) recovery. As the concentration of MES increased from about 0 to 100 mM, the recovery decreased from 47% to 17%. This decrease in PC3 cell recovery in the presence of increasing concentrations of MES is due to inhibition of ferrofluid aggregation and not due to any side effects of MES on the cells. This is because MES does not reduce the recovery rate of identical spiked PC3 cells obtained from hemolyzed samples. Hemolysis is obtained by lysing red blood cells with ammonium chloride and then removing plasma and ammonium chloride in a wash step. The hemolyzed sample contains only white blood cells (white cells), while whole blood also contains red blood cells and plasma. No ferrofluid aggregation is observed with hemolysis. Moreover, MES does not significantly affect cell morphology. These data indicate that low antigen density cells are isolated with lower efficiency than high antigen density cells, and inhibition of ferrofluid aggregation dramatically affects the isolation of low antigen density cells. When washed blood (blood cells from which plasma was removed) was used, no ferrofluid aggregation was observed. Therefore, a washed blood sample was used as a control without aggregation. Just as with whole blood, SKBR3 cell recovery was not reduced with washed blood. On the other hand, PC3 cell recovery was significantly reduced (2 to 5 times) when using washed blood. This data shows that ferrofluid agglutination has no effect on SKBR3 cell recovery but has a significant effect on PC3 cell recovery.

  In summary, the aggregation of tumor-specific ferrofluids with plasma components (IgM) present in the blood of many patients at various levels has a significant effect on the recovery of low antigen density cells. The recovery of such cells is affected by the degree of ferrofluid aggregation and increases with increasing aggregation. Ferrofluid aggregation increases the recovery of low antigen density cells by increasing the magnetic load on the cells. Ferrofluid aggregation can vary from one blood donor to another depending on the aggregating factor, ie the concentration of the aggregating agent. As a result, the recovery rate of tumor cells varies between people, even if they retain the same number of circulating tumor cells. Aggregating agents present in blood from the same person can also vary over time, thus varying the degree of ferrofluid aggregation and tumor cell recovery. The best way to prevent this variation is to prevent naturally occurring ferroparticle aggregation factors. However, this results in a decrease in the efficiency of tumor cell isolation and detection. One means of increasing tumor cell recovery under these circumstances is to improve the magnetic apparatus with a higher magnetic gradient that can efficiently pull weakly magnetically labeled cells and increase their recovery. I will. Another strategy to increase tumor cell recovery is to mimic natural ferroparticle aggregation with external reagents. This reagent is a specific multivalent reagent capable of recognizing ferrofluid and can be added to blood and ferrofluid. The specific reagent is similar to IgM but aggregates ferrofluids under a controlled reaction. There are two advantages to controlled aggregation: (1) the percentage of recovered tumor cells is increased, and (2) the percentage of recovered tumor cells is between patients when the sample has the same number of tumor cells. Does not fluctuate.

Example II
Preparation of desthiobiotin / EpCAM MAb for controlled aggregation Base ferrofluid is generated as described in US Pat. No. 5,698,271. Monoclonal antibodies against epithelial cell adhesion molecule (EpCAM) were conjugated to the base material by standard coupling chemistry as used in US patent application 09 / 248,388. The EpCAM MAb ferrofluid was then resuspended in 20 mM HEPES, pH 7.5, and N-hydroxysuccinimide-DL-desthiobiotin (NHS-desthiobiotin) (Sigma, catalog number H-2134). Was conjugated to desthiobiotin. NHS-desthiobiotin mother liquor is made at 1 mg / ml in DMSO. NHS-desthiobiotin (19 μg) was added to 1 mg EpCAM MAb ferrofluid and incubated for 2 hours at room temperature. Unreacted NHS-desthiobiotin is removed by washing three times with 20 mM HEPES, pH 7.5 containing 1 mg / ml BSA, 0.05% ProClin 300 using a high gradient magnet. After the final wash, desthiobiotin / EpCAM MAb ferrofluid was resuspended in Immunocon ferrofluid storage buffer and filtered through a 0.2 μm syringe filter.

Example III
Increased recovery of low antigen density PC3 tumor cells from spiked blood by aggregation of desthiobiotin / EpCAM ferrofluid and streptavidin In this example, prostate cancer cells (PC3) with low EpCAM antigen density were treated with normal blood And used as a model system for evaluating the recovery rate of the spiked cells. A known number of PC3 cells (˜5000) in 50 μl buffer (isotonic 7 mM phosphate, pH 7.4 with 1% BSA and 50 mM EDTA) were spiked into 1 ml of normal blood without plasma in a 12 × 75 mm polystyrene tube. did. Plasma-free blood was used in these experiments to prevent any interference with plasma components in the selection of target cells and was prepared by centrifugation of blood. 500 μl of Imicon diluted dilution buffer and 15 μl of streptavidin were added to the aliquots of the blood samples at different concentrations in PBS. After mixing the sample, desthiobiotin / EpCAM MAb ferrofluid (25 μl) from Example 1 was added to the sample, mixed well and incubated at room temperature for 15 minutes. The final concentration of ferrofluid was 5 μg / ml. After incubation, the tube containing the blood sample was placed in a quadrupole magnetic separator for 10 minutes to collect magnetically labeled cells. Uncollected sample was aspirated and the tube was removed from the magnetic separator. Magnetically collected cells were resuspended in 750 μl of dilution-wash buffer and re-separated in a magnetic separator for 5 minutes. Uncollected samples were discarded again and the tubes were removed from the magnetic separator before the collected cells were resuspended in 150 μl of dilution-wash buffer.

  The sample was then stained with antibody and tumor cell recovery was determined by flow cytometry as follows. 5 μl of phycoerythrin (PE) -conjugated MAb specific for tumor cells (Neu24.7) and 5 μl of peridinin chlorophyll protein (PerCP) -conjugated CD45 MAb are added to the sample, 15 Incubated for minutes. After incubation, 1 ml of dilution-wash buffer was added and magnetic separation was performed for 5 minutes to remove excess stained antibody. Magnetically collected cells were resuspended in 500 μl of dilution-wash buffer. Nucleic acid dye (10 μl) and 5 ml of 3 μM fluorescent beads (5000) were added to this sample. The samples were then analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using FL1 as the sensitivity limit. The fraction of fluorescent beads obtained with a flow cytometer was used to determine the amount of sample analyzed by flow cytometry, which was then used to calculate the recovery of spiked tumor cells. The percent recovery of tumor cells is shown in the table below.

  The sample remaining after flow cytometric analysis was divided into two parts. Biotin from the mother liquor in PBS was added to one portion of the sample to a final concentration of 2 mM and incubated for 15 minutes at room temperature to deaggregate streptavidin-mediated ferrofluid aggregates. These samples (5 μl) were spotted on a microscope slide slide, and a photograph of the collected cells was taken using a microscope equipped with a digital camera. The data suggests that increasing the concentration of aggregating agent (streptavidin) significantly increases the recovery of tumor cells (PC3), reaching a maximum at 2 μg / l concentration of streptavidin. These results correlated with free ferrofluid aggregates in solution and ferrofluid aggregates on the cells observed under the microscope. There was no ferrofluid aggregate at 0 μg / l streptavidin, and ferrofluid aggregates increased as the concentration of streptavidin increased. Streptavidin causes ferrofluid aggregation by the multivalent binding of streptavidin to desthiobiotin on different ferrofluid particles. All of these ferrofluid aggregates were reversibly deaggregated by the addition of excess biotin. The principle of this deaggregation of the ferrofluid with excess biotin is due to the substitution of streptavidin for desthiobiotin because it has a higher affinity for streptavidin than desthiobiotin.

Example IV
Recovery of spiked low and high density EpCAM antigen density cells from blood with or without desthiobiotin / EpCAM MAb ferrofluid aggregation Breast cancer cells (SKBR3) have about 7 times higher EpCAM antigen density compared to PC3 cells And selected as a model for high antigen density tumor cells in this example. A known number of SKBR3 or PC3 cells in cell buffer were separately spiked into 1 ml of plasma-free blood in 12 × 75 mm tubes. Ferrofluid dilution-wash buffer (500 μl) and 15 μl PBS containing streptavidin were added to the sample. After mixing the sample, 25 μl of desthiobiotin / EpCAM MAb ferrofluid was added and the blood sample was mixed well and incubated for 15 minutes. After incubation, the tube was placed in a quadrupole magnetic separator for 10 minutes to collect the labeled cells magnetically. The magnetically separated cells were analyzed by fluorometry for tumor cell recovery and by microscopy for cell observation as described in Example II.

  The above data revealed a significant difference in tumor cell recovery between low and high antigen density cells when the ferrofluid aggregating agent streptavidin was not added to the blood sample. Microscopic observation also showed no ferrofluid aggregates in solution or on cells without streptavidin. The addition of streptavidin to the blood sample increased the recovery of low antigen density PC3 cells in proportion to the increase in ferrofluid aggregates in solution and on the cells. On the other hand, there was only a slight difference in the recovery rate of high antigen density SKBR3 cells with or without streptavidin present in the blood sample. There were enough ferrofluid particles on SKBR3 cells, and even without ferrofluid aggregation, they were efficiently collected and all of them were recovered. The ferrofluid aggregates in solution and on the cells were completely disaggregated by adding excess biotin to the sample. In the case of low antigen density cells, there were not enough ferrofluid particles on the cells and they were not efficiently collected by magnetic methods. Ferrofluid aggregation with streptavidin increased the number of particles on these cells, facilitating collection and efficiently obtaining higher recoveries. It is also worth noting that ferrofluid aggregation has increased the recovery of low antigen density cells to near that obtained with high antigen density cells. In other words, there was no significant difference in the recovery of low and high antigen density tumor cells with the addition of the reversible ferrofluid agglutinating agent to the blood sample.

Example V
Inhibition of ferrofluid aggregation by endogenous aggregation factors and generation of controlled ferrofluid aggregation by exogenous aggregation factors In this example, inhibition of all endogenous ferrofluid aggregation factors and addition by exogenous aggregation factors are controlled. A method of producing ferrofluid aggregation is provided. Endogenous ferrofluid aggregation present in a sample can be inhibited by adding various inhibitors to the sample. These inhibitors act on different endogenous aggregation factors, preventing them from causing aggregation by either cross-linking or binding to ferrofluid. Inhibition excludes any change in ferrofluid aggregation between samples. This is because endogenous aggregation factors are present in various samples at various concentrations. Once intrinsic factor-ferrofluid aggregation is prevented, ferrofluid aggregation can be promoted by the addition of exogenous aggregation factors that can effectively facilitate target recovery. Exogenous aggregation can be consistently controlled in all samples and it can be easily reversed.

  Prior to adding the ferrofluid to the blood sample, the blood sample is preincubated with a buffer containing the inhibitor to inhibit endogenous ferrofluid agglutination factor. The antibody-linked ferrofluid contains bovine serum albumin and streptavidin on the surface of the ferrofluid particle in addition to the antibody. Thus, possible ferrofluid aggregation factors include IgM (specific to the crystal surface), human-anti-mouse antibody (HAMA), human-anti-bovine serum albumin antibody (HABAA), human-anti-streptavidin. Etc. If any of the above aggregation factors are present in the plasma, they recognize and bind to the ferrofluid and cause it to aggregate. It is already known that a patient's plasma sample has HAMA and HABAA therein. Obviously, any other component used to produce the ferrofluid can be a target for aggregation and needs to be handled accordingly.

  One of the inhibitors can be a reducing agent such as mercaptoethane sulfonate at 100 mM, which disables IgM-induced aggregation without affecting the ligand used to label the cells. The reducing agent could be added into the blood as a single reagent or could be placed in a blood collection tube. The second inhibitor can be bovine serum albumin, which can be included in the buffer at 10 mg / ml and neutralizes any HABAA. The third inhibitor may be of a suitable isotype that is compatible with non-specific mouse antibodies, particularly antibodies on the ferrofluid. This is included in the buffer at a concentration of 0.5-5 mg / ml to neutralize even the most severe HAMA. The fourth inhibitor can be streptavidin and is included in the buffer to neutralize any anti-streptavidin antibody present in the plasma if necessary. To date, however, there is no information regarding the presence of anti-streptavidin antibodies in plasma.

  Pretreatment of blood with the buffer and reducing agent can be for 15-30 minutes to neutralize all endogenous aggregation factors. After neutralizing all endogenous aggregation factors, exogenous ferrofluid aggregation factors are added to the sample followed by ferrofluid. The ferrofluid is linked to an antibody specific for the target as well as another ligand specific for the exogenous aggregation factor. After optimal labeling of target cells with ferrofluid and aggregation of ferrofluid induced with exogenous agglutination factor, the sample is subjected to magnetic separation to enrich the target. After removing all non-targets, the magnetically labeled target and free ferrofluid are resuspended in a small amount of buffer. Magnetically labeled targets such as cells are rendered permeable and stain intracellular antigens. The sample is then incubated with various staining reagents, depending on the desired analytical method, including flow cytometry or fluorescence or bright field microscopy. After the optimal incubation time, excess staining reagent is removed by a washing step used for magnetic separation. The magnetically labeled cells are then resuspended in a small amount of buffer. The final sample contains free ferrofluid aggregates and aggregates on target cells. The final sample can be used for flow cytometric analysis without further processing. This is because ferrofluid aggregation on the cell surface does not interfere with the analysis. However, ferrofluid aggregation on the cell surface interferes with microscopic analysis. In such cases, exogenous intervening-ferrofluid aggregation should be reversed. This is accomplished by resuspending the final sample in a buffer containing a deaggregation factor that binds to the exogenous aggregation factor. The deaggregation factor deaggregates all ferrofluid aggregates, making it easier to observe and analyze the cells. These methods allow visualization for efficient target recovery and morphology studies.

  Several patents and pending US patent applications are mentioned herein. The entire disclosure of each of these patents and patent applications is expressly incorporated herein and considered a part of this specification.

  While certain preferred embodiments of the invention have been described and described in detail above, it is not intended that the specification be limited to such embodiments. Various modifications can be made thereto without departing from the scope and spirit of the invention as set forth in the claims.

A series of micrographs depicting microscopic observations of blood donor-collected samples with high levels of endogenous aggregation factors compared to those with low levels of endogenous aggregation factors.

Claims (5)

A method of isolating a target biological part from a biological sample by means of the magnetic particles with reduced aggregation of colloidal magnetic particles,
(A) obtaining a biological specimen suspected of containing a non-target biological part and an endogenous aggregating factor together with the target biological part;
(B) contacting the biological specimen with a reagent effective to inactivate any endogenous aggregation factor present in the specimen;
(C) an immunomagnetic suspension comprising a mixture of the specimen and colloidal magnetic particles linked to a biospecific ligand having binding affinity for at least one characterization factor present on the target biological portion And then
(D) subjecting the immunomagnetic suspension to a magnetic field to obtain a target biological part-enriched fraction. A method for isolating a target biological part from a biological sample by the magnetic particles in which the aggregation of colloidal magnetic particles is controlled,
(A) obtaining a biological specimen suspected of containing a non-target biological part and an endogenous aggregating factor together with the target biological part;
(B) contacting the biological specimen with a reagent effective to inactivate any endogenous aggregation factor present in the specimen;
(C) an immunomagnetic suspension comprising a mixture of the specimen and colloidal magnetic particles linked to a biospecific ligand having binding affinity for at least one characterization factor present on the target biological portion Wherein the magnetic particles are further linked to a first exogenous aggregation promoting factor comprising one member of a specific binding pair;
(D) adding a second exogenous aggregation promoting factor to the immunomagnetic suspension to increase the aggregation of the particles, wherein the second aggregation promoting factor causes the other member of the specific binding pair to Containing; then
(E) subjecting the immunomagnetic suspension to a magnetic field to obtain a target biological part-enriched fraction. A method of isolating low antigen density tumor cells from a biological sample by means of the magnetic particles with controlled aggregation of colloidal magnetic particles comprising:
(A) obtaining a biological specimen suspected of containing non-tumor cells and endogenous aggregation factors with the tumor cells;
(B) an immunomagnetic suspension comprising a mixture of the specimen and colloidal magnetic particles linked to a biospecific ligand having binding affinity for at least one characterization factor present on the tumor cell Prepared, the magnetic particle is further linked to a first exogenous aggregation promoting factor comprising one member of a specific binding pair, the factor comprising one member of the specific binding pair;
(C) adding a second exogenous aggregation facilitating factor to the immunomagnetic suspension to increase the aggregation of the particles, the second agglutination facilitating the other member of the specific binding pair Containing; then
(D) The method wherein the sample is purified in a magnetic field to obtain a tumor cell-enriched fraction. A kit for inhibiting endogenous aggregation of colloidal magnetic particles in the processing of a substance for isolation of a target biological part from a biological substance, comprising:
(A) coated magnetic nanoparticles comprising a magnetic core material, a protein-based coating material, and an antibody that specifically binds to the first characterization factor of the target biological moiety, the antibody directly or indirectly Linked to the base coating material;
(B) at least one antibody having binding specificity for a second characterization factor of the target biological moiety;
(C) an endogenous aggregation inhibitor; and (d) a kit comprising a non-cell elimination agent to exclude anucleated cells from the analysis. A kit for isolating low antigen density tumor cells from a biological sample with controlled aggregation of colloidal magnetic particles comprising:
(A) a reagent effective to inactivate endogenous aggregation factors;
(B) coated magnetic nanoparticles comprising a magnetic core material, a protein-based coating material, and an antibody that specifically binds to a first characterization factor of the target biological moiety, the antibody directly or indirectly Linked to a base coating material; the magnetic particles are further linked to a first exogenous aggregation promoting factor, the factor comprising one member of a specific binding pair;
(C) at least one antibody having binding specificity for a second characterization factor of the tumor cell;
(D) a second exogenous aggregation promoting factor, the second aggregation factor comprises the other member of the specific binding pair; and (e) a non-cell elimination agent to exclude anucleated cells from the analysis. The kit containing.

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