KR101438209B1 - Enzyme-immunoassay complex and manufacturing method thereof - Google Patents

Abstract

The complex for enzyme immunoassay of the present invention can accumulate a large amount of enzyme as compared with other complexes of the prior art. In addition, the three-dimensional network structure of the nanofiber-based polymer improves the stability of the enzyme because it can effectively protect the enzyme from external impacts.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an enzyme immunoassay complex,

The present invention relates to a complex for enzyme immunoassay and a method for preparing the same, and more particularly, to an enzyme immunoassay which maximizes the amount of immobilized enzyme while minimizing signal generation due to nonspecific binding, And a method for producing the same.

Immunoassay is a generic term for biomolecular detection techniques. Generally, it is a technique to tell whether DNA, protein, or microorganisms such as bacteria exist by using specific binding such as DNA hybridization and antibody-antigen binding. Enzyme-Linked ImmunoSorbent Assay (ELISA) is one of these immunoassays that enables the detection and quantification of various antigens such as viruses and pathogens by using the specificity of the antibody against the antigen, the substrate selectivity of the enzyme and the catalytic reaction. It is a kind of immunoassay used for To detect the antigen to be analyzed, an enzyme-conjugated antibody is used in the enzyme-linked immunosorbent assay. Compared with radioactive labels that have been used for immunoassays since the 1960s, enzyme labels can be expected to have high efficiency due to the substrate selectivity of the enzyme itself and its catalytic reaction, Safety is also excellent. Based on these advantages, an enzyme-linked immunosorbent assay system, which can replace radioimmunoassay (RIA), was independently published by Perrlmann and Schuurs in 1971. Based on these results, Oraganon Teknika has succeeded in commercializing the world's first commercially available enzyme-linked immunosorbent assay system capable of detecting hepatitis B surface antigen (HBsAg) (Haas, H. and Hotz, G., J Virol., Methods, 2, 1980). To date, enzyme-linked immunosorbent assay is one of the most widely used technologies in diagnostic kits.

In the case of the conventional enzyme-linked immunosorbent assay, the enzyme-labeled antibody is based on a mechanism that indicates the amount of the antigen detected as an enzyme reaction signal. However, in the conventional enzyme-linked immunosorbent assay, there is a limitation in improving the sensitivity by binding one enzyme to one antibody. This is because some antigens requiring detection of trace amounts were difficult to judge whether they were harmful to the body or not. In addition, due to the instability of enzyme activity incidentally, it was difficult to obtain a consistent signal through the enzyme bound to the antibody depending on the surrounding environment even though the antigen was detected accurately through the antibody. Therefore, a high stability and high sensitivity immunoassay method are required for the consistent detection of the detection signal and the improved signal detection.

In this regard, various efforts have been made to achieve high sensitivity immunoassays. The most basic method is to increase the number of enzymes per unit antibody. However, when the amount of the enzyme is maximized, the size of the complex increases and the signal due to nonspecific binding is still present even when the target antigen is not present. Also, most of these complexes have never considered stability. There has been a desperate need for an alternative solution to these two problems.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide an enzyme immunoassay system which maximizes the amount of immobilized enzyme while minimizing signal generation due to nonspecific binding, An immunoassay complex, a method for producing the same, and an immunoassay system.

In order to solve the above-mentioned problems, the present invention relates to a crosslinked enzyme aggregate, which is supported inside a fiber interstices of a fiber matrix containing three-dimensional network fibers and has a diameter larger than an inlet size of the pores, A fibrous matrix conjugate of an enzyme-3-dimensional network structure comprising a shell comprising crosslinked enzymes surrounding the enzyme; And an antibody conjugated to the complex.

According to a preferred embodiment of the present invention, the three-dimensional network fibers may be microfibers or nanofibers.

According to another preferred embodiment of the present invention, the three-dimensional network fiber is made of polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, Polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and poly (styrene-co-maleic anhydride).

According to another preferred embodiment of the present invention, the shell may be a multilayer.

According to another preferred embodiment of the present invention, the enzyme is selected from the group consisting of: mottryptine, trypsin, subtilisin, papain, lipase, Hole Serradishesperoxidase, soybean peroxidase, chloroperoxidase, It is now possible to use a combination of peroxidase, tyrosinase, lactase, cellulase, silanease, lactase, sucrase, organophosphorohydrolase, chlorinestease, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase And may be any one selected from the group consisting of rhegogenase, hydrozogenase and glucose isomerase.

According to another preferred embodiment of the present invention, the enzyme may be a hole ceradylase peroxidase.

According to another preferred embodiment of the present invention, there is provided a method for producing a three-dimensional network fiber comprising the steps of: (1) adding an enzyme to a matrix comprising three-dimensional network fibers; (2) adding a precipitating agent to the matrix to prevent leakage of enzymes added to the pores of the matrix; (3) preparing an enzyme-fiber matrix complex by forming an enzyme aggregate by adding a cross-linking agent to form cross-linking between the enzymes; And (4) conjugating the antibody to the complex. The present invention also provides a method for producing an enzyme immunoassay complex.

According to another preferred embodiment of the present invention, the precipitating agent is at least one selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, , Potassium sulfate, potassium phosphate, and aqueous solutions thereof may be used singly or in combination.

According to another preferred embodiment of the present invention, the crosslinking agent is selected from the group consisting of a diisocyanate, a dianhydride, a diepoxide, a dialdehyde, a diimide, 1-ethyl-3-dimethylaminopropylcarbodiimide, a glutardialdehyde , Bis (imido ester), bis (succinimidyl ester), and diacid chloride.

According to another preferred embodiment of the present invention, the step of removing the precipitating agent and the crosslinking agent may be further included between steps (3) and (4).

According to another preferred embodiment of the present invention, the step (1) may be such that the enzyme is adsorbed or covalently bonded to the matrix surface.

The complex for enzyme immunoassay of the present invention can accumulate a large amount of enzyme as compared with other complexes of the prior art. In addition, the three-dimensional network structure of the nanofiber-based polymer improves the stability of the enzyme because it can effectively protect the enzyme from external impacts.

In addition, in the process of synthesizing enzyme immunoassay complexes, a variety of enzyme-3-dimensional network structure fibrous matrix complexes of various sizes are provided by changing nanofiber-based polymer synthesis and enzyme immobilization conditions. This provides universality in which signals can be amplified while minimizing non-specific binding as compared to other complexes that are produced in the immunodetection process, which causes non-specific binding with the substrate depending on the physical properties of the complex.

Therefore, when the complex for enzyme immunoassay of the present invention is used in the field of immunodetection, the signal can be improved as compared with the case of using a conventional single enzyme. By synthesizing complexes of various sizes, nonspecific binding can be minimized, And can provide optimized results. In addition, various platforms can provide optimized results that minimize non-specific coupling and improve signal quality.

1 is a schematic diagram showing the difference between a conventional ELISA using a single enzyme and an ELISA using the present invention.
FIG. 2A is a graph showing synthesis of polyaniline nanofibers of various sizes according to the addition amount of aniline, and FIG. 2B is a graph showing the activity of a fibrous matrix complex of an antibody-enzyme-3-dimensional network structure synthesized on polyaniline nanofibers of various sizes Graph.
FIG. 3A is a graph comparing enzyme activities of a fibrous matrix complex having a three-dimensional enzyme-3-dimensional network structure synthesized as described above, and FIG. 3B is a graph showing stability of enzyme activity.
FIG. 4A shows the result of ascertaining the synergistic effect of the signal due to the hIgG detection as a target at a specific concentration using a well plate-based immunoassay method using EAC and EAPC as a fibrous matrix complex having an enzyme-3-dimensional network structure, This is the result of confirming the immunoassay method based on nanoparticles.
FIG. 5A shows a result of a well plate-based immunoassay for a signal depending on the concentration of a target hIgG using EAC and EAPC as a fibrous matrix complex having an enzyme-3-dimensional network structure. FIG. It is the result confirmed by measurement method.
FIG. 6A shows the result of confirming the selective binding ability in the immune response using a well plate-based immunoassay method using EAC and EAPC as a fibrous matrix complex of an enzyme-3-dimensional network structure, and FIG. 6B shows the results of immunoassay The results showed that the selective binding ability was confirmed by the method.
7 is a perspective view and a cross-sectional view of a polyaniline nanofiber matrix composite of an enzyme-3-dimensional network structure according to a preferred embodiment of the present invention.
8 is a schematic diagram showing a process for immobilizing an enzyme in a fiber matrix.

Hereinafter, the present invention will be described in more detail.

As described above, in the case of the conventional enzyme-linked immunosorbent assay, since the amount of the enzyme bound to the antibody is limited and the signal that appears even after reacting with the substrate is weak, it is difficult to measure the low concentration of the antigen. The reliability of one signal was inevitable. In addition, although various studies have been carried out to increase the number of enzymes per unit antibody, there has been a problem in that the signal is generated due to nonspecific binding even in the absence of the target antigen due to the increase in the size thereof.

Accordingly, in the present invention, a crosslinked enzyme aggregate, which is supported in interspaces between fibers of a fiber matrix containing a three-dimensional network fiber and whose diameter is larger than an inlet size of the pores, and a cross-linked enzyme A fibrous matrix conjugate of an enzyme-3-dimensional network structure comprising a shell comprising; And an antibody conjugated to the complex. The present invention has been made to solve the above-mentioned problems. This allows a large amount of enzyme to be accumulated compared to other complexes of the prior art. In addition, the three-dimensional network structure of the nanofiber-based polymer improves the stability of the enzyme because it can effectively protect the enzyme from external impacts.

First, the complex for enzyme immunoassay of the present invention will be described. The enzyme immunoassay complex of the present invention comprises (1) adding an enzyme to a matrix comprising a three-dimensional network fiber; (2) adding a precipitating agent to the matrix to prevent leakage of enzymes added to the pores of the matrix; (3) preparing an enzyme-fiber matrix complex by forming an enzyme aggregate by adding a cross-linking agent to form cross-linking between the enzymes; And (4) conjugating the antibody to the complex.

First, as step (1), the enzyme is adsorbed and / or covalently bonded to the porous matrix containing the three-dimensional network fibers. If the surface of the three-dimensional network fiber contains a functional group capable of covalently binding to the enzyme (if it has been modified for this purpose), covalent bonding occurs between the enzyme and the fiber and is adsorbed if it does not.

The fiber used in the present invention can be used without limitations as long as a three-dimensional network is formed during spinning so that voids can be formed between the fibers and the fibers. The fibers can be used in various forms such as polyvinyl alcohol, polyacrylonitrile, nylon, Poly (styrene-co-maleic anhydride), polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and poly , And more preferably polyaniline nanofibers may be used in consideration of cost and efficiency, but the present invention is not limited thereto.

The kind of spinning for producing the fiber can also utilize a conventional polymerization and / or spinning process if it has a three-dimensional network structure between the fiber and the fiber, and both electrospinning and melt spinning can be used. The diameter of the fibers can also be applied to both nanofibers and microfibers, but it may be more advantageous to use nanofibers in view of the size of the enzyme aggregates described below and the size of pores formed between the fibers. However, carbon nanotubes do not fall within the scope of the present invention because they can not form a three-dimensional network even if they are woven with fibers.

The porous matrix according to one embodiment of the present invention may include a part or all of the three-dimensional network fibers, and the porosity refers to a space (gap) between the fibers and the fibers forming the three-dimensional network.

The fibers of the present invention form a network of three-dimensional networks through which a fiber matrix structure can be made, and the matrix structure can be an amorphous structure in which the fibers are intricately intertwined.

The enzyme included in the present invention can be used without limitation as long as it can be usually used in enzyme immunoassay and is preferably selected from the group consisting of motryptine, trypsin, subtilisin, papain, lipase, But are not limited to, cholinesterase, chymotrypsin, loxidase, chloroperoxidase, manganese peroxidase, tyrosinase, lactase, cellulase, silanease, lactase, sucrase, organophosphorohydrolase, May be any one selected from the group consisting of glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, hydrozogenase and glucose isomerase. On the other hand, more preferably, the activity of the hole ceradylase peroxidase is very excellent.

On the other hand, since the fiber matrix of the present invention can be applied to a fiber substantially containing or not containing any functional group (e.g., amino group) that can be covalently bonded to the surface of the fiber matrix, (Space) formed between the surface of the fiber or the fiber and the fiber. Therefore, when an external shock such as washing with water is simply applied to the surface of the matrix, most of the adsorbed enzyme is separated from the matrix, resulting in a marked decrease in the fixing rate of the enzyme.

In step (2), the matrix is subjected to enzyme precipitation in order to prevent the outflow of adsorbed and / or covalently bound enzymes in the pores of the matrix. The adsorbed enzyme is so small that it is hardly visible to the naked eye. When the precipitating agent is added to the matrix to precipitate the adsorbed enzyme, the adsorbed enzymes are aggregated with each other and become large in size. Eventually, the adsorbed enzyme is precipitated between pores (spaces) . This results in the formation of a crosslinked enzyme aggregate with a diameter greater than the inlet size of interstitial pores. If the precipitant is not added, the size of the aggregate will be smaller than the size of the pore opening, Thereby leaving the space between the pores.

The precipitating agent that can be used at this time can be used without limitation as long as it can precipitate the enzyme with little effect on the activity of the enzyme. Preferably, the precipitating agent is selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, But are not limited to, acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and aqueous solutions thereof.

Next, in step (3), a cross-linking agent is added to form an enzyme aggregate to form a cross-link between the enzymes. Specifically, since the size of the precipitated enzyme is relatively smaller than the size of the pores (spaces) formed between the fibers and the fibers, the enzyme precipitated by external impact such as washing with water can be leaked to the outside of the pores. However, when a cross-linking agent is added to form a cross-link between the enzyme and the precipitated enzyme, the cross-linked enzyme forms an aggregate, and the formed enzyme aggregate almost fills the inside of the gap. As a result, the size of the enzyme aggregate formed becomes larger than the size of the inlet of the pore, so that the cross-linked enzyme aggregate is not easily leaked to the outside of the pore even by an external impact such as flushing. As a result, since the enzyme aggregate is located within the pore even after a lapse of time, the enzyme aggregate can be provided in the fiber matrix for a long period of time even if a direct binding relationship such as covalent bond between the enzyme and the fiber is not formed. In addition, since the complex formed between the enzyme-3-dimensional network fiber matrix has a much larger amount of enzyme than that of the conventional complex, the use of the enzyme in a biosensor or a biofuel cell, Can be remarkably improved.

On the other hand, the effect of adding the crosslinking agent after the treatment with the precipitating agent is remarkably enhanced as compared with the case where only the crosslinking agent is added. This is because, when the cross-linking agent is added after the adsorption of the enzyme, the concentration of the enzymes becomes equal to the ambient concentration even if the inside of the pores formed between the fiber and the fiber is not sufficiently filled or the uterus is filled. The enzymes at the same concentration are cross-linked to form a larger lump than the openings of the pores in the pores in the nanofiber, so that there is a high probability that the cross-linked enzyme will leak out during washing. The covalent bond also has a high probability that the remaining enzymes other than the covalently bonded enzymes do not form large clumps and are subsequently washed.

However, in the case of the present invention, the enzymes forcibly deposit the pores in the nanofiber more densely through precipitation after the adsorption of the enzyme, and the enzymes filled in the core are formed into large lumps through cross-linking, It is expected that losses will be less than EAC during the wash.

Furthermore, in the case of polyaniline nanofibers which have little functional groups capable of covalently bonding to the surface of the fiber, it is very difficult for the enzyme to be immobilized on the surface of the fiber. However, in the present invention, enzymes precipitated from the surface of the fiber are cross-linked to form a shell surrounding the surface of the fiber, so that even when a covalent bond is not substantially formed between the fiber and the enzyme such as a hot dog, Of the enzyme can be immobilized by forming a shell.

The crosslinking agent that can be used in the present invention can be used without limitation as long as it can form cross-linking between enzymes without inhibiting the activity of the enzyme, but preferably a diisocyanate, a dianhydride, a diepoxide, a dialdehyde, At least one compound selected from the group consisting of diimide, 1-ethyl-3-dimethylaminopropylcarbodiimide, glutardialdehyde, bis (imido ester), bis (succinimidyl ester) and diacid chloride It is possible to use glutaraldehyde, but it is not limited thereto, and it is obvious to a person skilled in the art to use the crosslinking agent known in the art without limitation.

Preferably, after the step (3), the enzyme-3-dimensional network fiber matrix composite is washed with water to remove the added crosslinking agent and the precipitating agent. In the case of the matrix composite prepared by the conventional production method, the enzyme immobilized between the pores flows out of the pores through the washing step. However, the enzyme-3-dimensional network fiber matrix composite prepared by the present invention is formed in the pores Since the size of the enzyme aggregate is larger than the inlet of the pore, the enzyme aggregate can be fixed to the inside of the pore without spilling out to the outside despite the washing process.

Next, in step (4), the antibody is conjugated to a matrix containing the enzyme. The antibody to be used at this time can be variously used according to the target antigen, and it is also possible to conjugate one antibody or a mixture of antibodies.

The antibody is conjugated to the matrix by the functionality of the remaining cross-linking agent used in the course of making the enzyme-3-dimensional network fiber matrix complex.

The composite according to the present invention produced through this method comprises a cross-linked enzyme aggregate supported inside the fiber interstices of the fiber matrix containing the three-dimensional network fibers and having a diameter larger than the inlet size of the pores, A fibrous matrix complex of an enzyme-3-dimensional network structure comprising a shell comprising crosslinked enzymes; And an antibody conjugated to the complex.

7 is a perspective view and a cross-sectional view of a polyaniline nanofiber matrix composite of an enzyme-3-dimensional network structure according to a preferred embodiment of the present invention. In this case, a crosslinked enzyme aggregate having a diameter larger than the inlet size of the pores is carried inside the pores of the fiber matrix of the three-dimensional network structure. In addition, the surface of the three-dimensional network fibers is surrounded by a shell containing an enzyme aggregate. The EPCs at the bottom right of FIG. 7 are cross-sectional views of the surface of the three-dimensional network fiber wrapped by the shell including the enzyme aggregate, wherein covalent bonds are not substantially formed between the fibers and the enzyme aggregate forming the shell, Like a hot dog, a shell containing an enzyme aggregate surrounds the surface of the three-dimensional network fibers.

As a result, the enzyme-3-dimensional network fibrous matrix composite of the present invention has a sufficiently large size of the enzyme complex formed inside the pores to the extent that the pores formed between the fibers and the fibers do not flow out to the outside, Positive enzymes can be immobilized on the matrix to form complexes. In other words, the size of the enzyme complex formed inside the cavity is larger than the size of the inlet of the cavity, which is the passage through which the air flows out from the cavity formed between the fiber and the fiber, so that even when there is external stimulation such as flushing, , Which allows the complex to be maintained for a long period of time without forming a direct binding relationship between the enzyme and the matrix.

Furthermore, in the case of polyaniline nanofibers which have little functional groups capable of covalently bonding to the surface of the fiber, it is very difficult for the enzyme to be immobilized on the surface of the fiber. However, in the present invention, enzymes precipitated from the surface of the fiber are cross-linked to form a shell surrounding the surface of the fiber, so that even when a covalent bond is not substantially formed between the fiber and the enzyme such as a hot dog, Of the enzyme can form a shell and be immobilized, and such a shell can form a multilayer.

FIG. 1 is a schematic diagram showing an ELISA method using the complex of the present invention, which can have a remarkably improved effect as compared with the use of a conventional single enzyme.

Therefore, the enzyme immunoassay complex of the present invention can accumulate a large amount of enzyme as compared with other conventional complexes. In addition, the three-dimensional network structure of the nanofiber-based polymer improves the stability of the enzyme because it can effectively protect the enzyme from external impacts.

In addition, in the process of synthesizing enzyme immunoassay complexes, a variety of enzyme-3-dimensional network structure fibrous matrix complexes of various sizes are provided by changing nanofiber-based polymer synthesis and enzyme immobilization conditions. This provides universality in which signals can be amplified while minimizing non-specific binding as compared to other complexes that are produced in the immunodetection process, which causes non-specific binding with the substrate depending on the physical properties of the complex.

Therefore, when the complex for enzyme immunoassay of the present invention is used in the field of immunodetection, the signal can be improved as compared with the case of using a conventional single enzyme. By synthesizing complexes of various sizes, nonspecific binding can be minimized, And can provide optimized results. In addition, various platforms can provide optimized results that minimize non-specific coupling and improve signal quality.

≪ Preparation Example 1 > Synthesis of 3-dimensional polymer nanofiber and size change according to conditions

Specifically, for the enzyme immobilization, the polyaniline-based three-dimensional polymer nanofibers were prepared by oxidative polymerization using ammonium persulfate, an oxidizing agent, as an initiator. At this time, the growth of polyaniline can be controlled according to the amount of ammonium peroxodisulfate and the initial aniline concentration. When the initial aniline concentration is set as shown in FIG. 2 while the amount of ammonium peroxodisulfate is fixed, various sizes of polyaniline nanofibers . 10 ml of the prepared ammonium peroxodisulfate solution was added to 10 ml of aniline dissolved in HCl solution and mixed well. After that, the mixed solution was stirred at a rate of 200 rpm at room temperature for 24 hours. After the polymerization was completed, the solution was washed several times with distilled water to neutralize the acidic solution to raise the pH to neutrality and then stored at 4 ° C.

Dynamic light scattering was used to confirm the size of the synthesized polymer nanofibers. As a result, the size of the three-dimensional polymer nanofibers synthesized when the initial aniline concentration was lowest (0.5 wt%) was increased to 700 nm, and as the initial aniline concentration was increased, the size gradually decreased. When the concentration is 7.5 wt%, the size of the polymer nanofiber is reduced to 160 nm. Further, when the initial aniline concentration exceeded 10% by weight, a phenomenon of solidification occurred. This result implies that by adjusting the size of the fibrous matrix complex of the enzyme-3-dimensional network structure while changing simple conditions, it is possible to selectively provide the fibrous matrix complex of the enzyme-3-dimensional network structure according to its use. In order to carry out the enzyme-linked immunosorbent assay for the purpose of the present invention, the initial aniline concentration of 7.5 wt%, which is the smallest size among these results, was used.

PREPARATION EXAMPLE 2 Preparation of a polyaniline nanofiber matrix having a three-dimensional network structure

Specifically, for the enzyme immobilization, the polyaniline-based three-dimensional polymer nanofibers were prepared by dissolving ammonium persulfate, an oxidizing agent, as an initiator in a 1 M HCl solution to a concentration of 0.1 M, 17, and mix well. 10 ml of the prepared ammonium peroxodisulfate solution was added to a 10 ml solution containing aniline and HCl mixed well. After this, the well-mixed solution was stirred at room temperature for 24 hours at a speed of 200 rpm. After the polymerization was completed, the solution was washed several times with distilled water to neutralize the acidic solution to raise the pH to neutrality and then stored at 4 ° C. The synthesized polyaniline nanofibers are connected to each other like a coral complex to form a three-dimensional network structure and have many voids in the inside as well as the pores of the outer branch portions of the nanofibers according to the concentration of aniline.

COMPARATIVE EXAMPLE 1 Production of a fibrous matrix complex having an enzyme-3-dimensional network structure according to EA

A polyaniline nanofiber matrix composite having an enzyme-3-dimensional network structure was prepared according to EA (enzyme adsorption) in order to compare with the case of containing the precipitating agent of FIG. Specifically, the solution was mixed with 1 ml of a 10 mg / ml solution of HRP (Horseradish peroxidase) in the polyaniline nanofiber solution (initial aniline concentration: 7.5% by weight) prepared in Preparation Example 2 and stirred at 150 rpm for 1 hour so that the adsorption could proceed well. EA was fabricated as a fiber matrix composite with a three - dimensional network structure. The prepared EA solution was centrifuged and washed with PB buffer to remove residual enzymes.

<Comparative Example 2> Fabrication of a fibrous matrix complex having an enzyme-3-dimensional network structure according to EAC

A polyaniline nanofiber matrix composite having an enzyme-3-dimensional network structure was prepared according to EAC (enzyme adsorption and crosslinking) for comparison with the case of containing the precipitating agent of FIG. Specifically, the solution was mixed with 1 ml of a 10 mg / ml solution of HRP (Horseradish peroxidase) in the polyaniline nanofiber solution prepared in Preparation Example 2 (initial aniline concentration: 7.5% by weight), and stirred at 150 rpm for 1 hour so that adsorption could proceed well. After the addition of glutaric dialdehyde as a crosslinking agent to a concentration of 0.5%, EAC was prepared as a fibrous matrix complex having an enzyme-3-dimensional network structure by reacting in a refrigerator at 4 ° C for 1 hour for sufficient reaction of the crosslinking agent Respectively. The prepared EAC solution was centrifuged and washed using PB buffer to remove residual enzymes.

Example 1: Fabrication of a fibrous matrix complex of an enzyme-3-dimensional network structure according to EAPC

A polyaniline nanofiber matrix composite having an enzyme-3-dimensional network structure was prepared according to EAPC (enzyme adsorption and crosslinking) of FIG. Specifically, a solution of 10 mg / ml of HRP (Horseradish peroxidase) and 1 ml of GOx (glucose oxidase) was mixed with the polyaniline nanofiber solution prepared in Preparation Example 2 (initial aniline concentration: 7.5% by weight) Stir at 150 rpm for 1 hour. After that, the concentration of ammonium sulfate was adjusted to 50% (w / v) as a precipitating agent, and the mixture was stirred at 200 rpm for 30 minutes at room temperature to sufficiently precipitate the enzyme. After that, in order to cross-link the precipitated enzyme, the glutaraldehyde concentration was added as a crosslinking agent so that the concentration of glutaraldehyde was 0.5 wt%, followed by reaction in a refrigerator at 4 ° C for 1 hour for sufficient reaction of the crosslinking agent, EAPC was fabricated as a fiber matrix composite of network structure. The prepared EAPC solution was centrifuged and washed with PB buffer to remove residual enzymes.

Example 2 Antibody Immobilization of a Fabric Matrix Complex of an Enzyme-3-Dimensional Network Structure

In order to use the fibrous matrix complex of the enzyme-3-dimensional network structure prepared in Example 1 and Comparative Examples 1 and 2 for enzyme-linked immunosorbent assay, the hIgG antibody solution was mixed as a model protein and stirred at 200 rpm for 2 hours . In this case, the antibody reacts with the functional group activated by the crosslinking agent remaining around the enzyme, so that the concentration of the solution of the antibody solution and the fiber matrix complex of the enzyme-3-dimensional network structure is 1: 1 by mass ratio Respectively. The prepared antibody-enzyme-3-dimensional network structure of the fibrous matrix complex solution was deactivated using Tris buffer, and centrifugation and washing were repeated using a commonly used PBS buffer to remove the remaining functional groups Antibodies were removed.

Example 3 Enzyme activity and stability measurement of a fibrous matrix complex of enzyme-3-dimensional network structure

The enzymatic activity of each of the enzyme-3-dimensional network structure fiber matrix complexes prepared in Comparative Example 1 and Example 1 was measured using a UV / Vis spectrometer. Specifically, the concentration of the fibrous matrix complex of the enzyme-3-dimensional network structure was first prepared at a concentration of 10 μg / ml based on the amount of the polyaniline nanofibers. To investigate the enzymatic reactivity of HRP, TMB (3,3,5,5-Tetramethylbenzidine) was used as a substrate and DMSO (dimethyl sulfoxide) was used as an organic solvent for dissolving it. First, TMB was dissolved in DMSO at a high concentration and then diluted with phosphate buffer (PB buffer). This is because the enzyme is more stable on the general PB buffer than on the organic solvent in the reaction. After this, at 5:95 ratio, the fiber matrix complex solution of the enzyme-3-dimensional network structure was mixed with the TMB reaction solution, and the absorbance was measured in real time through the spectrometer and the activity of the enzyme was measured through this data.

FIG. 2B shows the activity of a fibrous matrix complex of an antibody-enzyme-3-dimensional network structure synthesized using polyaniline nanofibers of various sizes using a UV / Vis spectrometer. The activity (A655 / min) of Ab-HRP / EAPC is 0.430, 0.570, and 1.105 when the initial aniline concentration is 2.5, 5, and 7.5 wt%, respectively. 3A shows the activity of free enzyme, EA, EAC, and EAPC. The activity (A655 / min) of EA, EAC, and EAPC was 1 μg / ml of polyaniline nanofiber 0.005, 0.04, and 5.63, respectively. The activity of EAPC (Example 1) was 1000 times greater than EA and 140 times greater than EAC. FIG. 3B is a graph continuously measuring and recording enzyme activity at room temperature and stringent stirring conditions of 200 rpm. EA and EAC activity was 2% and 18%, respectively, after 60 days, whereas EAPC was 69 %. &Lt; / RTI &gt; It can be said that EAPC is much more active stabilized as a fiber matrix complex of enzyme-3-dimensional network structure using a precipitation technique than a simple liberated enzyme or a simple cross-linked EAC approach.

Example 4 Antibody Immobilization of Magnetic Nanoparticles

In order to use magnetic nanoparticles for enzyme-linked immunosorbent assay, hIgG antibody was immobilized as a model protein. First, the magnetic nanoparticles were washed twice with a sodium hydroxide solution, and then separated and washed with distilled water twice to remove impurities on the surfaces of the magnetic nanoparticles. To replace the carboxyl groups present in the magnetic nanoparticles with amine groups, 16.4 mg / mL EDC (ethyl (dimethylaminopropyl) carbodiimide) solution was added and stirred for 30 minutes at 4 ° C at 200 rpm. After washing with pH 6.0, 25 mM MES buffer, the hIgG antibody solution of the model protein was mixed and stirred at 200 rpm for 1 hour. At this time, the antibody reacted with the functional group generated by EDC solution and immobilized. The prepared antibody-magnetic nanoparticle solution was deactivated by using 0.1% BSA solution (0.1% BSA in 10 mM PBS (pH 7.4)) and magnetic separation and washing using a commonly used PBS buffer Lt; / RTI &gt; to remove residual antibodies.

Example 5 Analysis of Target Antigen Using Well Plate

Enzyme-linked immunosorbent assay was carried out using a well plate to confirm the characteristics of the antibody-enzyme-3-dimensional network structure of the fibrous matrix complex prepared in Example 2 for analyzing the target antigen. More specifically, the antibody (binding antibody) that binds to the target antigen in a well plate is diluted to 10 μg / ml in a sodium bicarbonate buffer (pH 9.4), and 50 μl is added to each well The binding antibody was adsorbed onto a well plate at 4 ° C for 12 hours. To prevent nonspecific binding of the complex to the well plate, 100 μl of 3 w / v% BSA solution was added to each well and reacted for 1 hour. Remove all of the BSA solution and wash each well 5 times with PBS-T [0.05% Tween-20 in 10 mM PBS (pH 7.4)]. In order to confirm the detection limit, add 50 μl of the target antigen solution (5-0.0001 μg / ml) diluted with PBS solution and incubate at 37 ° C for 1 hour to induce antigen-antibody binding. Wash 5 times with PBS-T solution to remove unbound target antigen. Thereafter, a solution of the antibody-enzyme-3-dimensional network structure-specific fibrous matrix complex that specifically binds to the target antigen was diluted to 1 μg / ml in PBS-T solution, and 50 μl of each solution was added thereto. Min to induce an antibody-antigen binding bridge between the fibrous matrix complex of the enzyme-3-dimensional network structure and the well plate. The cells were then washed 10 times with PBS-T solution to remove the remaining fibrous matrix complex of antibody-enzyme-3-dimensional network structure. After this, 50 μl of TMB solution as in Example 3 was added to quantify the target antigen, and the reaction was carried out with stirring for 30 minutes. Absorbance was measured at a wavelength of 655 nm through a plate reader.

In order to compare the diagnostic performance of the fibrous matrix complex of the enzyme-3-dimensional network structure, experiments were conducted using EAPC and EAC as described above. FIG. 4A shows the result of confirming the diagnosis of the target hIgG using EAPC (Example 1) and EAC (Comparative Example 2) as a fibrous matrix complex having an enzyme-3-dimensional network structure through a well plate-based immunoassay. Based on the concentration of the same polyaniline nanofibers, the EAPC signal was observed to be more than 6 times higher than that of EACC when the concentration of hIgG as the target antigen was 500 ng / ml. Based on these results, the detection limit and detection range of the fibrous matrix complex of the enzyme-3-dimensional network structure were confirmed by reacting various concentrations of the target antigen as shown in FIG. 5a. As a result, the detection limit of EAPC was 1 ng / mL hIgG (6.7 pM), and the detection range was 1 to 500 ng / ml.

<Example 6> Analysis of antigens using magnetic nanoparticles

The antibody-magnetic nanoparticles prepared in Example 4 were used for enzyme-linked immunosorbent assay (ELISA) to examine the properties of the antibody-enzyme-3-dimensional network structure of the fibrous matrix complex prepared in Example 2 for analyzing the target antigen Adsorption test was performed. Wash the prepared antibody-magnetic nanoparticles three times with PBS-T [0.05% Tween-20 in 10 mM PBS (pH 7.4)] solution. In order to confirm the detection limit, 1 ml of the target antigen solution (5-0.0001 μg / ml) diluted with PBS solution is added and stirred at 200 rpm for 1 hour to induce antigen-antibody binding. Wash 3 times with PBS-T solution to remove unbound target antigen. Thereafter, a solution of a fibrous matrix complex having an antibody-enzyme-3-dimensional network structure that specifically binds to the target antigen was diluted to a concentration of 1.5 μg / ml in a PBS-T solution, and 1 ml of the diluted solution was added thereto. Agitation to finally induce an antibody-antigen binding bridge between the antibody-magnetic nanoparticle and the fibrous matrix complex of the enzyme-3-dimensional network structure. And then washed 4 times with PBS-T solution to remove the remaining fibrous matrix complex of antibody-enzyme-3-dimensional network structure. After this, 1 ml of TMB, glucose and HRP solution was added to quantify the target antigen, and the reaction was carried out at 200 rpm for 30 minutes with stirring. The absorbance was measured at a wavelength of 655 nm through a UV / vis spectrometer.

FIG. 4B shows the result of confirming the diagnostic performance of hIgG as a target of infection using EAPC as a fibrous matrix complex having an enzyme-3-dimensional network structure through magnetic nanoparticle-based immunoassay. Based on these results, the detection limit and detection range of the fibrous matrix complex of the enzyme-3-dimensional network structure were confirmed by reacting various concentrations of the target antigen as shown in FIG. 5b. As a result, the detection limit of EAPC was 0.1 ng / mL hIgG (0.67 pM), and the detection range was 1 to 500 ng / ml.

Example 7 Confirmation of Selective Binding Capacity of Fibrous Matrix Complex of Enzyme-3-Dimensional Network Structure

FIG. 6A shows the EAPC of Example 2. The selective binding ability of the target antigen to three antigens such as hIgG, rIgG (rabbit immunoglobulin G) and mIgG (mouse immunoglobulin G) was confirmed by a well plate-based immunoassay And FIG. 6B is a result obtained by observing the magnetic nanoparticle-based disturbance test. As shown in the graph, only human immunoglobulin G selectively reacted.

The enzyme complex for enzyme immunoassay of the present invention and the preparation method thereof can be usefully utilized in the field of immunoassay.

Claims (13)

A cross-linked enzyme aggregate, which is supported inside the fiber interstices of the fiber matrix comprising the three-dimensional network fibers obtained by chemical synthesis rather than electrospinning and whose diameter is larger than the inlet size of the pores, and a surface of the three- A fibrous matrix conjugate of an enzyme-3-dimensional network structure comprising a shell comprising cross-linked enzymes; And
An antibody conjugated to said complex,
The shell is multi-layered,
Wherein the three-dimensional network fibers have an average diameter of 100 to 350 nm. delete The method according to claim 1,
Wherein the three-dimensional network fibers are selected from the group consisting of polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic- Wherein the conjugate is any one selected from the group consisting of polyacrylamide, polycaprolactone, collagen, polypyrrole, polyaniline and poly (styrene-co-maleic anhydride). delete The method according to claim 1,
The enzyme may be selected from the group consisting of chymotrypsin, trypsin, subtilisin, papain, lipase, horseradish peroxidase, soybean peroxidase ), Chloroperoxidase, manganese peroxidase, tyrosinase, laccase, cellulose, xylanase, lactase (see, for example, lactose, sucrose, organophosphate hydrolase, cholinesterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, glucose dehydrogenase, Wherein the enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase and glucose isomerase, wherein the enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase, and glucose isomerase. Station for measuring the complex. 6. The method of claim 5,
Wherein the enzyme is horseradish peroxidase. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; Polymerizing a solution containing 4 to 10% by weight of a fiber-forming monomer in the total solution (0) to prepare a three-dimensional network fiber having an average diameter of 100 to 350 nm;
(1) adding an enzyme to the matrix comprising the three-dimensional network fibers;
(2) adding a precipitating agent to the matrix to prevent leakage of enzymes added to the pores of the matrix; And
(3) preparing an enzyme-fiber matrix complex by forming an enzyme aggregate by adding a cross-linking agent to form cross-linking between the enzymes; And
(4) conjugating the antibody to the complex,
Wherein the enzyme is adsorbed or covalently bonded to the surface of the matrix in the step (1). 8. The method of claim 7,
The precipitating agent may be selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, Or a mixture thereof. 8. The method of claim 7,
The crosslinking agent may be selected from the group consisting of diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethylaminopropylcarbodiimide, glutardialdehyde, bis (imidoesters) A diester ester) and a diacid chloride. The method for producing a complex for enzyme immunoassay according to any one of claims 1 to 3, 8. The method of claim 7,
Further comprising the step of removing the precipitating agent and the cross-linking agent between the step (3) and the step (4). delete Adsorbing the antibody on a well plate;
Adding a sample containing the antigen to the well flight;
Adding a complex of any one of claims 1, 3, 5, and 6 to a wellfly to which a sample is added and reacting; And
Quantitatively analyzing the antigen in a wellfly to which the complex is added;
&Lt; / RTI &gt; Immobilizing the antibody on the magnetic nanoparticle by reacting the magnetic nanoparticle including a functional group capable of immobilizing the antibody with the antibody;
Preparing an antigen-antibody-magnetic nanoparticle by binding an antigen with an antibody-immobilized magnetic nanoparticle;
Mixing and reacting the antigen-antibody-magnetic nanoparticle with the complex of any one of claims 1, 3, 5, and 6; And
Quantitating the antigen in the antigen-antibody-magnetic nanoparticle comprising the complex;
&Lt; / RTI &gt;

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