KR20120017884A - Development of lateral flow assay using protein g coated magnetic bead and immunochromomatograpic strip and immunochromomatograpic kit - Google Patents

Abstract

PURPOSE: An antibody fixation method is provided to enhance fixation density of antibodies and to improve sensitivity. CONSTITUTION: An antibody fixation method of magnetic particles comprises: a step of adding chemical linker solution to magnetic particles for activation; a step of mixing protein G or A/G to the activated magnetic particles; and a step of adding the antibodies to the magnetic particles. The chemical linker is a solution containing EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS(N-hydroxysuccinimide), sulfo-NHS(N-hydroxysulfosuccinimide), CMC(1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), DCC(dicyclohexyl carbodiimide), or DIC(diisopropyl carbodiimide).

Description

Development method of lateral flow assay using Protein G coated magnetic bead and Immunochromomatograpic strip and Immunochromomatograpic kit}

The present invention relates to a method for producing a diagnostic strip using magnetic particles as a label, a diagnostic strip using the same, and a diagnostic kit using the same.

A diagnostic kit is a device that tests or examines the presence of a single or multiple substances in a liquid sample, such as a urine or blood sample. Specifically, the modern diagnostic business is being integrated into one point-of-care testing (POCT). POCT is a test that is performed outside the centralized laboratory and is available to the general public without expertise. Currently, the diagnosis area is expanding from the hospital to the field and the individual.

In particular, rapid diagnostic tests, represented by immunochromatographic analysis, are used to identify diseases or identify changes in health care, and qualitatively and quantitatively examine trace analytes in various fields such as food, biological process, and environment. Is developed in an easy way. The field of health care is also expanding its application to pregnancy, ovulation, infectious diseases, drug abuse, acute myocardial infarction and cancer.

Figure 1a shows the structure of a strip used in conventional immunochromatography. As shown in FIG. 1A, a typical immunochromatography strip 10 used for immunochromatographic analysis is an elongated rectangular support 11 made of an adhesive plastic material, and from one side to the other on the support. It comprises a sample pad 21, a conjugate pad 22, a signal detection pad 23, and an absorption pad 24, which are arranged substantially sequentially. The sample pad 21 absorbs a liquid sample (or analyte) to be analyzed and ensures a uniform flow of the liquid sample. The conjugate pad 22, which is disposed to partially overlap with the other end of the sample pad, includes a flowable conjugate that specifically binds to the analyte contained in the liquid sample, and thus through the sample pad 21. As the introduced liquid sample passes through the conjugate pad 22, specific binding between the analyte and the flowable conjugate occurs. The signal detection pad 23 disposed at a position next to the sample pad 21 and the conjugate pad 22 is usually a detection zone 23a and a control zone which are spaced apart from each other by a certain distance. zone) 23b. In this case, the detection area 23a is an area for checking whether an analyte is present in the liquid sample, and the control area 23b is an area for checking whether the liquid sample has normally passed through the detection area 23a. . The absorption pad 24 is disposed at a position next to the signal detection pad 23, that is, at a position adjacent to the other end of the support 11. The absorbent pad 24 absorbs the liquid sample that has passed through the signal detection pad 23 and assists in capillary flow of the liquid sample onto the immunochromatography strip 10.

In a conventional immunochromatographic assay system, the labeled antibody and polymerized detection antibody are accumulated in a dry state on the conjugate pad. When the sample is added, the conjugate dissolves and reacts with the analyte in the liquid phase. This reaction occurs when the sample solution moves along the fluid flow that occurs in capillary phenomena, and sandwich bonds form an immune conjugate between the conjugate and the analyte (antigen). The immunoconjugate is captured by immobilized antibodies that move up the membrane and react specifically with the analyte, generating a signal proportional to the analyte concentration. The intensity of the signal generated by this signaling mechanism is determined by the concentration of the immunoconjugate resulting from the specific adhesion reaction between the antigen and the antibody.

However, in the conventional case, as shown in FIG. 1B, a portion (X) where the antibody meets the antigen by using a method in which the labeling particle (1) used as a label for signaling is combined with the detection antibody (2) in a random manner. As the binding efficiency is significantly lowered at, there is a disadvantage that the antibody immobilization density is low and the reaction efficiency and sensitivity of the diagnostic kit cannot be increased.

The present invention has been made to solve the above problems, an object of the present invention is to provide a protein to a magnetic particle when the magnetic particle used as a labeling material and the antibody when combined with an analyte of a sample in a membrane-based diagnostic kit It is to provide an antibody immobilization method and diagnostic strip structure that can improve sensitivity by increasing the immobilization density and reaction efficiency of the antibody by projecting a stereo immobilization method that implements a structure in which G or protein A / G is immobilized. .

As a means for solving the above problems, the present invention comprises the steps of activating by injecting a chemical linker solution to the magnetic particles; and two steps of immobilizing the protein G or protein A / G by the activated magnetic particles; It is possible to provide a method for immobilizing the antibody of the magnetic particles comprising a; three steps of reacting the antibody to the magnetic particles.

In this case, in the first step, the chemical linker is a solution containing EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and sulfo-NHS (N-Hydroxysuccinimide), and the magnetic particles are MES (2- [N-morpholino] ethane sulfonic acid) It can be formed by mixing and activating in the state dissolved in the buffer. Chemical linkers used in this activation step are EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), sulfo-NHS (N-hydroxysulfosuccinimide), and CMC (1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (DIC), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) may be used a solution containing any one or two or more, and furthermore, the buffer is MES (2- [N-morpholino] ethane In addition to the sulfonic acid) buffer, any one selected from phosphate buffer, sodium acetate, and sodium phosphate may be used.

In addition, in the second step, when the protein G or protein A / G is mixed in a ratio inversely proportional to the size of the magnetic particles, when the size ratio of the magnetic particles is 1: 2, the input of the protein G or protein A / G The ratio can be entered at an 8: 1 ratio.

In addition, the step 2 may further comprise a step of reacting by incorporating and fixing the protein G or protein A / G to the magnetic particles, the glycine (Glycine).

In addition, the present invention may further comprise a step of removing the carboxyl groups remaining on the surface by adding a washing solution containing glycine (Glycine), BSA, Tween20 to the magnetic particles after the above three steps.

The antibody-magnetic particle complex formed by the above-described manufacturing step may be used in a diagnostic strip consisting of a sample pad, a conjugate pad, a signal detection pad, and an absorption pad disposed on the support. Of course, the above-described manufacturing method or the above-described diagnostic strip may produce a diagnostic kit including the same.

According to the present invention, in the membrane-based diagnostic kit, a clove which realizes a structure in which protein G or protein A / G is immobilized on a magnetic particle when the magnetic particle used as a label and the antibody is bound to an analyte of a sample in a membrane-based diagnostic kit By using an appropriate immobilization method, the immobilization density and reaction efficiency of the antibody are increased to improve the sensitivity, thereby implementing a diagnostic kit capable of quantitative analysis.

Figure 1a shows the structure of a strip used in conventional immunochromatography.
1B is a conceptual diagram illustrating a method in which a labeling particle used as a label for signal generation is conventionally combined with a detection antibody in a random manner.
Figure 2a is a flow chart showing the manufacturing procedure for the antibody immobilization method according to the present invention.
Figure 2b is a conceptual diagram illustrating the principle of the antibody immobilization method according to the present invention.
Figure 3 is an image showing the result of measuring the sensitivity by mounting the antibody-magnetic particle complex prepared by the random immobilization and the anticipated immobilization method using protein G on the strip sensor.
Figure 4 shows the change in the signal according to the antigen concentration when the conditions are optimized using protein G in accordance with the present invention.

Hereinafter, with reference to the accompanying drawings will be described in detail the configuration and operation according to the present invention. In the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and duplicate description thereof will be omitted. The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.

Figure 2a is a flow chart showing the manufacturing procedure for the antibody immobilization method according to the present invention.

Referring to the flowchart shown, the antibody immobilization method of the magnetic particles according to the present invention is a step of adding a chemical linker liquid to the magnetic particles to activate and incorporating the immobilized protein G or protein A / G in the activated magnetic particles It comprises two steps and three steps to react by adding the antibody to the magnetic particles.

The antibody immobilization step by step is basically made around the basic principle as shown in Figure 2b. That is, in the present invention, the protein 120 immobilized on the magnetic particles 110 is characterized in that it is implemented by using the protein G, or protein A / G, the immobilized immobilization. The reason for using the protein A or G according to the present invention is to give orientation to the magnetic particles.

Specifically, the protein G is a cell wall protein isolated from group G Streptococci. And has a large binding ability to the Fc portion of most immunoglobulin G. In addition, the protein A / G is a chimeric protein produced by gene fusion of the domains between the protein A and the Fc of the protein G, which has a greater binding capacity to the mouse monoclonal group than protein A or G and is less sensitive to pH To have. Therefore, since the protein G and the protein A / G, which are used as an immobilization material in the present invention, bind strongly to the Fc portion of the antibody 130, (Kd = ~ 8nM), the Fab region, which is a portion that binds to the antigen of the antibody, is exposed to the maximum. Advantages that can be immobilized are implemented. Therefore, in the present invention, the surface of the magnetic particles is first modified with protein G, A / G, and then implemented through a process of culturing at a constant concentration with the antibody.

Hereinafter, with reference to Figures 2a and 2b, an embodiment according to the present invention will be described through an experimental example.

1. Activation step of magnetic particles

In the present embodiment, in order to quantify the experimental data, the size of the magnetic particles is limited to 100 nm and 200 nm, and the experiment is performed by a protocol for immobilizing the antibody. 1 mg of particles were used for each experimental example.

First, activating the magnetic nanoparticles may be performed in the following order. The chemical linker EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) / sulfo-NHS (N-Hydroxysuccinimide) was added in excess of MES (2- [N-morpholino] ethane sulfonic acid) buffer (pH 6). Put it in the dissolved particles and mix and react for 15 minutes at room temperature. After the reaction was washed twice with PB (phosphate buffer) of pH7.5, the activated particles are suspended in PB.

Chemical linkers or buffers used in this step may be used in addition to those described above. For example, the chemical linker may be EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), sulfo-NHS (N-hydroxysulfosuccinimide), CMC (1-cyclohexyl-3- ( 2-morpholinoethyl) carbodiimide), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) can be carried out as a solution containing any one or two or more selected from.

In addition to the MES (2- [N-morpholino] ethane sulfonic acid) buffer, the buffer may be any one of phosphate buffer, sodium acetate, and sodium phosphate.

2. Immobilizing Proteins G and A / G on Magnetic Nanoparticles

In this process, protein G or A / G-mediated protein G or A / G is added to 100 μg for 100 nm magnetic particles and 50 μg to 200 nm magnetic particles.

That is, when the protein G or the protein A / G is mixed in a ratio inversely proportional to the size of the magnetic particles, when the size (diameter) ratio of the magnetic particles is 1: 2, the input ratio of the protein G or protein A / G is It is preferable to add in an 8: 1 ratio.

The range of the diameter of the magnetic particles according to the present invention can be applied in the range of 10nm ~ 1um, because the magnetic particles are less than 10nm measurement efficiency is not good, when the development of more than 1um on the membrane is not good.

Subsequently, the mixture is reacted at room temperature for 2 hours while mixing the above-described protein and magnetic particles.

In addition, after the reaction was washed with pH7.5 PB (phosphate buffer), the antibody / protein fixed particles are suspended in PB.

In this case, the protein G, A / G-fixed magnetic particles are added 40mM glycine and allowed to react for 30 minutes at room temperature. After the reaction was washed with pH 7.5 PB, a step of suspending the protein-fixed particles in PB may be added.

3. Antibody Reaction Step

75 μg of the antibody is added to the 100 nm magnetic particles to which the protein is immobilized, and 37.5 μg of the antibody is added to the 200 nm magnetic particles, and the mixture is allowed to react for 30 minutes at room temperature. After the reaction, the mixture was washed with pH 7.5 PB, and the antibody-immobilized particles were suspended in PB.

4. Removal of carboxyl group

Thereafter, as NHS ester quenching / blocking step remaining on the surface, 40mM glycine, 0.2% BSA, 0.01%, Tween 20 solution was added and blocked at room temperature for 30 minutes. After the reaction, the mixture was washed with pH 7.5 PB, and the particles immobilized on the antibody were suspended in PB. This step is essentially a step of removing carboxyl groups remaining on the surface of the magnetic particles.

Then, after 0.1M pH 7.5 PB, 0.01% BSA (Albumin, bovine serum, Fraction V, Approx.), 0.00005% Tween 20 solution is added to the particles well suspended and stored at 4 ℃ can be performed. .

Table 1 below shows the results of verifying whether or not the antibody is immobilized on the magnetic particles that are fixed by the above-described process.

{Table 1}

That is, referring to the table shown, according to the present invention, the surface of the magnetic particles are first modified with protein G and protein A / G, and then cultured at a constant concentration with an antibody, and the protein G according to the present invention in preparation for random fixation. In addition, the amount of antibody attached to the surface of the magnetic particles immobilized with protein A / G is about 1.5 to 2 times higher, which can lead to an improvement in sensitivity since it can guarantee the exact orientation of the immobilized antibody. .

Figure 3 is an image showing the result of measuring the sensitivity by mounting the antibody-magnetic particle complex prepared by the random immobilization and the anticipated immobilization method using protein G on the strip sensor.

Figure 3 shows detection of antigen-specific cardiac troponin I in (a) human serum and (b) Phosphate buffered saline (PBS) buffer solution, respectively. Detection in buffer solution There seems to be little difference in sensitivity between human serum and buffer solution except antibody-magnetic particle complex precipitated in sensor ① and ②.

In particular, in Fig. 3, ① and ② were 100 nm particles, ③ and ④ were 200 nm particles, respectively, and ① and ③ were random immobilization, and ② and ④ were oriented immobilization using protein G. The upper band of the two bands appearing brown indicates the control line (C), indicating the presence or absence of mouse IgG, and the lower band showing the cardiac specificity as the test line (T). The presence or absence of troponin I is shown. If the particles contain antibodies from mice, this can be confirmed by the darker control lines.

Figure 4 shows the change in the signal according to the antigen concentration when the conditions are optimized using protein G in accordance with the present invention. When protein G is mediated, it can be seen that when the antigen is 0.01 ng / ml, the signal is darker than the antigen.

Table 2 shows the results measured by the magnetoresistance type diagnostic device according to the concentration of cTnI of FIG.

{Table 2}

Referring to the results shown, it can be seen that the signal-to-noise ratio has a high sensitivity of 0.01 ng / ml or more when it is considered that 1.2 or more is effective.

The antibody-magnetic particle complex having a structure in which protein G or protein A / G is immobilized on the magnetic particles according to the present invention is implemented as a sample pad, a conjugate pad, a signal detection pad, and an absorption pad disposed on an upper portion of the support. It can be applied to increase the diagnostic efficiency by being included in the conjugate pad in the diagnostic strip. Of course, the above-described manufacturing method may be used to manufacture a diagnostic kit as a final product, and in the case of the diagnostic strip described above, it may be used in a diagnostic kit.

In the foregoing detailed description of the present invention, specific examples have been described. However, various modifications are possible within the scope of the present invention. The technical idea of the present invention should not be limited to the embodiments of the present invention but should be determined by the equivalents of the claims and the claims.

10: immunochromatography strip
11: support
21: sample pad
22: conjugate pad
23: signal detection pad
24: absorption pad
110: magnetic particles
120: Protein G or Protein A / G
130: antibody

Claims (8)

1 step of activating the chemical linker solution to the magnetic particles;
Incorporating and immobilizing protein G or protein A / G into the activated magnetic particles;
Injecting an antibody into the magnetic particles to react with each other;
Antibody immobilization method of a magnetic particle comprising a.
The method according to claim 1,
The first step,
The chemical linker is EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), sulfo-NHS (N-hydroxysulfosuccinimide), CMC (1-cyclohexyl-3- (2-morpholinoethyl) a solution containing any one or two or more selected from carbodiimide), dicyclohexyl carbodiimide (DCC) and diisopropyl carbodiimide (DIC),
The antibody is immobilized by mixing the magnetic particles in a dissolved state in a buffer of MES (2- [N-morpholino] ethane sulfonic acid) buffer or any one selected from phosphate buffer, sodium acetate and sodium phosphate. Way.
The method according to claim 2,
In the second step,
Incorporating the protein G or protein A / G in a proportion inversely proportional to the size (diameter) of the magnetic particles,
When the size (diameter) ratio of the magnetic particles is 1: 2, the injection ratio of the protein G or protein A / G is 8: 1 ratio of the antibody immobilization method of the magnetic particles.
The method according to claim 3,
Antibody-fixing method of the magnetic particles having a diameter range of 10nm ~ 1um of the magnetic particles.
The method according to claim 2,
In the second step,
The method of immobilizing the magnetic particles of the magnetic particles further comprises the step of mixing the protein G or protein A / G in the magnetic particles, and then adding and reacting glycine (Glycine).
The method according to any one of claims 1 to 5,
After step 3,
Method of immobilizing the magnetic particles of the magnetic particles further comprising the step of removing the carboxyl groups remaining on the surface by introducing a washing solution containing glycine (Blycine), BSA, Tween20 to the magnetic particles.
Including a sample pad and a conjugate pad, a signal detection pad and an absorption pad disposed on the support,
The conjugate pad comprises an antibody-magnetic particle complex having a structure in which protein G or protein A / G is immobilized to magnetic particles.
Diagnostic kit prepared using the antibody immobilization method of claim 1.

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