KR102658450B1 - Nanomagnetic antibody of Salmonella and the preparation method thereof - Google Patents

Abstract

The present invention relates to a nanomagnetic antibody for diagnosing Salmonella, wherein the nanomagnetic antibody is immobilized on the surface of deep superparamagnetic nanoparticles coated with carboxymethyl dextran. The present invention enables quantitative analysis using the magnetism of Salmonella or Salmonella-infected individuals and qualitative analysis through visual observation, and can be applied to the rapid and accurate analysis of various pathogens, including Salmonella.

Description

Nanomagnetic antibody of Salmonella and the preparation method thereof}

The present invention relates to a nanomagnetic antibody in which a salmonella antibody is bound to superparamagnetic iron oxide nanoparticles, a method for producing the same, and rapid diagnosis of salmonella using the same. Specifically, it relates to a nanomagnetic antibody in which a salmonella antibody is bound to deep superparamagnetic iron oxide. It relates to magnetic antibodies, methods for producing them, and methods for rapid diagnosis of Salmonella using the same.

The emergence of multi-drug resistant bacteria and super bacteria, and the emergence of unprecedented mutant viruses are expected to lead to the spread of infectious diseases worldwide and an increase in deaths from related diseases. However, the development of new or traditional antibiotics and antivirals against super bacteria and new viruses is not easy or is lacking.

For the diagnosis of pathogenic microorganisms and infectious diseases, in vitro diagnosis such as immunodiagnosis, genetic diagnosis, and base sequence analysis, in vivo diagnosis such as blood analysis and tissue analysis, and in vitro or in vivo imaging diagnosis are used.

Salmonella is one of the three major food poisoning bacteria that can cause food poisoning, along with intestinal Vibrio Parahaemolyticus and Staphylococcus aureus. Salmonella is a type of proteobacteria belonging to the Salmonella genus of the Enterobacteriaceae family and is a gram-negative bacillus that mainly lives in the digestive tract of humans and animals. Salmonella species are intracellular parasitic bacteria and infect cells such as epithelial cells, M cells, macrophages, and dendritic cells. As a facultative anaerobic cell, in an oxygenated environment, it uses oxygen to synthesize ATP, but in an oxygen-free environment, ATP can be synthesized through fermentation.

The main symptoms of salmonella infection include fever, headache, abdominal pain, vomiting, and diarrhea. Most Salmonella infections are caused by contamination of food with animal or plant excrement. The serotype is divided into typhoid fever type and non-typhoid fever type. Non-typhoid fungi, which cause gastrointestinal diseases, are common, and these non-typhoid fungi can infect other animals as well as humans, making them zoonotic.

Therefore, rapid identification of Salmonella is very important clinically and epidemiologically. In general, Salmonella infection is diagnosed by isolating and identifying the bacteria in the sample, but there is a problem in that diagnosis is inconvenient and rapid diagnosis is not possible. If it is possible to test suspected food poisoning patients or suspected substances on-site within a short period of time, prescriptions and follow-up measures can be quickly taken, and the diagnosis process can be shortened by screening only positive samples in advance. From this perspective, existing immunodiagnostic kits have the disadvantage of having to analyze a certain number of bacteria through culturing for several hours or more. Therefore, the convenience of diagnosis can be improved by improving the sensitivity of existing immunodiagnostic kits and simplifying the testing process. In general, the sensitivity of diagnostic tests using colorimetric methods or antigen-antibody reactions is known to be around 10 ⁵ to 10 ⁸ cfu/ml. Additionally, the PCR test method is known to be detected at around 10 to 10 ³ cfu/ml. Therefore, if the sensitivity of the immunodiagnostic test can be improved to the level of sensitivity of the PCR test, the accuracy of the test can be increased, and if pathogens can be pre-screened with PCR-level sensitivity, the cost, time, and labor required for the diagnostic test can be reduced. can

In general, paramagnetic nanoparticles are used for various purposes in the fields of disease diagnosis and treatment. Iron oxide (IO) is generally composed of magnetite (F ₃ O ₄ ) crystals and maghemite (F ₂ O ₃ ) crystals, and is classified into ferromagnetic iron oxide (Ferromagnetic) depending on its magnetic properties under high magnetic flux density. IO) and paramagnetic iron oxide (Paramagnetic IO). Superparamagnetic iron oxide (SPIO) exhibits a moderate susceptibility between paramagnetism and ferromagnetism, and SPIO is divided into standard SPIO (Standard SPIO, SSPIO, greater than 50 nm), polar SPIO (ultra-small SPIO, USPIO, 10 to 50 nm) and deep SPIO (Very-small SPIO, VSPIO, less than 10 nm).

Based on the above background technology, the present invention developed a nanomagnetic antibody that can be used to quickly detect Salmonella with high sensitivity. The present invention can be applied to early diagnosis of Salmonella by binding Salmonella antibodies to paramagnetic nanoparticles. As a result of research on iron oxide nanoparticles that can bind to Salmonella, a technology for binding Salmonella antibodies to VSPIO is provided.

Therefore, the problem to be solved by the present invention is very small superparamagnetic iron oxide (VSPIO), which has carboxymethyl dextran (CMD) adsorbed on the surface, and a Salmonella antibody bound to the CMD-VSPIO. Relates to nanomagnetic antibodies (NMAb) and methods for producing them.

The problem that the present invention seeks to solve is to propose a method of quickly diagnosing Salmonella from a Salmonella-infected specimen using a magnetic detector.

In addition, the problem to be solved by the present invention is to provide biocompatible polymer-coated VSPIO nanoparticles that can improve sensitivity to immunodiagnosis of Salmonella and an in vitro diagnosis method of Salmonella using the same.

In order to solve the above problems, the present invention provides a metal oxide nanomagnetic antibody for diagnosing Salmonella to determine the presence of Salmonella in an object and a method for producing the same. The present invention provides a metal oxide nanomagnetic antibody for diagnosing Salmonella. Specifically, a metal oxide nanomagnetic antibody for application in detecting Salmonella from individuals infected with Salmonella is provided.

Hereinafter, the term “CMD-VSPIO” used in this specification refers to superparamagnetic iron oxide nanoparticles whose surface is coated with carboxymethyl dextran (CMD). “NMAb” refers to nanoparticles with Salmonella antibodies immobilized on the surface of the CMD-VSPIO.

The NMAb may be a Salmonella diagnostic antibody immobilized on the surface of metal oxide nanoparticles coated with carboxymethyl dextran. In the present invention, it includes NMAb for detecting Salmonella with a magnetic detector, and it is preferable that the NMAb binds Salmonella antibodies to the surface of CMD-VSPIO with a high immobilization rate through covalent bonding or the like.

The metal oxide nanoparticles may be VSPIO (Fe ₃ O ₄ , or Fe ₂ O ₃ ) nanoparticles. The average particle diameter of the VSPIO nanoparticles may be about 3 to 10 nm, and preferably may be in the range of about 3 to 6 nm. The term 'about' can be understood to mean that the error range is up to ±1 nm. The average particle size can be measured by conventional methods in the industry, for example, optical microscopy, transmission electron microscopy (TEM), and scanning electron microscopy. , SEM), etc., and is not limited to the measurement method.

The metal oxide nanoparticles of the present invention may be coated with carboxymethyl dextran. The substances coated on the VSPIO for the purpose of application as NMAb for salmonella detection include low molecular weight compounds such as oleic acid, linoleic acid, and citric acid, chitosan, dextran, carboxydextran, pegylated starch, and hyaluronic acid. There are polymer compounds such as , polyacrylic acid, and mixtures thereof, but as a result of various studies, it has been confirmed that carboxymethyl dextran, which has high binding stability with VSPIO and high biocompatibility, is preferable. In particular, the present invention considered stable and uniform binding of VSPIO to antibodies, and as a result, metal oxide nanoparticles, preferably iron oxide nanoparticles, were formed by combining carboxymethyl dextran (CMD), which has a carboxymethyl group at each C2 position of the glucose monomer, with iron oxide. It was confirmed that stable and uniform antibody binding was possible by reacting with nanoparticles to have a carboxyl group only at the C2 position.

The carboxymethyl dextran may have a weight average molecular weight of 10,000 g/mole to 15,000 g/mole, preferably 11,000 g/mole to 14,500 g/mole. If the molecular weight of the carboxymethyl dextran is more than 15,000 g/mole, the viscosity of the reaction solution for CMD-VSPIO synthesis is high, making it difficult to control the size of the generated CMD-VSPIO, and when the molecular weight is less than 11,000 g/mole, the generated CMD- There is a disadvantage that the size of VSPIO becomes uneven.

The CMD-coated VSPIO nanoparticles may have a particle size of 3 to 30 nm, preferably 4 to 20 nm, and more preferably 5 to 10 nm. When it has the above size range, NMAb bound to Salmonella antibodies can be easily formed, the flow of NMAb in the immunodiagnostic kit is easy, and MNMAb can stably bind to Salmonella antigens, ultimately resulting in sensitivity. and the speed of diagnosis is improved.

One embodiment of the present invention provides an NMAb for diagnosing Salmonella containing the CDM-VSPIO. The NMAb can be used to diagnose Salmonella infection using a mixed-frequency magnetic detector.

One embodiment of the present invention includes the steps of a) treating metal oxide nanoparticles with carboxymethyl dextran to form metal oxide nanoparticles coated with carboxymethyl dextran, b) forming metal oxide nano particles coated with carboxymethyl dextran. activating the particles; and c) combining the activated carboxymethyl dextran-coated metal oxide nanoparticles with a Salmonella antibody.

The manufacturing method will be described in detail.

In step a), carboxymethyl dextran is applied to the surface of deep superparamagnetic iron oxide nanoparticles with a particle size of 3 to 10 nm so that the average particle size of the carboxymethyl dextran-coated metal oxide nanoparticles is 3 to 30 nm. It may include a coating step.

The manufacturing method of the metal oxide nanoparticles coated with carboxymethyl dextran is not particularly limited as long as carboxymethyl dextran can be coated on the surface of the metal oxide nanoparticles, and can be manufactured by conventional methods in the industry. , preferably can be produced by a one-bath synthesis method using coprecipitation of metal ions. The coprecipitation is preferably performed in water, but is not limited thereto. In one aspect, carboxymethyl dextran is added to a solution containing Fe ²⁺ and/or Fe ³⁺ ions, heated and stirred, and after stirring, a basic substance is added to obtain metal oxide nanoparticles coated with carboxymethyl dextran. . The basic material may be ammonia water or sodium hydroxide solution, and the basic material is not particularly limited. The metal oxide nanoparticles may be deep superparamagnetic iron oxide nanoparticles, and the average particle diameter may be 3 to 10 nm. The carboxymethyl dextran can be used by purchasing a commercially available product or can be manufactured and used through a known method. The heating and stirring may be carried out at a temperature of 70°C to 90°C, preferably at a temperature of 75 to 85°C.

Step b) is a step of activating the carboxymethyl dextran-coated metal oxide nanoparticles so that they can form a stable bond with the antibody. Specifically, step b) is a step to promote binding between CMD-VSPIO and Salmonella antibodies.

In step b), a buffer solution containing metal oxide nanoparticles coated with carboxymethyl dextran is mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and EDC hydrochloride (EDC). ·HCl), N-hydroxysuccinimide (NHS), and N-hydroxysulfosuccinimide (Sulfo-NHS), preferably treated with one or more selected from the group consisting of 1-ethyl-3 -Can be treated with a mixture of (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Metal oxide coated with carboxymethyl dextran when treated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) Nanoparticles can exhibit excellent activation. Preferably, EDC and NHS can be mixed and used at a weight ratio of 1:0.5 to 2.5, or 1:0.8 to 2, or 1:1 to 1.8, and when mixed at the above weight ratio, as in the present invention, the size is significantly smaller and the magnetism is significant. It can effectively act on the activation of nanoparticles with .

For example, the iron oxide nanoparticles are treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or EDC hydrochloride (EDC·HCl), or EDC·HCl or EDC and N-hyde Treatment with a mixture of hydroxysuccinimide (NHS), or a mixture of EDC and N-hydroxysulfosuccinimide (Sulfo-NHS), or a mixture of EDC·HCl and NHS, or a mixture of EDC·HCl and Sulfo-NHS You can activate it by doing this.

Step c) may include coupling the metal oxide nanoparticles coated with carboxymethyl dextran activated in step b) with the target Salmonella antibody. The coupling in step c) may include mixing the metal oxide nanoparticles coated with carboxymethyl dextran activated in step b) and the target Salmonella antibody at pH 4 to 6. Specifically, in step c), the activated VSPIO of step b) can be combined with a Salmonella antibody using a buffer solution whose pH is 0.5 to 1.5 lower than the isoelectric point of the Salmonella antibody that is the diagnostic target. The bond may preferably be a covalent bond. For example, if the isoelectric point of the antibody is pH 6.0, a buffer solution with a pH of 4.5 to 5.5 can be used. In the present invention, the sensitivity and specificity of NMAb to Salmonella antigens depend on the amount of antibody bound to NMAb and the reactivity of the binding site between the antigen and the antibody, so the pH of the coupling buffer can be considered as described above.

The antibody bound to CMD-VSPIO in step c) is a Salmonella antibody and may be a polyclonal antibody and/or monoclonal antibody capable of binding to CMD-VSPIO. The reaction rate of Salmonella antibodies to CMD-VSPIO in the NMAb prepared through the coupling method of the present invention is 56 to 92%, preferably 76 to 92%, and most preferably 86 to 92%.

In the CMD-VSPIO, the succinimidyl group that is not bound to an antibody can be used to block with albumin or the like, or to bind to a substance necessary for molecular imaging agents for analyzing or tracking specific molecules of individuals infected with Salmonella.

One embodiment of the present invention provides an immunodiagnostic kit for diagnosing Salmonella infection, manufactured by the above manufacturing method. In the present invention, the term “diagnosis” refers to confirming the status of Salmonella infection in an individual. For the purpose of the present invention, diagnosis is to confirm whether an individual is infected using an immunodiagnosis method in vitro. Preferably, the present invention is an immunodiagnostic system for in vitro diagnosis of Salmonella bacteria, and the presence or absence of Salmonella infection can be confirmed through magnetic signals and the naked eye.

Another embodiment of the present invention includes superparamagnetic iron oxide nanoparticles having an average particle diameter of 3 to 10 nm; And a carboxymethyl dextran coating layer present on the surface of the superparamagnetic iron oxide nanoparticles, wherein the coating layer provides superparamagnetic iron oxide nanoparticles coated with carboxymethyl dextran to which an antibody for Salmonella diagnosis is bound. The carboxymethyl dextran coating layer and the salmonella diagnostic antibody may be connected by amide bonding.

The present invention relates to a Salmonella antibody labeled with deep superparamagnetic iron oxide coated with carboxymethyl dextran and a method for producing the same. While the existing iron oxide contrast agent is USPIO-based, the present invention is based on VSPIO iron oxide with a particle size of 10 nm or less. do. The manufactured VSPIO is coated with CMD, which has excellent productivity and excellent binding stability to iron oxide, which has the effect of improving storage stability.

The iron oxide nanoparticles of the present invention have excellent binding to Salmonella antibodies. The manufactured NMAb is effective in early, rapid, and economical diagnosis of Salmonella due to its specificity for Salmonella antigens and improved sensitivity by magnetic detectors.

The following drawings attached to this specification illustrate preferred embodiments of the present invention, and serve to further understand the technology of the present invention along with the contents of the above-described invention, so the present invention is limited only to the matters described in such drawings. It should not be interpreted as such.
Figure 1 shows the X-ray diffraction pattern of carboxymethyl dextran-hyperparamagnetic nanoparticles (CMD-VSPIO).
Figure 2 shows the results of HR-TEM analysis of CMD-VSPIO (scale bar, 10 nm).
Figure 4 shows the amount of unreacted antibody remaining in the reaction solution according to reaction time in the coupling reaction between activated CMD-VSPIO and Salmonella antibody conducted under conditions where the pH of the reaction solution was 7.0 and 5.0, respectively.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as limited to the following embodiments. Embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the field to which the present invention pertains.

Example 1) Synthesis of CMD-VSPIO

5 mmol (98% purity, 1.013 g, Sigma-Aldrich Corp., St. Louis. MO. USA) of FeCl ₂ ·4H ₂ O and 10 mmol (97% purity, 2.787 g, Sigma-Aldrich Corp., St. Louis. MO. USA). Louis, MO. USA) FeCl ₂ ·6H ₂ O was added to a three-necked round flask, then 100 mL of deionized water was added and stirred with a mechanical overhead stirrer to prepare an Fe ion solution. . 0.1 g of carboxymethyl dextran (CMD, CM-Dextran sodium salt, BioXtra grade, Mwt 13,671 g/mol, Sigma life science, St. Louis. MO. USA) was added to the prepared Fe ion solution, and stirred at 1,000 rpm. The solution was heated to 80 degrees Celsius. 6 mL of NH ₄ OH was added in small amounts to the heated solution using a syringe pump (Model No. 300, New Era Pump System, Inc., Farmingdale, NY, USA), and then vigorously stirred for 3 hours. After the reaction flask was cooled to room temperature, the dark black iron oxide particles that settled at the bottom of the flask were separated using a permanent magnet, and the separated iron oxide particles were washed with deionized water until the pH of the washing solution became neutral. The washed iron oxide particles were freeze-dried and stored in a desiccator until use.

Experimental Example 1) XRD analysis of CMD-VSPIO

The XRD pattern of CMD-VSPIO prepared in Example 1 was analyzed using an X-ray powder diffractometer (D8 ADVANCE, Bruker AXS GmbH, Kalsruhe, Germany) using Cu Kα radiation, and the results are shown in Figure 1.

As shown in Figure 1, the diffraction peaks of CMD-VSPIO showed the unique diffraction peaks of Fe ₃ O ₄ crystals, which means that CMD-VSPIO prepared in Example 1 is a crystal of Fe ₃ O ₄ and CMD is Fe ₃ O ₄ It was confirmed that there was no effect on crystal formation.

Experimental Example 2) TEM analysis of CMD-VSPIO

The dispersion of CMD-VSPIO prepared in Example 1 in deionized water was added to a carbon-coated copper grid and dried at room temperature. The dried CMD-VSPIO sample was subjected to high-resolution TEM (HR- The results of TEM (high resolution-TEM) analysis are shown in Figure 2.

As shown in Figure 2, CMD-VSPIO was observed as oval or spherical particles with a long axis or particle diameter of 4 to 15 nm and an irregular surface.

Experimental Example 3) Isoelectric point measurement of Salmonella Typhimurium antibody

The zeta potential of the antibody was changed by changing the pH of the solution containing Salmonella antibody (Rabbit anti-vibrio species antibody, Cat. No. J142, EastCoastBio, North Brewick, ME, USA) using 0.25 M NaOH solution and 0.25 M HCl solution. was measured using a zeta potential meter (Particle Size Analyzer, Zetasizer nano zs, Malvern, Worcetershire, UK). The isoelectric point of the salmonella antibody measured according to the above measurement method was 5.4.

Example 2) Amide coupling of CMD-VSPIO and Salmonella antibody

Dissolve 1.07 g of MES monohydrate (2-Morpholinoethanesulfonic acid monohydrate, >99.5%, Sigma Chemical Co., St. Louis, MO, USA) in 400 mL of deionized water, adjust the pH of the solution using NaOH solution, A 10 mM MES buffer solution was prepared by adding deionized water so that the total volume of the solution was 500 mL. 10 mg of CMD-VSPIO was washed several times with 1 mL of 10 mM MES buffer (pH 6.1) (hereinafter referred to as MES buffer). 7.92 mg of EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, >98%, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) and 8.81 mg of NHS (N-Hydroxysuccimide, >98%, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) was dissolved in 10 mL of MES buffer to prepare an activation solution. To the CMD-VSPIO washed with MES buffer, 1 mL of activation solution was added to react 5 μmol of EDC and 7.5 μmol of NHS, and then incubated at 37 °C for 30 minutes. After completing the activation reaction, CMD-VSPIO was washed three times with MES buffer and immediately used for the coupling reaction with Salmonella antibody. A 1 mg/mL Salmonella antibody solution was prepared by adding 1 mL of 50% glycerol solution to a vial containing 1 mg of Salmonella antibody, and then 100 μL of the prepared antibody solution was added to 900 μL of 10 mM MES buffer (pH 4.9). ) or 900 μL of 10mM MES buffer (pH 5.9, containing 300mM NaCl). 10 mg of activated CMD-VSPIO was added to 1 mL of the prepared Salmonella antibody solution at pH 4.9 or pH 5.9, and then incubated at 37 °C for 2 hours. 1 mL of MES containing 1% BSA (≥98.0%, Sigma-Aldrich Corp., St. Louis, MO, USA) to block unreacted succinimidyl groups remaining in CMD-VSPIO after the coupling reaction. After adding the buffer, it was incubated at 25 °C for 16 hours. After completing the reaction, the reaction product was washed several times with MES buffer and stored at 4 °C until use.

Experimental Example 4) Analysis of reaction rate and degree of immobilization of Salmonella antibodies to activated CMD-VSPIO using BCA (Bicinchoninic acid) protein quantification method

Supernatant samples taken at regular time intervals from the coupling reaction solution of activated CMD-VSPIO and Salmonella antibody were dispensed in 10 μL portions into a 96-well plate. The 2 mg/mL BSA standard solution included in the protein assay kit (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific inc., Waltham, MA, USA) was diluted to 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL. A BSA (Bovine Serum Albumin) solution with a concentration of mL was prepared and dispensed in 10 μL each into a 96-well plate. After preparing a mixed solution of BCA reagent A and BCA reagent B included in the protein analysis kit at a volume ratio of 50:1, 100% of the mixed solution was added to each well of a 96-well plate containing the antibody sample solution and the diluted BSA standard solution. μL was added at a time and incubated at 37°C for 30 minutes. After measuring the absorbance of the antibody sample and BSA standard solution after the color development reaction at 560 nm, the concentration of unreacted Salmonella antibody was calculated using a calibration curve obtained using the absorbance according to the concentration of BSA. The amount of reacted antibody was calculated from the amount of unreacted antibody and the amount of antibody in the reaction solution before the coupling reaction. From the amount of reacted antibody, the antibody reaction rate and degree of antibody immobilization were calculated using the following equation, and the results are shown in Table 1.

Antibody reaction rate (%) = [(antibody amount in reaction solution before reaction (μg/mL) - antibody amount in supernatant after reaction (μg/mL))]/antibody amount in reaction solution before reaction (μg/mL)

Antibody immobilization degree (μg/g) = Weight of coupled antibody (μg/mL)/Weight of iron oxide nanoparticles (g/mL)

Immobilization of Salmonella antibodies to CMD-VSPIO pH 4.4 pH 6.4 Response rate (%) 95.09 46.15 Antibody immobilization degree (μg/g) 478.3 232.1

Experimental Example 5) Magnetism of nanomagnetic antibody

In order to analyze the relationship between the amount of nanomagnetic antibody of Salmonella prepared in Example 2 and the magnetic signal, the change in magnetic signal according to the amount of nanomagnetic antibody was measured using a nanoparticle magnetic analyzer (MayRay2000, Magsolution Inc., Daejeon, Republic of Korea). ) was measured using . As shown in Figure 3, as the amount of Salmonella nanomagnetic antibody increased, the magnetic signal was linearly proportional with a high correlation coefficient (R ² = 0.9913).

Claims (13)

delete delete delete delete delete a) treating deep superparamagnetic iron oxide nanoparticles having an average particle size of 3 to 10 nm with carboxymethyl dextran to form deep superparamagnetic nanoparticles coated with carboxymethyl dextran;
b) activating the deep polar superparamagnetic iron oxide nanoparticles coated with the carboxymethyl dextran;
c) A method for producing a nanomagnetic antibody for Salmonella diagnosis, comprising the step of combining the activated carboxymethyl dextran-coated deep superparamagnetic iron oxide nanoparticles with a Salmonella antibody,
In step b), deep superparamagnetic iron oxide nanoparticles coated with carboxymethyl dextran are mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hyde at a weight ratio of 1:0.5 to 2.5. Treated with Roxysuccinimide (NHS),
In step c), the deep polar superparamagnetic iron oxide nanoparticles coated with carboxymethyl dextran activated in step b) are coupled with the Salmonella antibody, and the coupling is performed with the deep polar superparamagnetic iron oxide nanoparticles activated in step b). A method for producing a nanomagnetic antibody for salmonella diagnosis, characterized in that mixing magnetic iron oxide nanoparticles and salmonella antibodies at pH 4 to 6. delete delete The method of claim 6, wherein the carboxymethyl dextran has a weight average molecular weight of 10,000 g/mole to 15,000 g/mole. The method of claim 6, wherein the carboxymethyl dextran activated in step b) combines the deep superparamagnetic iron oxide nanoparticles with the Salmonella antibody at a pH of 0.5 to 1.5 above the isoelectric point of the antibody. A method for producing nanomagnetic antibodies for salmonella diagnosis, characterized by mixing in a low buffer solution. A nanomagnetic antibody for diagnosing Salmonella infection, manufactured by the production method according to any one of claims 6, 9, and 10. A rapid diagnostic kit for diagnosing Salmonella comprising the nanomagnetic antibody according to claim 11. delete