WO2023043256A1 - Leukocyte-derived immunomagnetic particle and use thereof - Google Patents

Abstract

The present invention relates to a leukocyte-derived immunomagnetic particle and use thereof. More particularly, the present invention relates to an immunomagnetic particle, a method for detecting a pathogenic substance by using the immunomagnetic particle, and a method for diagnosing and treating an infectious disease, wherein the immunomagnetic particle comprises: a cell membrane derived from leukocytes capable of capturing pathogenic substances; and magnetic particles attached to the cell membrane. The leukocyte-derived immunomagnetic particle according to an aspect includes a cell membrane derived from leukocytes capable of capturing pathogenic substances so that side effects in a living body can be minimized, and due to the characteristics of the leukocytes from which the cell membrane is derived, various types of pathogenic substances can be detected. In addition, by including magnetic particles, the immunomagnetic particle can be simply separated by applying a magnetic field thereto, and thus pathogenic substances can be more effectively detected and removed.

Description

Leukocyte-derived magnetic immune particles and uses thereof

The present invention relates to leukocyte-derived magnetic immune particles and uses thereof. This patent application claims priority to Korean Patent Application No. 10-2021-0125199 filed with the Korean Intellectual Property Office on September 17, 2021, the disclosure of which is incorporated herein by reference.

Worldwide, pathogens such as pathogenic microorganisms or antigens derived therefrom infect water systems and humans, causing many diseases.

Conventional methods developed to detect pathogens present in blood have disadvantages such as long detection time or low detection efficiency. For example, a cell culture method and a polymerase chain reaction method (PCR), a gene detection method, are being developed to detect microorganisms (Korean Patent Publication No. 10-2018-0023545). The cell culture method is a method for separating and identifying pathogenic viruses and non-pathogenic viruses, and is judged by whether or not Cytopathic Effect (CPE) occurs. In general, it takes 1 to 4 weeks for the cytopathic effect to be seen. Therefore, the cell culture method is less effective in determining whether harmful microorganisms exist and preparing countermeasures therefor. In addition, the polymerase chain reaction method, which is one of the genetic diagnosis methods, is a method of amplifying a small amount of DNA or RNA and has excellent sensitivity, specificity, and rapidity compared to the cell culture method, and can overcome the disadvantages of the cell culture method. When harmful microorganisms are present or a large amount is lost during the nucleic acid extraction process, there is a disadvantage in that detection by the polymerase chain reaction method may be judged negative. That is, there is a possibility that microorganisms exist even if they are not detected by the polymerase chain reaction method. In addition, as a fundamental problem of the polymerase chain reaction method, quantitative analysis is difficult and the risk of false positives due to contamination is high.

On the other hand, the treatment of diseases caused by infection is still mostly dependent on the administration of antibiotics. However, the antibiotic administration method has fatal disadvantages of causing blood cell reduction, hypersensitivity reaction, and toxicity in the nervous system, heart, kidney, and liver. Recently, with the emergence of 'super bacteria' that are resistant to antibiotics, the success rate of treating infectious diseases based on antibiotic administration is also very low.

In order to solve these problems, there is a demand for the development of a method for detecting or removing safe pathogens, which has high pathogen detection efficiency and does not cause side effects when administered to the body.

In order to solve the problems as described above, the inventors of the present invention, leukocyte-derived cell membrane; and magnetic immune particles including magnetic particles attached to the cell membrane, and the magnetic immune particles can detect, separate, or remove pathogenic substances from samples such as blood, thereby diagnosing and treating infectious diseases. By confirming that it can be applied to, the present invention was completed.

One object of the present invention is a cell membrane derived from leukocytes; and magnetic immunization particles comprising magnetic particles attached to the cell membrane.

Another object of the present invention is to provide a composition or kit for detecting or removing pathogenic substances including the magnetic immune particles, or a method for detecting or removing pathogenic substances using the magnetic immune particles.

Another object of the present invention is to provide a composition or kit for diagnosing an infectious disease comprising the magnetic immune particles, or a method for diagnosing an infectious disease using the magnetic immune particles.

Another object of the present invention is to provide a composition for treating an infectious disease containing the magnetic immune particles, or a method or apparatus for treating an infectious disease using the magnetic immune particles.

Another object of the present invention is to provide a use of the magnetic immune particles for detecting or removing pathogenic substances, or for diagnosing or treating an infectious disease.

Another object of the present invention is a composition or kit for detecting or removing pathogenic substances of the magnetic immune particles; compositions or kits for diagnosis of infectious diseases; Alternatively, it is intended to provide a use for manufacturing a composition or device for treating an infectious disease, or a use for using the magnetic immune particle in a device for treating an infectious disease.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

One aspect is a cell membrane derived from leukocytes; and magnetic immunization particles comprising magnetic particles attached to the cell membrane.

The magnetic immune particles are magnetic particles attached to leukocyte-derived cell membranes capable of capturing pathogenic substances, and can capture various types of pathogenic substances such as microorganisms, viruses, antigenic proteins, and the like. In addition, the magnetic immune particles that capture the pathogenic substance may be separated or removed from a sample such as blood by a magnetic field. Accordingly, the magnetic immune particles can be used to detect, separate, or remove pathogenic substances from samples such as blood.

The technique proposed in the present specification is: 1) preparing magnetic immune particles attached to leukocyte-derived cell membranes capable of trapping pathogenic substances, and 2) contacting the magnetic immune particles with the sample to inject the magnetic immune particles into the sample. After the existing pathogenic substances are captured, 3) the magnetic immune particles that have captured the pathogenic substances are separated from the sample by a magnetic field to detect the pathogenic substances in the sample or to remove the pathogenic substances from the sample. do.

In this specification, the term "attachment" may refer to a form located outside or inside the cell membrane bilayer. For example, it may refer to a form in which magnetic particles are directly bonded to the outside or inside of a cell membrane bilayer or a form in which magnetic particles are absorbed into a cell membrane and trapped (or entrapped or encapsulated), but is not limited thereto.

The pathogenic substance may be one or more selected from the group consisting of pathogenic bacteria, fungi, viruses, parasites, prions, and toxins, but includes without limitation any pathogenic substances that can be captured by cell membranes. The pathogenic substance may cause an infectious disease in the body.

In one embodiment, the pathogenic bacteria may be any kind of gram-positive bacteria or gram-negative bacteria. More specifically, Enterococcus spp, Citrobacter spp, Staphylococcus spp, Klebsiella spp, Pseudomonas spp, Acinetobacter Acinetobacter spp , Salmonella spp, Streptococcus spp, Escherichia spp, Mycobacterium spp, Mycoplasma spp, Vibrio spp ), dysentery bacteria ( Shigella spp ), Campylobacter bacteria ( Campylobacter spp), Chlamydia ( Chlamydia spp), and at least one selected from the group consisting of the above bacteria acquired resistance to antibiotics, but is not limited thereto .

In addition, in one embodiment, the pathogenic fungi are a group consisting of Candida spp, Aspergillus spp, Trichophyton spp, and Cladophialophora spp It may include one or more selected from, but is not limited thereto.

Also, in one embodiment, the pathogenic virus is Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae , Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. It may include the above, but is not limited thereto. More specifically, the pathogenic viruses are RSV (Respiratory syncytial virus), ZIKV (Zika virus), HCov229E (Human Coronavirus 229E), HCoV-OC43 (Human coronavirus OC43), CMV (Cytomegalovirus), SARS-CoV-1 (Severe acute respiratory syndrome coronavirus 1), SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), mutant viruses of SARS-CoV-2 (Alpha mutation, Beta mutation, Delta mutation, etc.), Ebola virus, and dengue It may include one or more selected from the group consisting of viruses (Dengue virus), but is not limited thereto.

Also, in one embodiment, the parasite may be a malaria larvae.

Also, in one embodiment, the prions may be infectious protein pathogens that cause infectious diseases including scrapie, mad cow disease, and Critzevelt-Jakob disease.

Also, in one embodiment, the toxin may include any substance as long as it has pathogenicity such as causing a disease, and may be a pathogenic substance (eg, antigenic protein) derived from the pathogenic bacteria, fungi, viruses, or parasites. there is. For example, the toxin is LPS (Lipopolysaccharide), ZIKV (Zika virus) Envelope Protein, SARS-CoV-2 Spike Protein, or SARS-CoV-2 mutant virus Spike Protein (Alpha mutant Spike Protein, Beta mutant Spike Protein, or Delta mutant of Spike Protein) and the like.

In this specification, the term "capture" means that the pathogenic substance is bound to or linked to the magnetic immune particle. Specifically, the term "capture" may mean that the pathogenic substance is bound to or linked to the surface or inside of the cell membrane of the magnetic immune particle. In this specification, the term "entrapment" may be used interchangeably with terms such as "capture", "adsorption", and "absorption".

The leukocyte may be at least one selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, lymphocytes, and macrophages, but is not limited thereto. The lymphocytes may include B cells, T cells, natural killer cells (NK cells), or a combination thereof.

According to one embodiment, the magnetic immune particles are separated from a mixture of two or more types of leukocytes, for example, a mixture of two or more leukocytes selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, lymphocytes, and macrophages. It can be made using cell membranes. In this case, the ability to capture, detect, or remove pathogenic substances may be further increased compared to magnetic immune particles prepared using a cell membrane isolated from a single type of leukocyte.

The leukocytes or leukocyte-derived cell membranes are primates such as humans and monkeys, rodents such as rats and mice, ungulates such as horses, cows, pigs, sheep, and goats, mammals such as horses, canines, and felines, birds, fish, and reptiles. , amphibians, crustaceans, and may be derived from cells of one or more species selected from the group consisting of insects, but is not limited thereto. Since the leukocyte-derived cell membrane does not cause immune rejection when administered to the body, safety is excellent when administered to a living body.

As used herein, the term "cell membrane" refers to a cell membrane present in a cell itself, or a cell membrane separated from a cell (specifically, a cell membrane maintained in its original form) through conventional methods such as ultrasonic treatment, use of osmotic pressure difference, extrusion, and the like. Cell membrane, part or piece of cell membrane, membrane formed by reassembling part or piece of cell membrane, etc.)

The leukocyte-derived cell membrane is lectin, toll like receptor (TLR), pattern recognition receptor (PRR), cluster of differentiation (CD) molecule, NET (Neutrophil extracellular) trap), and cytokine receptors, but may express at least one selected from the group consisting of, but is not limited thereto. The cell membrane expresses at least one selected from the group consisting of lectins, toll-like receptors, pattern recognition receptors, differentiation cluster molecules, NETs, and cytokine receptors to capture or absorb magnetic particles or pathogenic substances (uptake, endocytosis), or , can bind or adhere to magnetic particles or pathogenic substances.

The leukocyte-derived cell membrane may be overexpressed with CR1 (complement receptor 1), CR3 (complement receptor 3), or a combination thereof. Specifically, the leukocyte-derived cell membrane may refer to a cell membrane separated from a leukocyte having a cell membrane in which CR1, CR3, or a combination thereof is overexpressed. Here, overexpression may refer to a state in which the expression of CR1, CR3, or a combination thereof is further increased compared to the cell membrane of normal leukocytes.

Leukocytes having cell membranes in which CR1, CR3, or a combination thereof are overexpressed can be prepared according to a method known in the art. For example, a method of increasing the expression of CR1, CR3, or a combination thereof in leukocytes using genetic engineering may be used. Specifically, a method of transfecting a foreign gene encoding CR1, CR3, or a combination thereof into leukocytes to increase the copy number of the gene in leukocytes can be used, and the method can be any method known in the art, such as For example, CR1, CR3, or a gene encoding a combination thereof is operably linked to a vector, independent of the host, or by introducing a vector capable of replicating and functioning in leukocyte cells into leukocyte cells, Not limited to this.

It is possible to prepare leukocytes having a cell membrane in which CR1, CR3, or a combination thereof are overexpressed through the above-described method, etc., and according to the method according to an embodiment of the present invention, the cell membrane is separated from the leukocyte, and the separated cell membrane By attaching the magnetic particles to the surface, it is possible to prepare magnetic immune particles including leukocyte-derived cell membranes in which CR1, CR3, or a combination thereof are overexpressed.

According to one embodiment, in the case of magnetic immune particles including leukocyte-derived cell membranes, CR1, CR3, or a combination thereof, which are surface molecules of the cell membrane, greatly enhance the ability of the magnetic immune particles to capture (detect or remove) pathogenic substances. contribution was confirmed. Therefore, in the case of magnetic immune particles including leukocyte-derived cell membranes in which CR1, CR3, or a combination thereof are overexpressed, the ability to capture (detection or remove) pathogenic substances can be remarkably increased.

As used herein, the term "magnetic particle" refers to a particle capable of responding to a magnetic field, which can be easily absorbed into a cell, or can be bound, attached, introduced, entrapped, encapsulated, or entrapped outside or inside a cell membrane. refers to particles in Specifically, the magnetic particles include iron (Fe), nickel (Ni), cobalt (Co), manganese (Mn), bismuth (Bi), zinc (Zn), strontium (Sr), lanthanum (La), cerium ( Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho) , Erbium (Er), Thulium (Tm), Ytterbium (Yb), Ruthenium (Lu), Copper (Cu), Silver (Ag), Gold (Au), Cadmium (Cd), Mercury (Hg), Aluminum (Al) At least one magnet selected from the group consisting of gallium (Ga), indium (In), thallium (Tl), calcium (Ca), barium (Ba), radium (Ra), platinum (Pt), and lead (Pd). It may contain elements, but is not limited thereto.

The magnetic element may be oxidized or surface modified. Specifically, iron may be oxidized and included in the magnetic immune particle in the form of iron oxide. The surface modification is surface modification with a metal, surface modification with a functional group such as a carboxy group or an amine group, surface modification with a protein such as an antibody, streptavidin, or avidin, surface modification with a carbohydrate, surface modification with a polymer, It may be surface modification by lipids, but is not limited thereto. The magnetic particles may be stabilized by the modification.

The magnetic particles may be prepared and used through a known method, or may be purchased and used commercially.

The magnetic particles may be used as is or may be used in a state of being dispersed or suspended in an appropriate solvent (eg, buffer (PBS, saline, Tris-buffered saline, etc.), but is not limited thereto.

Since the magnetic particles have a small particle size, each particle may have a single magnetic domain. As a result, superparamagnetism, which has magnetic properties, can be exhibited only when an external magnetic field is present. By making the magnetic immune particles with magnetic particles exhibiting superparamagnetism, the magnetic immune particles can be simply and easily separated by applying an external magnetic field. Separation by applying a magnetic field is excellent in stability and sensitivity because it is not affected by surrounding environments such as pH, temperature, and ions.

The magnetic particles may be selected from all particles having a particle size capable of attaching to, entering, encapsulating, or being encapsulated in a cell membrane capable of trapping the pathogenic substance and being magnetic.

The magnetic particles may be included in a solution. The magnetic particles may be attached to the cell membrane while being contained in a solution. In one embodiment, the solution may include a medium used for culturing or differentiating cells, a buffer, or a combination thereof, and may be the same as the medium of the magnetic immune particles.

The magnetic immune particle may include an outer surface including the cell membrane and an inner core including the magnetic particle.

In the magnetic immune particle, the cell membrane may have a vesicle shape. As used herein, the term "vesicle" refers to a step in which a cell membrane separated by a step of extracting (separating) a cell membrane from a cell by a known technique such as ultrasonic treatment, use of osmotic pressure difference, or extrusion, self-assembly, or reassembling by extrusion It may refer to particles of the form formed by.

An example of a manufacturing process of magnetic immune particles according to one embodiment is schematically shown in FIG. 1 . Specifically, according to the Type 1 method of FIG. 1, leukocytes extracted from a subject are administered to a solution (eg, blood, aqueous solution, purified water, buffer, medium, etc.) containing magnetic particles, and cellular uptake By allowing the magnetic particles to be absorbed (or contained or integrated) into cells by development, leukocyte cells (eg, magnetic immune cells) or leukocyte cell analogues containing the magnetic particles therein can be generated. The leukocyte cells (eg, magnetic immune cells) containing the magnetic particles therein may exhibit magnetism or be affected by a magnetic field by the magnetic particles contained therein while performing the original functions of the leukocyte cells.

In addition, according to the Type 2 method of FIG. 1, magnetic immune particles may be formed using cell membranes separated (or purified) from white blood cells. Specifically, cell membranes are separated (or purified) from leukocyte cells through a conventional method (eg, use of osmotic pressure difference, ultrasonic treatment, extrusion, etc.), and the obtained leukocyte-derived cell membrane and magnetic particles are extruded to obtain magnetic properties. By including the particle inside the leukocyte-derived cell membrane, magnetic immune particles containing magnetic particles inside the leukocyte-derived cell membrane can be generated. More specifically, in order to separate (or purify) cell membranes from leukocytes, leukocyte cells are administered to a low osmotic solution, so that the low osmotic solution moves into the white blood cells and expands the white blood cells, and at the same time, the organelles within the white blood cells move outside the cells. Pores can be formed in cell membranes that allow escape to the environment. The intracellular organelles that have escaped through the pores are separately removed using centrifugation, only the cell-derived membrane is separated (or purified), and the separated (or purified) leukocyte-derived cell membrane is ultrasonically treated to reduce the cell membrane to a smaller size. can be split into The leukocyte-derived cell membrane and magnetic particles may be mixed and extruded to generate magnetic immune particles containing magnetic particles inside the cell membrane.

In addition, as shown in FIG. 1, magnetic immune particles can be formed using a cell membrane isolated (or purified) from leukocyte cells according to the Type 3 method. Specifically, cell membranes are separated (or purified) from leukocyte cells through a conventional method (eg, using osmotic pressure difference, extrusion, etc.), and after mixing the leukocyte-derived cell membrane and magnetic particles obtained therefrom, ultrasonic treatment is performed to obtain the magnetic particles from the leukocytes. Magnetic immune particles containing magnetic particles inside the leukocyte-derived cell membrane can be generated by inclusion in the derived cell membrane.

Since the magnetic immune particles contain cell membrane components (lipid bilayer, membrane proteins, receptors, etc.) of white blood cells in their outer membrane, they can perform functions similar to those of white blood cells. In addition, the magnetic immune particles may show magnetism by the magnetic particles contained therein or be affected by a magnetic field.

Another aspect provides a composition for detecting pathogenic substances including the magnetic immune particles.

Another aspect provides a composition for removing pathogenic substances including the magnetic immune particles.

As used herein, the term "detection of a pathogenic substance" may mean determining whether a pathogenic substance is present in a sample or detecting a pathogenic substance present in a sample.

As used herein, the term "removal of pathogenic substances" means separating and removing pathogenic substances present in a sample from the sample.

The magnetic immune particles use a leukocyte-derived cell membrane capable of trapping a pathogenic substance, and may bind to a pathogenic substance or capture the pathogenic substance into the cell membrane according to the characteristics of the leukocyte cell from which the cell membrane is derived. In addition, magnetic immune particles that capture pathogenic substances may be collected or concentrated in a magnetic field region by applying a magnetic field, and through this, may be separated from a sample. According to this principle, the composition including the magnetic immune particles can be used to detect pathogenic substances in a sample, remove pathogenic substances from a sample, or diagnose or treat an infectious disease of a subject.

In the case of magnetic immune particles using a conventional targeting material (eg, antibody), the targeting material capable of binding to the pathogenic material is formed on the surface or inside of the magnetic particle to bind to the pathogenic material. Therefore, it is impossible to effectively target a pathogenic substance without accurate information on a specific antigen for the pathogenic substance to be targeted, and it is difficult to target several types of pathogenic substances at the same time. In addition, since an antibody must be used, synthesis is very difficult and expensive.

Since the magnetic immune particles can use leukocyte cells isolated from living organisms or cell membranes derived therefrom, various types of unknown pathogenic substances (microbes or viruses, etc.) are captured at once using the system and characteristics of the leukocyte cells themselves. Since it has the advantage that it can be used for detection or removal of various pathogenic substances.

The composition is a contaminant (bacteria, fungi, viruses, other microorganisms, toxins (eg, endotoxins, etc.), contaminants present in extremely small amounts in various foods, sanitary products, environmental samples, etc. including drinking water and beverages. compound) can be detected or removed at high speed, so it can be used for safety evaluation of food, sanitary products, environmental samples, etc. In addition, the composition can detect or remove pathogenic substances present in a biological sample.

Another aspect provides a composition for diagnosing an infectious disease comprising the magnetic immune particles. The composition can be used to diagnose an infectious disease of an individual by detecting a pathogenic substance captured by the magnetic immune particle.

Another aspect provides a composition for treating an infectious disease comprising the magnetic immune particles. The composition can be used to treat an infectious disease of an individual by detecting and removing pathogenic substances present in the body by the magnetic immune particles included in the composition. Specifically, the composition for treating an infectious disease can detect and remove the causative agent of the infectious disease in a sample isolated from the subject, and the sample from which the causative agent of the infectious disease has been removed is injected back into the subject, so that the subject Infectious diseases can be cured.

As used herein, the term "diagnosis of an infectious disease" may mean determining whether an individual currently or previously has an infectious disease, or determining whether an individual is infected with a pathogenic substance capable of causing an infectious disease.

As used herein, the term "treatment of an infectious disease" refers to any activity in which the symptoms caused by an infectious disease are improved or beneficially changed by reducing or removing the causative agent of an infectious disease in a subject.

The infectious disease may be at least one selected from the group consisting of systemic or local infection, inflammation, sepsis, and poisoning by toxins, but is not limited thereto. In addition, diseases caused by infection with the aforementioned pathogenic substances are included without limitation. Specifically, the infectious diseases include malaria infection, mycobacterial tuberculosis, pneumonia, food poisoning, tetanus, typhoid fever, diphtheria, syphilis, Hansen's disease, chlamydia infection, smallpox, influenza, mumps, measles, chickenpox, Ebola, rubella, coronavirus infection, It may be at least one selected from the group consisting of scrapie, mad cow disease, and Kritsvelt-Jakob disease, but is not limited thereto.

The composition may further include opsonins. The opsonin may be at least one selected from the group consisting of Mannose binding lectin (MBL), Ficolin-1 (FCN-1), and Immunoglobulin G (IgG) antibodies. The IgG antibody is an antibody specific to a target pathogenic substance, and its type may vary depending on the target pathogenic substance. For example, the IgG antibody may include an anti-SARS-SARS-CoV-2 spike protein antibody.

According to one embodiment, the ability of the magnetic immune particles included in the composition to capture pathogenic substances (detection or removal) can be further improved by the opsonin. In particular, compared to other opsonins such as FCN-2 and C3b, specific opsonins such as MBL, FCN-1, or IgG can further improve the ability to capture (detection or remove) pathogenic substances of the magnetic immune particles. can

The composition may further include solvents, solvents, binders, lubricants, disintegrants, buffers, suspending agents, isotonic agents, viscosity modifiers, carriers, dispersants, lubricants, excipients, stabilizers, pH adjusters, preservatives, and the like. Not limited.

Of the terms or elements mentioned in the composition, the same ones mentioned in the description of the magnetic immune particles are understood to be the same as those mentioned in the description of the magnetic immune particles above.

Another aspect provides a method for detecting a pathogenic substance comprising mixing the magnetic immune particles and a sample in contact with each other, and applying a magnetic field to the mixed sample.

Another aspect provides a method for removing a pathogenic substance, comprising mixing the magnetic immune particles and a sample in contact with each other, and applying a magnetic field to the mixed sample.

Another aspect is a method for diagnosing an infectious disease comprising contacting and mixing the magnetic immune particles and a sample, and applying a magnetic field to the mixed sample, or a method for providing information necessary for diagnosing an infectious disease. to provide.

Another aspect is the method of contacting and mixing the magnetic immune particles with a sample separated from the individual, and applying a magnetic field to the mixed sample to remove the magnetic immune particles that have captured pathogenic substances from the sample. Methods for treating infectious diseases are provided.

In the above method, the sample is a biological sample (eg, blood (eg, whole blood), body fluid such as plasma, serum, lymph, cerebrospinal fluid, or cells, separated from a living body of an animal (including or not including a human), or tissue), drinking water (e.g., ground water, tap water, bottled water, purified water, mineral water, etc.), various foods, various sanitary products that directly act on the living body, tableware, kitchen utensils, environmental samples (e.g., soil, seawater, river water, etc.), etc. It may be one or more selected from the group consisting of, but is not limited thereto, and may be any subject requiring detection and/or removal of pathogenic substances. The sample may be in the form of a fluid itself or a suspension suspended in an appropriate medium (eg, purified water, sterile buffer, etc.).

The contacting and mixing of the magnetic immune particles and the sample may be performed outside the body, and may include culturing the magnetic immune particles and the sample together outside the body.

The in vitro culturing step may be performed under conventional conditions using a medium, buffer solution, saline, drinking water, biological sample itself, etc. commonly used for cell culture. For example, 1 second to 96 hours, 1 second to 48 hours, 1 second to 24 hours, for example, 1 second to 12 hours, 1 second to 6 hours, 1 second to 24 hours at a temperature condition of 0 to 40 ° C. or 2 to 38 ° C. It may include incubating for 120 minutes, or 1 second to 60 minutes.

Also, the step of applying the magnetic field may be performed outside the body.

The method may further include, after applying a magnetic field to the mixed sample, separating (or removing) the magnetic immune particles from the sample using the applied magnetic field (magnetic force). At this time, the separated magnetic immune particles may be bound to the pathogenic substances in the sample or may be in a state of capturing the pathogenic substances in the sample. This step may be performed outside the body, and pathogenic substances may be removed from the sample through this step.

In the treatment method, after the step of applying a magnetic field to the mixed sample or the step of separating (or removing) the magnetic immune particles from the sample using the applied magnetic field (magnetic force), the sample from which the magnetic immune particles are removed is treated. A step of injecting the sample into the living body of the individual who provided the sample again outside the body may be further included. At this time, the magnetic immune particles removed from the sample may be bound to the pathogenic substance in the sample or may be in a state of trapping the pathogenic substance in the sample. Accordingly, the sample injected back into the body of the individual may be a sample from which pathogenic substances and magnetic immune particles are all removed.

The method may further include analyzing pathogenic substances captured by magnetic immune particles separated (or removed) from the sample. The analysis may be performed by means commonly used for analysis of the pathogenic substances (eg, bacteria, fungi, viruses, parasites, prions, toxins (eg, endotoxins), antigenic proteins, etc.) .

In the above method, in order to facilitate measurement of the magnetic immune particles, the magnetic immune particles may be obtained using cells (cell membranes) and/or magnetic particles labeled with a detectable labeling substance. The labeling material may be any material (small molecule compound, protein, poly/oligo peptide, etc.) detectable by a conventional method, and may be, for example, at least one selected from the group consisting of a fluorescent material and a light emitting material.

The method may further include injecting at least one selected from the group consisting of opsonin, blood, and plasma before applying the magnetic field. The opsonin may be at least one selected from the group consisting of MBL, FCN-1, and IgG antibodies. The blood or plasma may be blood or plasma separated from an individual who provided the sample or another individual (eg, another person). For example, the blood or plasma was isolated from the individual before or after the individual who provided the sample suffered from a disease, or was isolated from another individual (eg, another person) who had been infected with a specific pathogenic substance, or was isolated from a specific pathogenic substance. It may be isolated from other individuals (eg, other people) who have been vaccinated against. Since the blood or plasma is rich in antibodies or opsonins, the ability to capture (detection or remove) pathogenic substances of the magnetic immune particles can be further improved.

In the method, the step of injecting at least one selected from the group consisting of opsonin, blood, and plasma may be performed at any stage before applying the magnetic field. For example, prior to mixing the magnetic immune particles and the sample by injecting and mixing at least one selected from the group consisting of opsonin, blood, and plasma into the magnetic immune particle or the sample, or mixing the magnetic immune particle with the magnetic immune particle In the step of contacting and mixing the samples or after mixing, at least one selected from the group consisting of the opsonin, blood, and plasma may be additionally injected and mixed.

The subject may be a human or a non-human animal (eg, mammal, etc.).

The method may include a kit for detecting or removing pathogenic substances including the magnetic immune particles; kits for diagnosis of infectious diseases; Alternatively, it may be implemented as a device for treating an infectious disease using the magnetic immune particles.

A schematic diagram of the kit or device is shown in FIG. 10, but is not limited thereto.

Specifically, the kit or device may include a reaction unit and a magnetic field forming unit.

The reaction part may refer to a part into which a reactant in which the magnetic immune particles and the sample are mixed, cultured, or reacted by contacting each other, or where a reaction occurs by contacting the magnetic immune particle and the sample, is introduced. In the kit or device, the magnetic immune particles may be included in the reaction unit, react with the sample in advance and applied to the reaction unit in the form of a reactant, or may be provided separately from the reaction unit, and may be prepared in an appropriate medium ( For example, it may be provided in the form of a dispersion dispersed in a buffer).

The magnetic field forming part may refer to a part forming a magnetic field. The magnetic field forming unit may be included in the reaction unit, provided separately from the reaction unit, or integrally provided with all or part of the reaction unit. When the magnetic field forming unit exists separately from the reaction unit, the reaction unit and the magnetic field forming unit may be connected by a channel through which fluid can move. The magnetic particles attached to the magnetic immune particles are moved to the magnetic field forming unit by the magnetic field formed by the magnetic field forming unit, so that the magnetic immune particles can be separated. In this case, the separated magnetic immune particles may capture pathogenic substances. For example, the magnetic field generator may include one or more means capable of applying a magnetic field such as a magnet (eg, an electromagnet by electromagnetic induction, a permanent magnet, etc.).

The number of integral or separately provided reaction units and/or magnetic field generators is also not particularly limited, and may be one or more. For example, when the reaction unit and the magnetic field forming unit are provided separately, one or more, for example, 1 to 10 reaction units are connected to 1 to 10 magnetic field forming units, or when the reaction unit and the magnetic field forming unit are integrally formed, integral reaction It may be provided with 1 to 10 parts and magnetic field forming parts, but is not limited thereto.

The kit or device includes a reaction unit into which a sample (fluid state such as blood), magnetic immune particles (eg dispersion state), other additional components (eg opsonin, blood or plasma other than a sample), or a reaction product thereof are injected. It may include one or more injectors connected thereto. The other side of the injector that is not connected to the reaction unit may be directly connected to an object or connected to a separated sample, and as a result, the sample may be injected through the injector. In addition, magnetic immune particles may be injected through the other side of the injection unit that is not connected to the reaction unit. In addition, additional components such as opsonin and blood or plasma other than the sample may be injected through the other side of the injection unit that is not connected to the reaction unit.

The kit or device may further include one or more discharge units connected to the magnetic field forming unit and discharging the magnetic immune particles captured by the magnetic field. The discharge unit may further include a detection unit including a detection means capable of detecting pathogenic substances captured by the discharged magnetic immune particles.

In the kit or device, in the magnetic field generator, the magnetic immune particles may be separated from the sample by the magnetic field and separately collected or concentrated. The magnetic immune particles may include all magnetic immune particles that may or may not capture pathogenic substances. In this case, the magnetic immune particles may be filtered, collected, concentrated, or removed in the magnetic field forming unit or in a collecting unit connected to the magnetic field forming unit without being discharged.

The kit or device may further include a sample outlet.

The sample discharge unit may refer to a portion from which a sample separated by applying a magnetic field to the sample is discharged. The sample outlet may be one or more. A sample moved to one place by application of a magnetic field in a fluid flow state, for example, a sample from which magnetic immune particles have been removed, is discharged through the sample discharge unit and can be separately concentrated or separated. The magnetic immune particles may include all magnetic immune particles that may or may not capture pathogenic substances. Accordingly, the sample discharged through the sample outlet may be free of both pathogenic substances and magnetic immune particles.

In the kit or device, the sample discharge unit may be connected to the injection unit or to the subject. Therefore, by repeating the process of removing the pathogenic substances in the sample by injecting the sample from which the magnetic immune particles are removed, discharged through the sample discharge unit, through the injection unit, the pathogenic substances that are not completely removed can be removed more effectively. . In addition, the sample from which the magnetic immune particles are removed, discharged through the sample discharge unit, may be injected into the subject again. The magnetic immune particles may include all magnetic immune particles that may or may not capture pathogenic substances. Accordingly, the sample injected again into the subject may be a sample from which both pathogenic substances and immune particles have been removed.

According to one embodiment, the kit or device may be implemented in the form of a fluidic device. In particular, in the case of a device for treating an infectious disease, it may be implemented by injecting a sample from which pathogenic substances and magnetic immune particles are removed back into the subject.

Among the terms or elements mentioned in the methods, kits, and devices, the same ones mentioned in the description of the magnetic immune particles and compositions are understood to be the same as those mentioned in the description of the magnetic immune particles and compositions above.

Another aspect provides a use of the magnetic immune particle for detecting or removing a pathogenic substance, or for diagnosing or treating an infectious disease.

Another aspect is a composition or kit for detecting or removing pathogenic substances from the magnetic immune particles; compositions or kits for diagnosis of infectious diseases; Alternatively, it provides a use for preparing a composition or device for treating an infectious disease, or a use for using the magnetic immune particles for a device for treating an infectious disease.

Among the terms or elements mentioned in the above use, the same ones mentioned in the description of the magnetic immune particles, compositions, methods, kits, and devices are the same as those mentioned in the description of the magnetic immune particles, compositions, methods, kits, and devices above. It is understood that as mentioned.

Since the magnetic immune particle according to one aspect includes a leukocyte-derived cell membrane, in vivo side effects can be minimized, and various types of pathogenic substances can be detected by characteristics of the leukocyte from which the cell membrane is derived. In addition, since it contains magnetic particles, it is possible to simply separate magnetic immune particles by applying a magnetic field, so that pathogenic substances can be more effectively detected and removed. In addition, when the magnetic immune particles are used for treatment, the possibility of injecting the magnetic immune particles into the body can be minimized, and side effects in vivo can be remarkably reduced.

1 is a schematic diagram showing a method of generating magnetic immune particles according to an embodiment.

2 is a diagram showing the ability to capture (detection or remove) pathogenic bacteria (MRSA and ESBL-EC) of magnetic immune particles according to an embodiment (HL60(N): neutrophil-derived magnetic immune particles; U937(M) : macrophage-derived magnetic immune particles; hWBC: leukocyte-derived magnetic immune particles).

3 is a diagram showing the ability (detection or removal) of magnetic immune particles according to an embodiment to capture viruses (CMV and RSV) (HL60(N): neutrophil-derived magnetic immune particles; U937(M): macrophages). derived magnetic immune particles; hWBC: leukocyte-derived magnetic immune particles).

4 is a diagram showing the ability to capture (detection or remove) virus-derived antigen proteins (ZIKV E Protein and SARS-CoV-2 S Protein) of magnetic immune particles according to an embodiment (HL60(N): derived from neutrophils). magnetic immune particles; U937(M): macrophage-derived magnetic immune particles; hWBC: leukocyte-derived magnetic immune particles).

5 shows the antigenic proteins derived from SARS-CoV-2 virus (USA-WA1/2020) and SARS-CoV-2 mutant viruses (Alpha, Beta, and Delta) of leukocyte-derived magnetic immune particles according to an embodiment. It is a diagram showing the capture ability (detection or removal ability) for

6 is a diagram showing the decrease in the ability to capture (detection or remove) various pathogens of leukocyte-derived magnetic immune particles in which cell membrane surface molecules (CR1, CR3) are inactivated according to an embodiment (WBC-MNVs: leukocyte-derived Magnetic immune particles; CR1 blocked WBC-MNVs: magnetic immune particles derived from leukocytes in which CR1 is inactivated; CR3 blocked WBC-MNVs: magnetic immune particles derived from white blood cells in which CR3 is inactivated; CR1&CR3 blocked WBC-MNVs: CR1 and CR3 inactivated white blood cell-derived magnetic immune particles; MNPs: magnetic particles that do not contain a cell membrane surface).

7 is a graph illustrating the ability of leukocyte-derived magnetic immune particles to capture pathogenic bacteria when the leukocyte-derived magnetic immune particles are injected together with opsonin (MBL or FCN-1) into blood containing pathogenic bacteria according to an embodiment ( It is a diagram showing the results of analyzing the detection or removal ability).

8 is a diagram illustrating leukocyte-derived magnetic immune particles according to an embodiment when injected together with opsonin (MBL, FCN-1, or C3b) into a TBS buffer containing a virus or virus-derived antigen protein, the leukocyte-derived magnetic immune particles It is a diagram showing the result of analyzing the capture ability (detection or removal ability) for the virus or virus-derived antigen protein.

9 is a diagram illustrating the opsonization of leukocyte-derived magnetic immune particles (MBL, FCN-1, FCN-2, or IgG), it is a diagram showing the results of analyzing the ability (detection or removal ability) of the leukocyte-derived magnetic immune particles for SARS-CoV-2 S protein.

10 is a schematic diagram schematically illustrating a method for collecting, concentrating, and removing pathogenic substances in a magnetic immune particle-based sample according to an embodiment.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of leukocyte-derived magnetic immune particles

Red blood cells were removed from the separated blood sample to obtain a white blood cell sample. The obtained white blood cells were subjected to low osmotic treatment at 4° C. for 1 hour and then centrifuged at 4° C. for 5 minutes (Centrifuge 5424R, Eppendorf, Germany) to separate and purify only cell membranes from the white blood cells. The obtained leukocyte-derived cell membrane was sonicated (Q700 Ultra-Sonicator, Qsonica, USA) at 4° C., 20 kHz, and 150 W for 10 minutes to break the cell membrane into smaller units. Although the leukocyte sample is a mixture of various types of leukocytes, it is not limited thereto, and various types of leukocytes or a mixture thereof may be used to prepare the leukocyte-derived magnetic immune particles. For example, one or more white blood cells selected from among neutrophils, eosinophils, basophils, monocytes, lymphocytes, and macrophages may be used to prepare the white blood cell-derived magnetic immune particles.

Meanwhile, as the magnetic particles, iron oxide magnetic particles (Carboxyl-Adembeads, Ademtech, France) whose surface is modified with carboxylic acid were used.

Magnetic immune particles were prepared by extruding the prepared leukocyte-derived cell membrane with the magnetic particles using an Avanti mini extruder (Avanti Polar Lipids, Alabaster, AL, USA). Specifically, as in the type 2 method shown in FIG. 1, the prepared cell membrane and the magnetic particles were mixed and extruded to prepare magnetic immune particles incorporating the magnetic particles into the cell membrane.

Experimental Example 1: Evaluation of capture ability (detection or removal ability) of leukocyte-derived magnetic immune particles for pathogenic bacteria

In this experimental example, in order to confirm whether the leukocyte-derived magnetic immune particles prepared in Example 1 can capture (detect or remove) pathogens in blood, human blood samples are randomly inoculated with pathogenic bacteria, and then the leukocyte-derived magnetic immune particles are randomly inoculated. A magnetic field was applied by injecting the particles to measure the change of the inoculated bacteria in the culture medium.

Specifically, the gram-positive bacterium MRSA (Methicillin Resistant Staphylococcus aureus) or the gram-negative bacterium ESBL-EC (Extended-Spectrum Beta-Lactamases Producing Escherichia coli) was added to 1 mL of anticoagulated human blood (Red Cross, South Korea) sample. , 10 ⁴ CFU / mL concentration was inoculated and incubated for 10 minutes at 37 ℃. The leukocyte-derived magnetic immune particles were injected into the cultured blood sample so that the final concentration of the magnetic immune particles reached 150 μg/mL. Then, after 20 minutes of reaction at 37 ° C., the magnetic immune particles in the blood sample are fixed to a specific position using a magnet for 15 minutes to prevent magnetic immune particles from being included in the supernatant, and then the supernatant is collected. to confirm the CFU (colony forming unit) of bacteria in the supernatant. Specifically, the supernatant (100 μL) was diluted in 900 μL of physiological saline, spread on LB agar medium using a microbial analyzer (EDDY JET2, IUL micro, USA), and incubated at 37 ° C. for 24 hours, CFU of bacteria generated on LB agar medium was measured using a microbial colony counter and zone reader (IUL micro, USA). As a sample obtained by inoculating and culturing the bacteria in a blood sample in the same manner as described above, a sample in which the leukocyte-derived magnetic immune particles were not injected was used as a control. Based on the CFU value of the bacteria measured in the control group, the reduction level of the CFU value of the bacteria measured in the experimental group is calculated as a percentage (%), and the capture rate (or removal rate, %) was evaluated. In addition, neutrophil-derived magnetic immune particles using neutrophil (HL60)-derived cell membranes and macrophage-derived magnetic immune particles using macrophage (U937)-derived cell membranes prepared in the same manner as in Example 1 were used as comparative groups.

As a result, as shown in FIG. 2, it was confirmed that the leukocyte-derived magnetic immune particles can capture and detect or remove pathogenic bacteria in the blood, and capture remarkably good antibiotic-resistant bacteria such as MRSA and ESBL-EC. rate (about 40 to 70%).

In particular, compared to the neutrophil-derived magnetic immune particles and the macrophage-derived magnetic immune particles, the white blood cell-derived magnetic immune particles prepared in Example 1 using cell membranes separated from various types of white blood cell mixtures are MRSA and ESBL-EC. It was confirmed that the capture ability (detection or removal ability) for pathogenic bacteria such as

Experimental Example 2: Evaluation of virus capture ability (detection or removal ability) of leukocyte-derived magnetic immune particles

In this experimental example, in order to check whether the leukocyte-derived magnetic immune particles prepared in Example 1 can capture (detect or remove) pathogens in blood, human blood samples are randomly inoculated with viruses, and then the leukocyte-derived magnetic immune particles was injected and a magnetic field was applied to measure the change of the inoculated virus in the culture medium.

Specifically, 1 mL of anticoagulated human blood (Red Cross, South Korea) sample was inoculated with CMV (cytomegalovirus) or RSV (respiratory syncytial virus) at a concentration of 10 ⁴ PFU/mL and incubated at 37° C. for 10 minutes. . The leukocyte-derived magnetic immune particles were injected into the cultured blood sample so that the final concentration of the magnetic immune particles reached 150 μg/mL. Then, after 20 minutes of reaction at 37 ° C., the magnetic immune particles in the blood sample are fixed to a specific position using a magnet for 15 minutes to prevent magnetic immune particles from being included in the supernatant, and then the supernatant is collected. The amount of viral RNA in the supernatant was measured. Nucleic acids were extracted from viruses present in the supernatant using the QIAmp viral RNA mini kit (QIAGEN, Germany), and SYBR PCR master mix (Toyobo, Japan) and real time PCR (CFX connect, BIO-RAD, USA) were used. The extracted nucleic acid was amplified and the amount of RNA was measured. As a sample cultured by inoculating the virus in a blood sample in the same manner as described above, a sample in which the leukocyte-derived magnetic immune particles were not injected was used as a control. Based on the RNA amount of the virus measured in the control group, the reduction level of the RNA amount of the virus measured in the experimental group is calculated as a percentage (%) to capture the leukocyte-derived magnetic immune particles for the virus (or removal rate, %) was evaluated. In addition, neutrophil-derived magnetic immune particles using neutrophil (HL60)-derived cell membranes and macrophage-derived magnetic immune particles using macrophage (U937)-derived cell membranes prepared in the same manner as in Example 1 were used as comparative groups.

As a result, as shown in FIG. 3, it was confirmed that the leukocyte-derived magnetic immune particles can capture and detect or remove pathogenic viruses in the blood (capture rate: about 45 to 80%). In particular, compared to the neutrophil-derived magnetic immune particles and the macrophage-derived magnetic immune particles, the white blood cell-derived magnetic immune particles prepared in Example 1 using cell membranes separated from various types of white blood cell mixtures have the same characteristics as CMV and RSV. It was confirmed that the ability to capture (detection or remove) pathogenic viruses was more excellent.

Experimental Example 3: Evaluation of capture ability (detection or removal ability) of leukocyte-derived magnetic immune particles for virus-derived antigens

In this experimental example, in order to confirm whether the leukocyte-derived magnetic immune particles prepared in Example 1 can remove virus-derived antigens in blood, human blood samples are randomly inoculated with virus-derived antigens, and then the leukocyte-derived magnetic immune particles are After injection, a magnetic field was applied to measure changes in antigens derived from the inoculated virus in the culture medium.

Specifically, in 1 mL of anticoagulated human blood (Red Cross, South Korea) sample, ZIKV (Zika virus) Envelope Protein, SARS-CoV-2 Spike Protein, or SARS-CoV-2 mutant virus Spike Protein (Alpha mutant Spike Protein, Beta-mutated Spike Protein, or Delta-mutated Spike Protein) was inoculated at a concentration of 1 μg/mL, and the leukocyte-derived magnetic immune particles were injected at a concentration of 150 μg/mL. Thereafter, the magnetic immune particles in the blood sample are fixed to a specific position using a magnet for 15 minutes to prevent magnetic immune particles from being included in the supernatant, and then the supernatant is collected to determine the concentration of the virus-derived antigen in the supernatant. measured. The concentration of the virus-derived antigen was measured by enzyme-linked immunosorbent assay (ELISA), and for measurement, Zika virus (strain Zika SPH2015) Envelope Protein (ZIKV-E) ELISA Kit (Sinobio, China) or SARS-CoV-2 Spike A protein ELISA kit (ab274342, abcam, USA) was used. Based on the concentration of the virus-derived antigen in the blood sample before injecting the magnetic immune particles, the reduction level of the virus-derived antigen concentration measured in the supernatant was calculated as a percentage (%), and the leukocyte-derived antigen for the virus-derived antigen The capture rate (or removal rate, %) of the magnetic immune particles was evaluated. In addition, neutrophil-derived magnetic immune particles using neutrophil (HL60)-derived cell membranes and macrophage-derived magnetic immune particles using macrophage (U937)-derived cell membranes prepared in the same manner as in Example 1 were used as comparative groups.

As a result, as shown in FIG. 4, it was confirmed that the leukocyte-derived magnetic immune particles can capture and detect or remove virus-derived antigen proteins in the blood (capture rate: about 30 to 80%). In particular, compared to the neutrophil-derived magnetic immune particles and the macrophage-derived magnetic immune particles, the white blood cell-derived magnetic immune particles prepared in Example 1 using cell membranes separated from various types of white blood cell mixtures have ZIKV E Protein and SARS - It was confirmed that the capture ability (detection or removal ability) for virus-derived antigen proteins such as CoV-2 S Protein was more excellent.

In addition, as shown in FIG. 5, it was confirmed that the white blood cell-derived magnetic immune particles had a remarkably excellent ability to capture (detection or remove) antigen proteins derived from the SARS-CoV-2 mutant virus (removal rate: about 60 to 80%). %), Through this example, it was confirmed that the leukocyte-derived magnetic immune particles can detect or remove virus-derived antigen proteins with high efficiency regardless of whether or not the virus has mutated.

Experimental Example 4: Identification of Molecules Contributing to Pathogen Capture Present on the Surface of Leukocyte-Derived Magnetic Immune Particles

In this experimental example, among various surface molecules including various receptors present on the surface of the cell membrane of the leukocyte-derived magnetic immune particles prepared in Example 1, which molecules contribute to pathogen capture of the leukocyte-derived magnetic immune particles are identified. To do this, leukocyte-derived magnetic immune particles in which specific surface molecules are inactivated on the surface of cell membranes were prepared and pathogen removal rates were analyzed.

Specifically, in the leukocyte-derived magnetic immune particles prepared in Example 1, CR1 (complement receptor 1) and/or CR3 (complement receptor 3), cell membrane surface molecules, were inactivated with appropriate antibodies, respectively, and CR1 and/or The leukocyte-derived magnetic immune particles in which CR3 was inactivated were obtained.

In addition, human plasma was inoculated with various pathogens (MRSA, ESBL-EC, RSV, CMV, ZIKV E protein, HCoV OC43 ( Human coronavirus OC43 ), or SARS-CoV-2 S protein), respectively, and various types of autoimmune After each particle was injected, the pathogen removal rate (%) of each magnetic immune particle was measured. The specific experimental method is the same as the method performed in Experimental Examples 1 to 3, and the types of injected magnetic immune particles are as follows:

1) Leukocyte-derived magnetic immune particles (WBC-MNVs) prepared in Example 1 above

2) In the leukocyte-derived magnetic immune particles prepared in Example 1, CR1, a cell membrane surface molecule, is inactivated (CR1 blocked WBC-MNVs)

3) In the leukocyte-derived magnetic immune particles prepared in Example 1, CR3, a cell membrane surface molecule, is inactivated (CR3 blocked WBC-MNVs)

4) In the leukocyte-derived magnetic immune particles prepared in Example 1, the cell membrane surface molecules CR1 and CR3 are inactivated (CR1&CR3 blocked WBC-MNVs)

5) magnetic particles (MNPs) that do not contain cell membrane surfaces.

As a result, as shown in FIG. 6, when CR1 or CR3, which is a cell membrane surface molecule, in the leukocyte-derived magnetic immune particles prepared in Example 1 is inactivated, the pathogen removal rate of the leukocyte-derived magnetic immune particles is significantly reduced. It was confirmed that, in particular, when both CR1 and CR3, which are cell membrane surface molecules, were inactivated, the pathogen removal rate of the leukocyte-derived magnetic immune particles was the most reduced. Through this, it was found that in the leukocyte-derived magnetic immune particles, CR1 and/or CR3 present on the cell membrane surface greatly contributed to the capture of pathogens.

Therefore, through this Example, it was found that the pathogen trapping ability (detection or elimination ability) can be remarkably increased in the case of leukocyte-derived magnetic immune particles prepared using the leukocyte-derived cell membrane in which CR1 and/or CR3 are overexpressed. .

Experimental Example 5: Confirmation of enhancement of pathogen trapping ability (detection or removal ability) of leukocyte-derived magnetic immune particles due to the addition of opsonin

In this experimental example, the effect of improving the pathogen trapping ability (detection or removal ability) of the leukocyte-derived magnetic immune particles prepared in Example 1 due to the addition of opsonin was confirmed.

Specifically, various pathogens (MRSA, ESBL-EC, RSV, CMV, ZIKV E protein, HCoV OC43, or SARS-CoV-2 S protein) are inoculated into TBS buffer, human blood sample, or human plasma sample, respectively, In addition, after injecting various opsonins (MBL (mannose binding lectin), FCN-1 (Ficolin-1), FCN-2, IgG (Immunoglobulin G), or C3b), respectively, the leukocyte-derived magnetic immune particles are injected, The pathogen capture rate (or removal rate, %) of the magnetic immune particles was measured. The specific experimental method is the same as the method performed in Experimental Examples 1 to 3, except that the opsonin was injected into TBS buffer, human blood sample, or human plasma sample. The IgG used above is an anti-SARS-SARS-CoV-2 spike protein antibody.

As a result, as shown in FIGS. 7, 8, and 9, the leukocyte-derived magnetic immune particles prepared in Example 1 were added to the TBS buffer, human blood sample, or human plasma sample containing the pathogen, along with MBL, When FCN-1 or IgG opsonin was injected, it was confirmed that the pathogen trapping ability (detection or elimination ability) of the leukocyte-derived magnetic immune particles was remarkably improved. However, it was confirmed that the injection of FCN-2 or C3b opsonin did not significantly increase the pathogen trapping ability (detection or elimination ability) of the leukocyte-derived magnetic immune particles. From this, it was found that specific opsonins such as MBL, FCN-1, or IgG, rather than all types of opsonins, can improve the ability of the leukocyte-derived magnetic immune particles to capture pathogens (detection or removal).

In addition, as shown in FIG. 9, compared to the case where the leukocyte-derived magnetic immune particles were injected into TBS containing pathogens, the leukocyte-derived magnetic immune particles were injected into human blood samples or human plasma samples containing pathogens. case, it was confirmed that the pathogen trapping ability (detection or elimination ability) of the leukocyte-derived magnetic immune particles was further improved, and this result remained the same even when a specific opsonin such as MBL, FCN-1, or IgG was additionally injected. confirmed. It is understood that these results are due to the abundant presence of antibodies or opsonics in blood or plasma.

Through this embodiment, in diagnosing or treating an infectious disease by detecting or removing pathogens in the blood of an individual using the leukocyte-derived magnetic immune particles, a specific opsonin such as MBL, FCN-1, IgG, and/or When blood or plasma is additionally injected, the pathogen trapping ability (detection or elimination ability) of the leukocyte-derived magnetic immune particles is further improved, so that the effect of detecting or removing pathogens in the blood of an individual and of diagnosing or treating infectious diseases is further improved. knew it could be.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (20)

1. cell membranes derived from leukocytes; and

   Magnetic immunization particles comprising magnetic particles attached to the cell membrane.
2. The magnetic immune particles according to claim 1, wherein the leukocyte is at least one selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, lymphocytes, and macrophages.
3. The method of claim 1, wherein the magnetic particle is iron (Fe), nickel (Ni), cobalt (Co), manganese (Mn), bismuth (Bi), zinc (Zn), strontium (Sr), lanthanum (La) , cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), Ruthenium (Lu), Copper (Cu), Silver (Ag), Gold (Au), Cadmium (Cd), Mercury (Hg), Aluminum 1 selected from the group consisting of (Al), gallium (Ga), indium (In), thallium (Tl), calcium (Ca), barium (Ba), radium (Ra), platinum (Pt), and lead (Pd) A magnetic immune particle comprising more than one kind of magnetic element.
4. The magnetic immune particle according to claim 3, wherein the magnetic element is oxidized or surface-modified with a metal, functional group, protein, carbohydrate, polymer, or lipid.
5. The magnetic immune particle according to claim 1, wherein the magnetic particle is contained in a solution.
6. The magnetic immune particle according to claim 1, wherein the magnetic immune particle includes an outer surface including the cell membrane and an inner core including the magnetic particle.
7. The magnetic immune particle according to claim 1, wherein the cell membrane has a vesicle shape.
8. The method of claim 1, wherein the cell membrane is lectin, toll like receptor (TLR), pattern recognition receptor (PRR), cluster of differentiation (CD) molecule, NET (Neutrophil extracellular trap), and magnetic immune particles expressing at least one selected from the group consisting of cytokine receptors.
9. The magnetic immune particle according to claim 1, wherein the cell membrane is overexpressed with CR1 (complement receptor 1), CR3 (complement receptor 3), or a combination thereof.
10. The magnetic immune particle according to claim 1, wherein the magnetic immune particle is for detecting or removing pathogenic substances.
11. The magnetic immune particle according to claim 10, wherein the pathogenic substance is one or more selected from the group consisting of pathogenic bacteria, fungi, viruses, parasites, prions, and toxins.
12. A composition for detecting or removing pathogenic substances comprising the magnetic immune particles of any one of claims 1 to 11.
13. A composition for diagnosis of an infectious disease comprising the magnetic immune particles of any one of claims 1 to 11.
14. 14. The method of claim 13, wherein the infectious disease is systemic or local infection, inflammation, sepsis, and at least one member selected from the group consisting of toxin poisoning, infectious disease diagnostic composition.
15. A method for detecting or removing pathogenic substances, comprising the steps of contacting and mixing the magnetic immune particles of any one of claims 1 to 11 with a sample, and applying a magnetic field to the mixed sample.
16. 16. The method of claim 15, further comprising injecting opsonin prior to applying the magnetic field,

    The opsonin is MBL (Mannose binding lectin), FCN-1 (Ficolin-1), and IgG (Immunoglobulin G) at least one selected from the group consisting of antibodies, a method for detecting or removing pathogenic substances.
17. Providing information necessary for diagnosing an infectious disease, comprising mixing the magnetic immune particles of any one of claims 1 to 11 in contact with a sample separated from an individual, and applying a magnetic field to the mixed sample. How to.
18. 18. The method of claim 17, further comprising injecting opsonin prior to applying the magnetic field,

    The opsonin is one or more selected from the group consisting of Mannose binding lectin (MBL), Ficolin-1 (FCN-1), and Immunoglobulin G (IgG) antibodies.
19. Contacting and mixing the magnetic immune particle according to any one of claims 1 to 11 with a sample separated from the subject; applying a magnetic field to the mixed sample to remove the magnetic immune particles that have captured the pathogenic substance from the sample; and injecting the sample from which the magnetic immune particles capturing the pathogenic material are removed back into the subject.
20. The method of claim 19, further comprising injecting at least one selected from the group consisting of opsonin, blood, and plasma before applying the magnetic field,

    Wherein the opsonin is at least one selected from the group consisting of Mannose binding lectin (MBL), Ficolin-1 (FCN-1), and Immunoglobulin G (IgG) antibodies.

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