JP2009247320A - Cell separation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To recover a trace of septicemia-causing bacteria inexpensively in high efficiency, easily and surely separating and removing a large amount of hematocytes in the blood of a patient of septicemia and to contribute to the improvement in sensitivity of a genetic test of septicemia-causing bacteria. <P>SOLUTION: A method for separating and removing animal cells from a specimen includes a step to immobilize a compound having affinity to a lipid bilayer and containing a hydrophobic part and a hydrophilic part on a carrier, and a step to immobilize the animal cells on the carrier by contacting a specimen containing the animal cells and a pathogen with the carrier. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for separating animal cells from a specimen. More specifically, the present invention relates to a method for separating blood cells and bacteria with high accuracy using a carrier having affinity for a lipid bilayer membrane.

  Among infectious diseases caused by bacteria, improvement of diagnostic and therapeutic methods for bacteremia and sepsis is strongly desired from the viewpoint of the severity and rapidity of the symptoms.

  For the treatment of bacteremia and sepsis, it is first to administer antibiotics and antifungal agents against the causative bacteria. For this purpose, it is important to correctly diagnose sepsis and identify the causative bacteria. Currently, a blood culture method is generally performed in which blood collected from a patient is mixed with an appropriate medium and incubated to confirm the growth of bacteria. In the blood culture method, since it cannot be determined unless the number of bacteria is increased to some extent, it takes 1 to several days to determine. At the time of blood collection, it is not known whether the causative bacteria are aerobic, anaerobic or fungus, and it is difficult to select a medium suitable for each, leading to a decrease in the positive rate. In addition, because sepsis requires prompt treatment, drugs such as antibiotics are often administered without waiting for the identification of the causative bacteria, and even if bacteria are included in the collected blood Often not detected without bacteria. Due to these causes, the positive rate of blood culture methods is very low.

  Furthermore, since only the presence or absence of bacteria in blood is known in blood culture, additional processing is required to identify the causative bacteria. In general, identification culture is often performed by transplanting a blood culture specimen into various selective media. In addition, a method for detecting a specific antibody from a culture solution or a nucleic acid analysis method for detecting a gene sequence peculiar to a bacterium may be used. When these operations are taken into account, it takes at least 1 to 2 days for selective separation of bacteria from sample collection, 1 day for enrichment, 1 day or more for identification operation, and 3 to 4 days in total. In spite of the need for quick and reliable diagnosis, the current situation is that the conventional examination diagnosis method has not been able to cope with it sufficiently.

  Technological development has been carried out with the aim of speeding up such a test method, and a technique for detecting and identifying the bacteria by targeting various bacterial genes from blood has also been developed. In order to detect and identify the causative bacteria directly from blood or through some culture, it is possible to significantly reduce the time compared to the blood culture method (for example, Patent Document 1). The nucleic acid detection method is a method of amplifying and detecting nucleic acid extracted from a specimen. In general, for the extracted nucleic acid, a PCR reaction is performed in a reaction solution containing a primer set for amplifying a specific sequence possessed by a specific bacterial species, and nucleic acid amplification is performed to confirm the amplification product. (For example, patent document 2). On the other hand, since sepsis requires rapid identification of the causative bacteria, it is desirable to detect a wide range of bacterial species simultaneously. For this reason, a method has also been developed in which a gene site possessed by a wide range of bacteria is amplified by nucleic acid by PCR and hybridized with a probe having a gene sequence specific to the bacterial species (for example, Patent Document 3).

  However, at present, no technology has been established that exceeds the performance of blood culture methods. The reason why the blood culture method is still common despite the influence of time and administered drug is that the number of bacteria present in the blood is too small.

  Genetic testing has the advantage of being detected and identified very quickly compared to blood culture, but is less sensitive than blood culture and is still rarely used in general diagnostic methods. This is due to the small amount of blood that can be input for genetic testing. In blood culture, since several ml to 20 ml of blood is cultured, it is highly possible to detect if several bacteria having activity are contained therein. Since the average concentration of bacteria in patients with bacteremia and sepsis is said to be several bacteria / ml on average, it is considered that there is a high possibility that several bacteria are contained in the blood culture solution. On the other hand, since only a few hundred μl to 1 ml of blood can be processed by genetic testing, the number of bacteria contained therein is very small. In addition, in nucleic acid amplification, gene amplification is often not possible up to a detectable amount unless starting from a certain number of bacteria, and therefore it is highly possible that amplification is not sufficient with several bacteria. In addition, nucleic acid amplification is performed in genetic testing, but it is desirable to suppress the total amount of nucleic acid to be input to this amplification process to about several hundred ng. If the amount of nucleic acid is too large, the efficiency of amplification is greatly reduced. Since blood contains many cells having human genomic nucleic acids such as leukocytes, the amount of blood available for genetic testing is limited to about 1 ml at most. In order to achieve the same sensitivity as blood culture, it is desirable to extract genes from blood several times the current level, but this increases the amount of nucleic acid derived from the human genome, and nucleic acid amplification is not performed normally. Therefore, it is necessary to increase the amount of the sample to be put into the test by separating only the bacterial cells contained in the blood.

In general, in order to separate bacteria in a solution, a filtration method using a nonwoven fabric or centrifugation is performed. However, the filtration using a nonwoven fabric is not useful because red blood cells and white blood cells, which occupy most of the blood components, are immediately clogged, and finer bacteria than these are not allowed to permeate. In addition, in the centrifugal separation, since the density of both blood cells and bacteria is very similar, it is difficult to accurately separate them. In order to overcome such a problem, for example, a solid phase separation method in which target cells are affinity-separated using a carrier on which an antibody that binds to a cell surface antigen is immobilized is effective (for example, Patent Document 4). . Such a carrier on which an antibody is immobilized is affinity separation based on the difference in antigens that are clearly different depending on the cell type, so high resolution (strict separation) is expected, but there are problems in terms of cost and separation efficiency. is there. Specifically, in addition to the fact that the antibody itself is very expensive, generally the antigen species and the expression level of the antigen differ depending on the cell type, so in order to trap blood cells contained in the blood almost completely Therefore, it is necessary to use a large amount of plural kinds of antibodies. Furthermore, since an antibody is a protein having a large molecular weight, the occupation density on the carrier is low and there is a limit to increasing the density, so it is difficult to ensure a sufficient binding force between the cell and the carrier. Therefore, due to the problems described above, it is not realistic to apply the antibody-immobilized carrier as a means for trapping blood cells from the blood of a septic patient.
JP 05-049477 A Japanese Patent Application Laid-Open No. 06-077071 JP 2004-3131181 A JP 2005-253465 A

  The present invention is a cell separation method for solving the above problems in the background art (particularly, the problem in affinity separation methods using antibody-immobilized carriers). That is, an object is to collect a large amount of blood cells contained in the blood of a septic patient at a low cost, in a simple and reliable manner, and to recover sepsis-causing bacteria contained in a very small amount with high efficiency. Furthermore, the purpose is to provide high sensitivity for genetic testing of septic bacteria.

  The present inventors contact a carrier having a substance having affinity for a lipid bilayer membrane with a specimen containing animal cells and a pathogen, and fix the animal cells on the carrier, thereby making the animal cells highly efficient. It was found that pathogens can be recovered with high efficiency.

In the present invention, the substance having affinity for a lipid bilayer specifically refers to a hydrophilic-hydrophobic composite compound. It is considered that the hydrophilic-hydrophobic complex compound is adsorbed so as to be oriented at the hydrophilic-hydrophobic interface of the lipid bilayer membrane by a surface active action. Not limited to blood cells, animal cells are outermost layer lipid bilayers, hydrophilic groups of the outer surface of the lipid bilayer membrane is mainly choline ^{_{(-CH 2 CH 2 N + (}} CH 3) 3) small, such as Consists of molecules. Since there is almost no steric hindrance, it is considered that the hydrophilic-hydrophobic complex compound is efficiently adsorbed on the lipid bilayer membrane. On the other hand, except for some bacteria such as mycoplasma and phytoplasma, almost all bacteria have cell walls. Bacteria are roughly classified into two types, Gram-positive bacteria and Gram-negative bacteria. The cell wall of Gram-positive bacteria has a thick peptidoglycan layer with a thickness of about 100 nm. In the cell wall of Gram-negative bacteria, the outermost layer is LPS (Lipopolysaccharide). In both gram-positive and gram-negative bacteria, the lipid bilayer membrane (inner membrane) is in an encapsulated state. In addition, both peptidoglycan and LPS are considered to be hydrophilic substances as estimated from the molecular structure. In other words, since the cell wall on the surface of the bacteria is a hydrophilic barrier with great steric hindrance, it becomes difficult for the hydrophobic part of the compound to reach the internal lipid bilayer, and it is difficult to anchor to the cell wall of the cell. I can expect things. The present inventors paid attention to the difference in the outermost surface structure between bacteria and blood cells, and reached the cell affinity separation method of the present invention.

  Substances having affinity for lipid bilayers are described in K. Kato, et.al, Biotechnol. Prog. , 20, 897-904 (2004), as known as a BAM (biocompatible anchor for cell membrane) reagent. As a specific molecular structure of a BAM reagent, a compound in which an active ester group such as an NHS (N-hydroxysuccinimide) ester group is modified at the end of a polyethylene glycol chain of a molecule in which a polyethylene glycol chain and an oleyl group are linearly bonded It is introduced in. In the above document, it has been confirmed that the BAM molecule can be modified to the mouse cell membrane by modifying the fluorescent dye molecule or protein to the NHS group of the BAM molecule.

  A carrier on which the BAM reagent is immobilized is also known (WO2003 / 074691). In this document, as an inexpensive and simple method for immobilizing floating cells, phospholipid vesicles, etc. on a solid surface, a method of immobilizing cells by contacting the cells with a carrier having a hydrophobic chain and a hydrophilic chain Is described. Specifically, a cell array or the like is constructed by binding the NHS group of the BAM molecule to the amino group of the solid phase. However, although these known documents describe techniques for cell surface modification and cell immobilization, there is no description of affinity separation techniques as in the present invention.

  Further, in completing the present invention, a technique for improving the bacteria recovery rate while maintaining a high blood cell removal rate has been devised.

  The first is to subject the carrier on which the hydrophilic-hydrophobic complex compound is fixed to a surface treatment, that is, an anionization treatment. Even if the carrier to which the hydrophilic-hydrophobic complex compound is immobilized and the bacterium may interact somewhat, all the bacteria have a negative charge. Therefore, by increasing the negative charge of the carrier, The repulsion is increased and the undesirable interaction of bacteria with the carrier can be reduced.

  Secondly, the density of the hydrophilic-hydrophobic complex compound acting on the cells per unit area of the carrier is adjusted. This is because even if the bacterium interacts with the hydrophilic-hydrophobic complex compound, if the hydrophilic-hydrophobic complex compound is adjusted to the minimum necessary amount, the interaction with the bacterium is reduced accordingly. One of the methods is to make the hydrophilic-hydrophobic complex compound and the hydrophilic molecule react with the carrier competitively. Simply reducing the reaction concentration of the hydrophilic-hydrophobic complex compound to an extreme would reduce the number of hydrophilic-hydrophobic complex compounds per unit area of the carrier. However, since the surface of the substrate is exposed, there is a possibility that the part may interact with bacteria. Therefore, non-specific adsorption can be prevented if a hydrophilic molecule such as a PEGylation reagent is present in the exposed portion. As another method, if a trace amount of a component that binds to the hydrophilic-hydrophobic complex compound, specifically serum (mainly albumin) is present in the sample, the hydrophilicity on the carrier that can bind to the cell- Since the number of hydrophobic complex compounds can be reduced, it is possible to prevent undesirable interactions with bacteria.

  When a magnetic material is used as a carrier for immobilizing the hydrophilic-hydrophobic composite compound, it is possible to use a particle, network, fiber, plate or porous material. When a magnetic substance is not used, the carrier for fixing the hydrophilic-hydrophobic composite compound may be planar (for example, a substrate). If a fluid flow type device is applied, it is also possible to fix and use a hydrophilic-hydrophobic composite compound on the wall surface of the flow path or the wall surface of the chamber.

  In the present invention, a carrier having a substance having affinity for a lipid bilayer immobilized thereon binds to a lipid bilayer that is necessarily present on the surface of almost all types of blood cells, so only one type of carrier needs to be used. Since it is not necessary to prepare a plurality of types of carriers unlike an antibody solid carrier, the cost can be reduced. In addition, since the substance having affinity for the lipid bilayer membrane can be applied with a substance having a molecular weight smaller than that of the antibody, the substance on the carrier can be made denser than the antibody. Therefore, sufficient binding force to cells can be ensured rather than antibodies. From the above effects, the cell separation method of the present invention can achieve affinity separation between bacteria and blood cells at low cost and high efficiency. Furthermore, the recovery rate of pathogenic bacteria can be improved by increasing the anionicity of the carrier.

  A step of immobilizing a compound containing a hydrophobic part and a hydrophilic part, which has affinity for a lipid bilayer membrane, on a carrier; and contacting a specimen containing animal cells and a pathogen with the carrier; The animal cell can be separated and removed from the specimen by a method comprising the step of fixing the cell to the carrier.

  In the present invention, the substance having affinity for the lipid bilayer membrane is, for example, a compound containing a hydrophobic portion and a hydrophilic portion, that is, a hydrophilic-hydrophobic composite compound. Specifically, it can also be said to be a surfactant molecule that can be immobilized on a solid phase. In the case of a general surfactant, it has the ability to form micelles, so the cell membrane is destroyed.However, the surfactant molecules are constrained by fixation, and micelles cannot be formed. It can be fixed.

As a specific chemical structure of a substance having affinity for the lipid bilayer membrane, it is desirable to have a hydrophobic group that can be anchored to the hydrophobic portion of the lipid bilayer membrane that is a cell membrane. It may be a single chain or a plurality of chains, and is appropriately selected from saturated or unsaturated hydrocarbon chains and complex lipid chains which may have a substituent. For example, a saturated or unsaturated hydrocarbon chain having 6 to 22 carbon atoms, such as a saturated alkyl group such as a myristyl group (C ₁₄ ), a palmityl group (C ₁₆ ), a stearyl group (C ₁₈ ), or an oleyl group Examples thereof include unsaturated hydrocarbons such as (CH ₃ (CH ₂ ) ₇ CH═CH (CH ₂ ) ₈ ). Among them, those having 14 to 18 carbon atoms including linole group, linoleyl group and the like are exemplified as preferable examples. Further examples include phospholipids and cholesterol. Further, these hydrophobic chains may have a heteroatom such as N, S, O, or a substituent. Among these, an oleyl group which is a part of phospholipid constituting the biological membrane is preferable. On the other hand, the hydrophilic chain is selected from proteins, oligopeptides, polypeptides, polyacrylamides, polyethylene glycols, dextran and other polysaccharides, or polymers and copolymers of glycolic acid derivatives, lactic acid derivatives, and p-dioxane derivatives. The From the viewpoint of biocompatibility, polyethylene glycol (PEG) is preferred. Such a hydrophilic chain may further have a functional group or may be chemically modified. For example, succinic acid is bonded to a terminal to form a carboxyl group, or an activity such as NHS (N-hydroxysuccinimide). It may have an ester group. When the hydrophilic chain is chemically modified in this manner, the hydrophilic-hydrophobic complex compound can be bound to the solid phase. The length of the hydrophilic group, for example, in the case of a polyethylene glycol _{chain, - (CH 2 CH 2 O} ) n - group requires a certain length. This is because the membrane protein exists in a state of protruding from the outer surface of the lipid bilayer membrane and causes the steric hindrance by the amount of protrusion. Specifically, the required length is desirably n is 10 or more.

In the present invention, the carrier on which the substance having affinity for the lipid bilayer membrane is fixed is further subjected to surface treatment, that is, anionization treatment. The anionic group modified by the anionic treatment is not limited as long as it is anionic. In particular, a carboxyl group (—COOH), a sulfone group (—SO ₃ H), or a sodium salt or potassium salt of these functional groups can be preferably applied. If the functional group present on the substrate is an amino group, the functional group that reacts with the amino group is an active ester such as an NHS (hydroxysuccinimide) ester group, a formyl group, a carboxyl group, an isocyanate group, etc. And a homo- or hetero-crosslinking agent having an anionic group at the other end can be used. Alternatively, the reaction of a cyclic acid anhydride such as succinic anhydride to carboxylate the surface can also be suitably applied. In addition, a crosslinking agent having a functional group that can be bonded to the base at the other end and another reactive group such as a maleimide group at the other end is competitive with the hydrophilic-hydrophobic composite compound. A method of making the surface anionic by reacting the maleimide group with an anionic reagent after reacting with a solid phase is also conceivable, but is not limited thereto. In addition, it is possible to achieve an anionic treatment by reacting various functional groups on the substrate other than amino groups with various appropriate crosslinking agents capable of reacting with the functional groups. .

  The anionization method can be performed by anionic treatment after immobilizing a substance having affinity for the lipid bilayer membrane on the base material, and an anionic property with the substance having affinity for the lipid bilayer membrane. It is also possible to react with the base material competitively.

  According to Coulomb's law, charged particles having the same sign increase in force generated in proportion to their charges. However, the surface potential of the carrier obtained by the anionic treatment is -10 to- It is desirable to be within the range of 100 mV. If the surface potential is too low, a sufficient repulsive force with bacteria cannot be ensured. Conversely, if the surface potential is too high, blood cells have a negative charge, so that a sufficient binding force to the carrier cannot be ensured. In addition, as long as the substance having affinity for the lipid bilayer membrane has a negative charge in a desired range at the time of fixing to the carrier, there is no need to darely perform the anionic treatment.

In the present invention, a hydrophilic treatment may be mentioned as a method for adjusting the density of the hydrophilic-hydrophobic composite compound of the carrier. The hydrophilization treatment in the present invention is effective for preventing an undesirable interaction with bacteria on the surface of a substrate exposed by lowering the fixing density of the hydrophilic-hydrophobic complex compound on the carrier. It is a technique. Specifically, a PEGylation reagent can be preferably used. By reacting a reaction liquid in which a PEGylation reagent and a hydrophilic-hydrophobic complex compound are mixed with a substrate, the density of the hydrophilic-hydrophobic complex compound on the carrier can be adjusted effectively. It becomes possible. The mixing molar ratio in the reaction solution of the hydrophilic-hydrophobic complex compound and the PEGylation reagent is 0 to 1 for the PEGylation reagent with respect to the substance 1 having affinity for the lipid bilayer membrane. Preferably, it exists in the range of 0.1-0.5. The Pegi configuration _{reagent, CH 3 O (CH 2 CH} 2 O) n -X-NHS, CH 3 O (CH 2 CH 2 O) n -X-CHO, CH 3 O (CH 2 CH 2 O) n - X-SH (X is various linkers; —CH ₂ —, —CO—CH ₂ —CH ₂ —COO—, —CO—CH ₂ —CH ₂ —CH ₂ —COO—, etc.) can be preferably used. However, it is not limited to these.

  In the present invention, as another method for adjusting the density of the hydrophilic-hydrophobic complex compound of the carrier, a component that binds to the hydrophilic-hydrophobic complex compound, specifically, serum (mainly albumin) is extremely present in the sample liquid. It only needs to be present in trace amounts. Since the density of the hydrophilic-hydrophobic complex compound on the carrier capable of binding to cells is reduced, it becomes possible to prevent undesirable interactions with bacteria. The serum concentration in the specimen that begins to inhibit the binding between the hydrophilic-hydrophobic complex compound and blood cells is about 3%. Moreover, since about 10% of albumin is contained in serum, the concentration of albumin contained in 3% of serum in the sample is about 0.3% with respect to the sample. Although it seems to depend on the type of carrier and hydrophilic-hydrophobic complex compound, if the sample has a serum concentration of less than 3% and an albumin concentration of less than 0.3%, the binding force of the carrier to blood cells is not reduced. The number of hydrophilic-hydrophobic complex compounds per unit area of the carrier can be reduced. In other words, it is possible to prevent undesirable interactions with bacteria. Further, blood containing blood cells and bacteria is subjected to powerful centrifugal force (1000 to 20000 rpm), and all cells contained in the blood are completely settled on the bottom of the container, and the supernatant is removed to remove PBS (phosphate buffered saline). The operation of replacing with an appropriate buffer such as is performed a plurality of times. As a result, it is possible to adjust the serum concentration and albumin concentration within the above ranges without losing almost all cells contained in the blood.

  In the present invention, the shape of the carrier (base material) for immobilizing a substance having affinity for the lipid bilayer is a particle that can increase the surface area in order to achieve a high removal rate of blood cells. It is possible to use a shape (may be a mesh, a fiber, a plate, or a porous piece). Furthermore, if the base material is made of a magnetic material such as iron oxide (III), chromium iron oxide (ferrichrome, FeCr), cobalt iron oxide, metal magnetic material, barium ferrite (BaFe), etc., it can be easily collected with a magnet. It can be simplified and automated. On the other hand, the shape of the carrier (base material) for immobilizing the substance having affinity for the lipid bilayer membrane does not necessarily need to be in the form of particles as described above. For example, it may be planar, and if a fluid flow device is applied, it is possible to fix and use a hydrophilic-hydrophobic composite compound on the wall surface of the flow path or the wall surface of the chamber. With the carrier having such a shape, high resolution can be achieved by placing the carrier horizontally and dropping a specimen containing bacteria and blood cells from the top. The reason is that blood cells (white blood cells are 10 to 20 μm) are larger than bacteria (several μm), so that the gravity applied to the blood cells is larger. Therefore, when the specimen is dropped and allowed to stand for a certain period of time, the number of blood cells in contact with the surface of the carrier on which the hydrophilic-hydrophobic composite compound is fixed becomes larger than that of bacteria. Particularly in the fluid flow system, the sample is introduced from the inlet of the chamber shown in FIG. 1 and allowed to stand until an appropriate time for the blood cell to sink, and then the sample is sucked and discharged from the chamber outlet. The sample can collect bacteria almost completely and has the effect that the number of blood cells is drastically reduced (FIG. 2).

  The animal cell in the present invention is a cell whose outermost surface is a lipid bilayer and includes not only leukocytes but also erythrocytes and platelets. Since the subject of the present invention is that a large amount of leukocyte-derived human genome is brought into the PCR template, it is sufficient that leukocytes having nuclei can be removed. However, since erythrocyte lysate is known to inhibit PCR, it is effective if the erythrocytes can also be removed before the nucleic acid extraction step, since the purification load in the nucleic acid extraction step can be reduced.

  Moreover, although pathogenic bacteria and viruses can be mentioned as pathogens, in the present invention, it refers to pathogenic bacteria. However, with regard to viruses, if affinity separation is carried out with a fluid flow device as shown in FIGS. 1 and 2, the virus is overwhelmingly smaller than bacteria and is in a dispersed state with little gravity, so that a high affinity can be obtained. It is believed that separation can be achieved.

  In the examples of the present invention, the BAM reagent (SUNBRIGHT OE-020CS; manufactured by NOF Corporation) shown in FIG. 3 was used as the substance having affinity for the lipid bilayer membrane. Qualitative evaluation (Example 1) of affinity separation between bacteria and blood cells using the BAM reagent was performed using a glass bottom culture dish as the BAM reagent carrier, and quantitative evaluation (Example 2) was performed using surface amino acids. The magnetic beads were used as the BAM reagent carrier.

<Example 1; qualitative evaluation>
Whether or not cells (white blood cells / bacteria) bind to the BAM coated glass was verified by direct microscopic observation. The cells used in the experiment were K562 cells cultured at 37 ° C. for 3 days in RPMI 1640 medium containing 10% FBS for leukocytes. Before being used in the experiment, K562 cell culture solution was used that was substituted with PBS three times (1000 rpm, 3 minutes). After centrifugal replacement, it was diluted with PBS to prepare a concentration of 10 ⁶ cells / ml. The bacteria used were Staphylococcus aureus for Gram-positive bacteria and Escherichia coli for Gram-negative bacteria. Both bacteria were used after culturing at 37 ° C. overnight on a normal bouillon agar plate medium. Bacteria after culturing were prepared by colony picking and suspending in PBS to a concentration of 10 ⁶ cells / ml.

(I) BAM binding carrier production method (BAM-coated glass bottom culture dish)
A glass bottom culture dish (glass portion dia. 10 m; manufactured by MatTek) was used as a BAM binding carrier for direct observation. The glass part is physically adsorbed with BSA (Alubumin from bovine serum powder, Fatty acid free, low endotoxin; Sigma), and then the NHS group of the BAM reagent shown in FIG. 3 is covalently bonded to the amino group of BSA. Was fixed to the glass bottom portion. In order to confirm the effect of BAM, a glass bottom culture dish with only BSA coating was used as a reference for the fact that cells were not fixed (nonspecific adsorption did not occur).

In the coating process, first, 200 μl of 0.1% BSA / PBS was dropped on the glass bottom portion of the glass bottom culture dish, left overnight at room temperature, then rinsed 5 times with pure water, and dried with N ₂ spray. . Next, a 10 mM BAM / DMSO solution was diluted 100 times with PBS to 100 μM, and 200 μl of the solution was dropped on the BSA-coated glass bottom portion and allowed to react at room temperature for 1 hour. Thereafter, it was rinsed 5 times with pure water and dried with N ₂ spray to obtain a BAM-bound carrier.

(Ii) BAM binding carrier and cell binding conditions (BAM coated glass bottom culture dish)
The cell / PBS solution was dropped onto the BAM-coated glass bottom culture dish prepared above, left standing at room temperature for a certain period of time, and then washed with PBS to observe whether the cells were fixed on the BAM-coated surface.

  To explain in detail, 100 μl of cell / PBS solution is dropped on the BAM coated glass bottom and left for 30 minutes. After removing the solution, 200 μl of new PBS is dropped and washed by pipetting three times. did. Subsequently, 100 μl of PBS was dropped and the glass bottom surface coated with BAM was observed with a Keyence Biozero microscope.

(Iii) BAM binding carrier and cell binding results (BAM coated glass bottom culture dish)
A photograph of the glass bottom surface at the time of observation is shown in FIG. Looking at the incubation result of K562, which is a white blood cell (FIG. 4), K562 cells were removed by washing on the BSA-coated surface, but were not removed by washing on the BAM-coated surface, and remained almost bound and remained. . On the other hand, S. aureus and E. coli were washed on the BSA-coated surface, but binding could not be observed on the BAM-coated surface. Therefore, it was confirmed that blood cells bind to the BAM molecule, but gram-positive bacteria and gram-negative bacteria do not bind to bacteria. The reason why bacteria do not bind to the BAM-coated surface is considered to include some factors that did not ensure sufficient pressure on the BAM-coated surface necessary for binding because the gravity applied to the bacteria was small in this experimental system. However, it was clear that the BAM molecule has affinity for K562 cells having a lipid bilayer on the outermost surface, and has little affinity for bacteria whose resurface is the cell wall.

<Example 2; quantitative evaluation>
In the qualitative evaluation, since quantitative discussion was difficult, using a carrier in which a BAM reagent was fixed to magnetic beads so that the contact conditions on the BAM coated surface were the same for leukocytes and bacteria, the blood cell removal rate, Quantitative measurement of fungal recovery was performed. Here, using a commercially available anti-hemocyte antibody-immobilized bead as a comparative example, the affinity separation performance of BAM-modified magnetic beads and further BAM-modified magnetic beads subjected to anionization treatment was compared.

(I) Preparation method of carrier 1 (BAM-modified magnetic beads without anionization treatment) Surface-aminated magnetic beads (trade name: M-PVA N12, particle size 1 to 3 μm, manufactured by Chemagen) 100 μl (5 mg) in a microtube And remove the stock solution. Subsequently, 500 μl of 0.1% BSA / PBS solution was added and stirred at room temperature for 3 hours, followed by rinsing 5 times with pure water. Next, 500 μl of a 100 μM BAM solution (10 mM BAM / DMSO diluted with PBS) was added, stirred at room temperature for 1 hour, and then rinsed 5 times with pure water. Finally, it was suspended in 500 μl of ultrapure water to obtain BAM-modified magnetic beads. The surface potential of this carrier was −4.0 mV (the measurement apparatus used was Zetasizer 3000; Malvern Instruments Ltd.).

(Ii) Method for producing carrier 2 (succinic anhydride-treated BAM magnetic beads) 500 μl of BAM magnetic beads produced with carrier 1 are placed in a microtube and pure water is removed. Subsequently, 500 μl of a 1 mM succinic anhydride solution (10 mM succinic anhydride / DMSO diluted with PBS) was added, stirred at room temperature for 30 minutes, and then rinsed 5 times with pure water. Finally, it was suspended in 500 μl of ultrapure water to obtain an anionic BAM-modified magnetic bead introduced with a carboxyl group. The surface potential of this carrier was −17.6 mV (the measurement apparatus used is Zetasizer 3000; Malvern Instruments Ltd.).

(Iii) Preparation method of carrier 3 (sulfonated BAM magnetic beads) Surface aminated magnetic beads (trade name: M-PVA N12, particle size 1 to 3 μm, manufactured by Chemagen) 100 μl (5 mg) was taken in a microtube, Remove the stock solution. Subsequently, 500 μl of 0.1% BSA / PBS solution was added, stirred at room temperature for 3 hours, and then rinsed 5 times with pure water. Next, 500 μl of BAM / NHS-PEG-maleimide crosslinker solution (3.75 μl of 10 mM BAM / DMSO + 1.25 μl of 10 mM MAL-dPEG ™ NHS ester (FIG. 5, Toyobo) / DMSO + 495 μl PBS) was added. And then rinsed 3 times with pure water to obtain beads having maleimide groups and BAM introduced on the surface. Subsequently, 500 μl of 100 μM sodium 3-mercapto-1-propanesulfonate (Sigma) / PBS was added, stirred at room temperature for 1 hour, rinsed 5 times with pure water, and finally suspended in 500 μl of pure water. An anionic BAM-modified magnetic bead having a sulfonated maleimide group was obtained. The surface potential of this carrier was −38.2 mV (the measurement apparatus used was Zetasizer 3000; Malvern Instruments Ltd.).

(Iv) Method for Producing Carrier 4 (Dextran Sulfate Treated BAM-Beaded Magnetic Beads) 500 μl of BAM-made magnetic beads produced with Carrier 1 are placed in a microtube and pure water is removed. Subsequently, 500 μl of dextran sulfate sodium solution (10 mM sodium dextran sulfate (molecular weight 5000, manufactured by Wako Pure Chemical Industries, Ltd.) / H ₂ O) was added, and the mixture was stirred at room temperature for 60 minutes, and then rinsed 5 times with pure water. Finally, it was suspended in 500 μl of ultrapure water to obtain anionic BAM-modified magnetic beads whose surface was covered with a hydrophilic polymer having a sulfone group. The surface potential of this carrier was −46.4 mV (the measurement apparatus was Zetasizer 3000; Malvern Instruments Ltd. was used).

(V) Preparation procedure before use of carrier 5 (anti-CD33 antibody-bound magnetic beads): Comparative Example 50 μl of a cell / PBS sample was placed in a microtube, and 200 μl of FcR blocking reagent (Miltenyi Biotech) was added. Mixed. Further, 200 μl of CD33 antibody-bound magnetic beads (particle size 50 nm, manufactured by Miltenyi Biotech) were added and stirred and mixed, and then incubated at 4 ° C. for 15 minutes to label the magnetic beads on the cell sample (total The liquid volume is 450 μl). The surface potential of the carrier in the FcR blocking reagent was −6.3 mV (the measurement apparatus used was Zetasizer 3000; Malvern Instruments Ltd.).

(Vi) Binding conditions for each carrier and cells (BAM-modified magnetic beads, anti-hemocyte antibody magnetic beads)
The prepared BAM-modified magnetic beads and the prepared anti-CD33 antibody-bound magnetic beads are mixed with cells / PBS and allowed to stand at room temperature for a certain period of time. By measuring the cell number, the blood cell removal rate and the bacteria recovery rate were calculated by comparing with the initial sample cell concentration. In addition, the cell used for experiment used 1 type of white blood cells and 1 type of bacteria, and used K562 cells cultured at 37 ° C. for 3 days in RPMI 1640 medium containing 10% FBS. Prior to use in the experiment, K562 cell culture medium was used after centrifugal replacement with PBS (1000 rpm, 3 minutes). After centrifugal replacement, it was diluted with PBS to prepare a concentration of 10 ⁶ cells / ml. On the other hand, for bacteria, Staphylococcus aureus cultured overnight at 37 ° C. in a normal bouillon agar plate medium was used. Bacteria after culturing were prepared by colony picking and suspending in PBS to a concentration of 10 ⁶ cells / ml.

  To describe the detailed binding conditions, for each type of BAM magnetic beads, 200 μl of beads are put into a microtube for each type, and after collecting the beads on the wall of the tube with a magnetic stand (Magna-Sep; manufactured by Invitrogen), store water. Removed and suspended in 50 μl cells / PBS. Since it settles due to its own weight, it was left at room temperature for 20 minutes while stirring with a pipette every 5 minutes, and the beads were collected on the wall surface of the tube with the magnetic stand, and the transparent residual liquid was collected.

  For the anti-CD33 antibody-bound magnetic beads, 2 ml of cell separation buffer (PBS pH 7.2, 0.5% BSA, 2 mM EDTA, degassed) was injected using an LD column (Miltenyi Biotech) for 10 minutes. The column was rinsed by allowing it to stand. Next, 450 μl of the magnetic bead-labeled cell sample obtained in the above step and 1 ml of cell separation buffer were injected twice, and the eluate from the column was used for counting the number of cells (cells contacted with anti-CD33 magnetic beads). Time was about 20 minutes).

  Regarding the evaluation method, K562 cells were counted using CELLOMETERTM (manufactured by Nexus), a cell counter, and Staphylococcus aureus was seeded with a colony count method (a sample diluted in a normal bouillon agar medium with a spreader). After overnight culture, the number of cells was counted using colonies.

(Vii) Results of affinity separation between each carrier and cells (BAM-modified magnetic beads, anti-hemocyte antibody magnetic beads)
FIG. 6 shows the result of affinity separation of K562 white blood cells by each carrier, and FIG. 7 shows the result of affinity separation of S. aureus by each carrier.

  First, for K562 white blood cells (FIG. 6), the carrier 5 anti-CD33-immobilized magnetic beads have a K562 cell removal rate of 67.2%, while the carrier 1, 2, and 4 except for the carrier 3 have BAM-modified magnetic properties. All the beads showed a very high K562 cell removal rate of 99% or more. Note that the amount of K562 cells in the remaining liquid was directly counted because it was so small that it could not be measured with a calculation board. In the sepsis diagnosis that is the subject of the present invention, the human genome that does not inhibit PCR is about several hundred ng, and the leukocyte removal rate corresponding to it is preferably 99% or more. Based on this, it is suggested that the antibody-fixed beads of the carrier 5 cannot secure the sensitivity of PCR in sepsis diagnosis.

For S. aureus (FIG. 7), the recovery rate of S. aureus is proportionally increased in proportion to the zeta potential (R ² = 0.8224, proportional expression y = −0.4162x + 68.376). This means that the repulsive force between negative charges is increased by Coulomb's law because Staphylococcus aureus is negatively charged particles and beads are also negatively charged particles. Therefore, it was found that the anionization treatment of the carrier is effective for improving the bacteria recovery rate. In this example, the recovery rate of 88.4% was the highest, but in order to make the recovery rate (y) 100%, the zeta potential (x) must be −75 mV or more from the proportional formula. I understand. In order to achieve 100% recovery, it is necessary to construct an anionic treatment method according to the zeta potential.

  In this example, the state in which the BAM-conjugated magnetic beads (carrier 1) were bound to K562 cells could be photographed, and a photograph thereof is shown in FIG. When the magnet is brought close to the BAM-conjugated magnetic bead-bound K562 cell in the state of FIG. 8 under the microscope observation, the BAM-conjugated magnetic bead moves toward the magnet without being detached from the K562 cell and without breaking the K562. I was able to confirm that. Even particles as large as 1 to 3 μm were not peeled off from the cells, so it was confirmed that they were bound quite firmly.

It is a figure which shows the fluid flow device which the substance which has affinity to a lipid bilayer couple | bonded with the chamber bottom face. It is a figure which shows the mode of affinity separation of bacteria and a blood cell using the fluid flow device with which the substance which has affinity for a lipid bilayer couple | bonded with the chamber bottom face. It is a figure which shows the BAM reagent (SUNBRIGHT OE-020CS) used in the Example. It is a figure which shows the BAM application | coating surface after K562 cell incubation. It is a figure which shows MAL-dPEG (trademark) NHS ester (PEG chain length 12). It is a figure which shows the affinity separation result (K562 blood cell) with various carriers. It is a figure which shows the affinity isolation | separation result (Staphylococcus aureus) with various support | carriers. It is a figure which shows a mode that the BAM-ized bead and K562 cell have couple | bonded.

Explanation of symbols

1. 1. Fluid flow device 2. Chamber inlet channel Chamber (A substance having affinity for lipid bilayer is bound to the bottom)
4). 4. Chamber outlet channel 5. Substance having affinity for lipid bilayer membrane Carrier (base material)
7). Blood cells (white blood cells)
8). Pathogen (bacteria)
9. Gravity on blood cells 10 Gravity on the pathogen (gravity is greater for blood cells)

Claims (17)

A step of immobilizing a compound containing a hydrophobic part and a hydrophilic part, which has affinity for a lipid bilayer membrane, on a carrier; and contacting a specimen containing animal cells and a pathogen with the carrier; Fixing the cells to the carrier;
A method for separating and removing animal cells from a specimen, characterized by comprising:   The method according to claim 1, wherein the hydrophilic portion has a functional group capable of binding to a solid phase.   The method according to claim 1, wherein the hydrophilic portion is a polyethylene glycol chain. The method according to claim 3, wherein the polyethylene glycol chain is a — (CH ₂ CH ₂ O) _n — unit, and n is 10 or more.   The method according to claim 1, wherein the hydrophobic portion is a hydrocarbon chain having 6 to 22 carbon atoms.   The carrier on which the compound having affinity for the lipid bilayer membrane is immobilized is surface-treated so as to have a difference in binding force to animal cells and binding force to pathogens. Method.   The method according to claim 6, wherein the surface treatment is an anionic treatment.   The method according to claim 7, wherein the carrier obtained by the anionization treatment has a zeta potential in the range of −10 to −100 mV.   The method according to claim 6, wherein the surface treatment is a hydrophilic treatment.   The method according to claim 9, wherein the hydrophilization treatment is performed with a PEGylation reagent.   The method according to claim 1, wherein the specimen containing animal cells and pathogen contains serum in a range of up to 3% of the specimen.   The method according to claim 1, wherein the specimen containing animal cells and pathogen contains albumin in a range of up to 0.3% relative to the specimen.   The method according to claim 1, wherein the carrier is in the form of particles, meshes, fibers, plates, or porous bodies.   The method according to claim 13, wherein the carrier is a magnetic substance.   The method of claim 1, wherein the carrier is planar.   The method of claim 1, wherein the animal cell is a blood cell.   The method according to claim 1, wherein the pathogen is a pathogenic bacterium.