KR101945997B1 - Method for detection of antigen using fluorescence resonance energy transfer immunoassay on membrane - Google Patents

Abstract

The method for detecting an antigen according to an embodiment of the present invention comprises the steps of (a) contacting a sample containing an antigen to be detected with the other end of a diagnostic unit formed of a membrane containing an antibody against the antigen to be detected, Process; (b) irradiating the antigen-antibody complex formed by the step (a) with light to cause fluorescence resonance energy transfer and obtaining a spectrum therefrom; And (c) analyzing the spectrum obtained in the step (b) to determine the presence or absence of the antigen to be detected and measuring the concentration.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for detecting an antigen using fluorescence resonance energy transfer immunoassay on a membrane,

The present invention relates to a method for detecting an antigen using a fluorescence resonance energy transfer immunoassay, and more particularly, to a method for detecting an antigen by using fluorescence resonance energy transfer And an antigen detection method using the same.

An enzyme-linked immunosorbent assay (ELISA), which is widely used for the detection of conventional antigens, is widely used as an assay method which is excellent in sensitivity, fast, and economical by using immunization technique of antigen / antibody reaction Has been practically used. A known indirect enzyme immunoassay is a method in which a substance as an antigen is coated on the bottom and then a cell culture supernatant is added to bind the antibody present in the supernatant to the antigen and then to another antibody-enzyme conjugate To determine whether the cell line is producing the desired antibody.

However, this method has many drawbacks compared to the simplicity of the experiment. First, a direct competitive enzyme immunoassay or an indirect competitive enzyme immunoassay is used to quantitatively analyze a substance to be detected. In the direct competitive enzyme immunoassay, a competition reaction occurs between the substance to be detected and the substance-enzyme conjugate to be detected In the indirect competitive enzyme immunoassay, a detection target substance-enzyme conjugate coated with the produced monoclonal antibody and a fusion cell line producing an antibody in which a competition reaction with the detection target substance is well produced should be selected. However, in the indirect competitive enzyme immunoassay, Since there is no step to confirm competitiveness, it can not be guaranteed that the finally produced antibody has the ability to compete against the substance to be detected. Secondly, indirect enzyme immunoassay is suitable only when the substance to be detected is a substance having a high molecular weight such as a protein. This is because a substance having a high molecular weight is easily coated when enzyme immunoassay is used. It can be used for a small molecule (eg, a fungal toxin such as aflatoxin, ocratoxin, gellulene, a biomarker such as neopterin, bipoterin, Etc.) are not suitable for indirect enzyme immunoassay because they are not well coated. Therefore, when a substance to be detected is a low-molecular substance, it is impossible to directly coat low-molecular substances using indirect enzyme immunoassay, and a protein having a large molecular weight, that is, a substance capable of being coated, . Thereafter, when the cell culture supernatant is reacted, an antibody recognizing the antigen can be bound, but an antibody recognizing the carrier and an antibody recognizing the antigen and the carrier may be present. . In addition to the ELISA method, antibody detection methods include lateral flow immunoassay (LFA), electrochemical immunoassay, piezoelectric immunosensors, and fluorescence polarization immunoassays exist.

In particular, among the low molecular antigens to be detected, mycotoxin is a secondary metabolite produced by the fungus, which causes diseases and abnormal physiological actions in humans and livestock, and mainly occurs in foods in which cereals, nuts and molds tend to breed Occurs. Therefore, fungal toxin contamination in foods is an inevitable phenomenon, and inspection of agricultural products and their products is essential.

Fungal toxins are classified into Aspergillus, Penicillium, and Fusarium fungi according to their production. Representative examples include Aflatoxin, Ochratoxin (OTA) and zearalenone. These fungal toxins are highly pathogenic substances, and generally have low solubility in water, It is well soluble in polar organic solvents such as chloroform, methanol, acetonitrile and the like. In particular, despite the growing interest in the detection of mycotoxins due to the rapid increase in the imports and exports of the world's agricultural products, the conventional analysis method has been limited in terms of equipment, manpower, etc. Therefore, it is urgently required to introduce a method for efficiently analyzing this problem.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

Korean Patent Publication No. 10-2014-0058305 (Apr.

SUMMARY OF THE INVENTION The present invention has been made to overcome the above problems, and it is an object of the present invention to provide a method for detecting fluorescence of an antibody in various antigens / antibody complexes more effectively using a membrane, And a method for detecting an antigen capable of detecting an antigen.

In order to accomplish the above object, an antigen detection method according to an embodiment of the present invention comprises the steps of: (a) detecting a sample containing an antigen to be detected at the other end of a diagnostic device formed of a membrane containing an antibody against the antigen to be detected Contacting the antibody with the antibody; (b) irradiating the antigen-antibody complex formed by the step (a) with light to cause fluorescence resonance energy transfer and obtaining a spectrum therefrom; And (c) analyzing the spectrum obtained in the step (b) to determine the presence or absence of the antigen to be detected and measuring the concentration.

Wherein the diagnostic device comprises: a reaction membrane including a sample contacting portion capable of contacting the sample and capable of injecting the sample, a test line containing the antibody, and a control line for confirming whether or not the sample is input; And an absorbent pad formed to absorb the contacted sample.

The reaction membrane may be separated from one sample contacting portion and branched into two or more branches.

The antigen may be a fungal toxin, a biomarker or a polycyclic aromatic carbon having an absorption wavelength at 300 to 400 nm.

The fungal toxin may be aflatoxin, Ochratoxin (OTA) or gellulenone.

The biomarker may be neopterin, biopterin, nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH).

The polycyclic aromatic carbon may be benzopylene, dibenzoanthracene, pyrene or benzofluorene.

The step (a) may further include a step of drying the diagnostic unit after the antigen-antibody reaction.

The step (a) may further include washing the buffer solution after the antigen-antibody reaction.

The fluorescence spectrum analysis can be performed by comparing and analyzing the fluorescence intensity at a wavelength showing the maximum fluorescence intensity among the fluorescence spectra of the antigen and the maximum fluorescence intensity among the fluorescence spectra of the antibody.

The fluorescence spectrum analysis was carried out by _measuring the ratio of the fluorescence intensity (I _antigen ) at the wavelength representing the maximum fluorescence intensity among the fluorescence spectra of the antigen to the fluorescence period (I _antibody ) at the wavelength representing the maximum fluorescence intensity among the fluorescence spectra of the antibody I _< / RTI &_gt; _antigen / I _antibody .

The antigen detection method according to the present invention has the following effects.

First, it is easy to observe fluorescence spectra even in the case of a sample having a high color by contacting a sample containing an antigen to be detected on the membrane, so that an antigen in a sample such as coffee or wine can be easily detected.

Secondly, when a membrane diagnostic system is composed of multiple stages, it is possible to determine the presence or absence of each antigen even in a single sample collection in the case where various antigens are present in the sample.

Third, in a conventional competitive ELISA method, the intensity of a signal that can be detected decreases as the concentration of the antigen increases. However, as the concentration of the antigen increases, the detection signal becomes larger as the fluorescence resonance energy transfer method is used. Do.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a representative embodiment of an immunochromatographic assay strip.
Figure 2 is a simplified representation of a membrane diagnostic device used in the antigen detection method according to the present invention.
FIG. 3 is a view schematically showing an OTA detection process in the antigen detection method according to the present invention.
FIG. 4 is a diagram illustrating various types of membrane diagnostic apparatuses that can be used in the antigen detection method according to the present invention.
5 is a diagram illustrating an experimental procedure according to an antigen detection method according to the present invention.
FIG. 6 is a graph showing the results of spectrum measurement according to presence or absence of an antigen according to the antigen detection method according to the present invention. FIG.
FIG. 7 is a graph showing the results of spectrum measurement according to the antigen concentration according to the antigen detection method according to the present invention. FIG.
8 is a graph showing the fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
FIG. 9 is a graph showing the ratio of fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
10 is a graph showing the results of spectrum measurement of a coffee sample to which OTA is added according to the antigen detection method according to the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms as used herein include plural forms as long as the phrases do not expressly express the opposite meaning thereto. Means that a particular feature, region, integer, step, operation, element and / or component is specified, and that other specific features, regions, integers, steps, operations, elements, components, and / And the like.

Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly used predefined terms are further interpreted as having a meaning consistent with the relevant technical literature and the present disclosure, and are not to be construed as ideal or very formal meanings unless defined otherwise.

Hereinafter, a method of detecting an antigen according to a preferred embodiment of the present invention will be described with reference to the accompanying drawings.

Fluorescence resonance energy transfer (FRET) is a phenomenon in which, when fluorescent materials having two different wavelength regions are adjacent to each other, a resonance phenomenon occurs in which one fluorescent donor is transferred to another fluorescent material And another fluorescence (acceptor or quencher) is generated. The FRET phenomenon is a physical phenomenon in which energy is transferred through a non-radiative process from one excited dye molecule to another dye molecule by long-distance dipole-dipole interaction, where the energy-giving molecule is called a donor, The molecule is called the acceptor or quencher.

When the donor molecule is excited by using light of a specific wavelength, if the acceptor molecule is nearby, energy of the donor molecule is transferred to the acceptor molecule to decrease the fluorescence of the donor molecule and fluorescence of the acceptor molecule appears, or Only fluorescence of the donor molecule is reduced. This energy transfer phenomenon occurs when the donor and acceptor are spatially separated from each other by a distance of 1 to 10 nm. The fundamental requirement for energy transfer is that the fluorescence spectrum of the donor molecule and the absorption spectrum of the acceptor molecule overlap each other to be. In the case of proteins such as antibodies, the presence of tryptophan (Trp) leads to a maximum absorption wavelength at 260 nm to 290 nm, approximately 280 nm, and from 300 nm to 400 nm when light is generated at this wavelength band, Lt; / RTI > signal. Thus, as described above, it can be seen that the absorption wavelength of an antifungal such as antigens having fluorescence properties such as aflatoxin, oclatoxin, gellulenone and the like and the fluorescence spectrum of an antibody as a protein are overlapped with each other, It is possible to provide a method of detecting antigens having fluorescence properties.

Immunochromatographic assay (ICA) is a method which can qualitatively and quantitatively inspect the analyte in a short time using the property that a biological substance or a chemical substance attaches to each other in a specific manner. The analytical strip is placed inside the plastic housing Immunoassay kit in the form of mounted and assembled is generally used. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a representative embodiment of an immunochromatographic assay strip. 1, the analytical strip used in the immunochromatographic assay comprises a sample pad 40, a conjugate pad 30, a signal detection pad 20 and an absorbent pad (not shown) on an adhesive plastic support 60 50). The sample pad 40 absorbs a liquid sample (or an analytical sample) and ensures a uniform flow of the liquid sample. The conjugate pad 30 includes a fluidic conjugate that specifically binds to an analyte contained in the liquid sample. A liquid sample introduced through the sample pad 40 is introduced into the conjugate pad 30 ), A specific binding between the analyte and the flowable conjugate is achieved. The signal detection pad 20 generally comprises a detection zone 21 and a control zone 22. [ The detection region 21 is an area for confirming whether or not an analyte is present in the liquid sample. The verification region 22 confirms whether or not the liquid sample has normally passed through the detection region 21 . Above the signal detection pad 20, the absorption pad 50 is located. The absorbent pad (50) absorbs the liquid specimen that has passed through the signal detection pad (20) and helps the capillary flow of the liquid specimen in the analytical strip. The analyte strip is attached to the adhesive plastic support 60 in the order of the sample pad 40, the conjugate pad 30, the signal detection pad 20 and the absorption pad 50 in this order, (40) to the absorption pad (50) via the signal detection pad (20), and performs immunoassay through signal detection at the signal detection pad (20). In another modified method, the conjugate and the signal detecting substance are integrated into one porous pad. The sample pad, the conjugate pad, the signal detection pad, and the absorption pad are arranged to be overlapped with each other or arranged at a predetermined interval on the plastic support. In the latter case, the liquid sample moves to the sample pad, the conjugate pad, and the signal detection pad by capillary phenomenon using another medium. When the signal detection pad composed of a membrane as described above is detected by a chromatographic method, there is an advantage that samples such as dark coffee, tea, and wine can be used.

In other words, the present inventors have developed an antigen detection method capable of detecting the presence or absence of an antigen by causing the fluorescent resonance energy transfer phenomenon by reacting each antibody on the membrane using each antibody that specifically binds to the antigen . The method for detecting an antigen according to an embodiment of the present invention comprises the steps of (a) contacting a sample containing an antigen to be detected with the other end of a diagnostic unit formed of a membrane containing an antibody against the antigen to be detected, (C) analyzing the spectra obtained in the step (c); and (c) analyzing the spectra obtained in the step (c) by analyzing the fluorescence resonance energy transfer by irradiating the antigen- And determining the presence or absence of the antigen to be detected and measuring the concentration.

The antigens that can be detected using the antigen detection method according to the present invention include, but are not limited to, fungal toxins having an absorption wavelength at 300 to 400 nm, biomarkers Or polycyclic aromatic carbons. Particularly, specific examples of the fungal toxin include aflatoxin, Ochratoxin (OTA) or zearalenone, and specific examples of the biomarker include neopterin, bipoterin biopterin, nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). Specific examples of the polycyclic aromatic carbon include benzopyran, dibenzoanthracene, pyrene or benzofluorene.

In particular, the tryptophan residue of the antibody protein exhibits the maximum absorption characteristic at 280 nm and the maximum emission characteristic at 330 nm. The fluorescence spectrum is analyzed by excitation using light of 280 nm wavelength and emission spectrum observation at 330 nm . That is, when a fungal toxin is present in a sample, a quenching phenomenon occurs due to the binding between the fungal toxin and the antibody. As the fungal toxin is higher in the sample, the degree of quenching becomes larger. The fluorescence intensity reduction phenomenon becomes more prominent. In contrast, in the absence of mycotoxins in the sample, such quenching does not occur and thus the fluorescence intensity at 330 nm is greater.

Therefore, analysis of the fluorescence spectrum can be performed by analyzing the fluorescence intensity at a wavelength indicating the maximum fluorescence intensity of the antigen or the fluorescence intensity at a wavelength representing the maximum fluorescence intensity among the fluorescence spectrum of the antibody.

Alternatively, the analysis of the fluorescence spectrum may be performed by measuring the fluorescence intensity at a wavelength representing the maximum fluorescence intensity among the fluorescence spectra of the antigen with respect to the fluorescence intensity (I _antibody ) at a wavelength representing the maximum fluorescence intensity among the fluorescence spectra of the antibody a it may be carried out by measuring the ratio (I _antigen / I _antibody), of (I _antigen).

OTA as a fungal toxin, for example, describes the antigen detection process in more detail. Figure 2 is a simplified representation of a membrane diagnostic device used in the antigen detection method according to the present invention. FIG. 3 is a view schematically showing an OTA detection process in the antigen detection method according to the present invention. OTA, for example, the antigen detection process will be described. In the present invention, when a sample is brought into contact with an anti-OTA antibody capable of binding to OTA, which is an antigen to be detected, the anti-OTA antibody is contacted to the diagnosis unit formed with a membrane after a certain period of time. In the case of anti-OTA antibody, it exhibits the maximum absorption characteristic at 280 nm and the maximum emission characteristic at 330 nm. At this time, the analysis of the fluorescence spectrum can be performed by exicitation using light at a wavelength of 280 nm and observation of emission spectrum at 330 nm. FIG. 4 is a diagram illustrating various types of membrane diagnostic apparatuses that can be used in the antigen detection method according to the present invention. As shown in FIG. 4, when the membrane is divided into several fractions and an antibody capable of reacting with each antigen is placed in each branch, diagnosis is performed, and it is possible to detect the presence of various antigens by a single diagnosis Do.

Hereinafter, the present invention will be described in more detail with reference to examples. 5 is a diagram illustrating an experimental procedure according to an antigen detection method according to the present invention. Absorbent pad (AP222), PALL, Grade 222) was attached to the upper part of the nitrocellulose membrane (180 membrane card, Millipore, HF180MC100), and then cut using an automatic belt loop cutter (CuTex, TBC-50) (3.8 mm) to prepare a strip. Anti-OTA was dissolved in 2mM borate buffer (pH 8.5) to prepare 0.44mg / ml. (MES, Sigma, M8250) + 0.3% Tween-20 (Tween 20, Sigma, P1379) was prepared by dissolving the antigen (ochratoxin A, Sigma, O1877) in 100% methanol (methanol, Millipore, 106007) ) Buffer. 1 μl of the antibody is dropped on the prepared strip and dried at a temperature of about 37 ° C for about 15 minutes using a thermostat. Separately, 10 μl of antigen and 90 μl of buffer are mixed and the reaction solution (sample to be detected) is prepared in a microplate well. After the strips are dried, the strips are placed in a buffered microplate well and allowed to react at room temperature for about 15 minutes. Prepare 100 μl of buffer in a separate microplate well. After completion of the reaction with the antibody on the strip and the antigen, place the strip in a buffered microplate well and allow to react at room temperature for about 15 minutes. After all reactions have been completed, they are again dried at 37 ° C for about 15 minutes in a thermo-hygrostat. The dried strips were measured for fluorescence through a micro-reader (Infinite 200 PRO NanoQuant Microplate Readers from TECAN). FIG. 6 is a graph showing a result of spectrum measurement according to the antigen detection method according to the present invention. FIG. As shown in FIG. 6, when OTA is not present, it is seen that the peak at 330 nm decreases and the peak at 440 nm which is the emission spectrum increases.

Also, in order to confirm the spectrum detection result according to the antigen concentration, the experiment was conducted by changing the concentration of the antigen to 1 to 1000 ng / ml in the same manner. FIG. 7 is a graph showing the results of spectrum measurement according to the antigen concentration according to the antigen detection method according to the present invention. FIG. (a) is a graph analyzing the spectrum in the entire wavelength region. (b) is a graph showing a spectrum around 330 nm, and (c) is a graph showing an analysis of a spectrum in a graph in the vicinity of 440 nm. As shown in FIG. 7, as the concentration of OTA increases, the emission spectrum near 330 nm decreases and the light of 330 nm generated by the antibody is absorbed again by the antigen. Also, it can be seen that the emission spectrum around 440 nm increases as the concentration of OTA increases.

Also, FIG. 8 shows the fluorescence intensity at 330 nm and the fluorescence intensity at 440 nm according to the OTA concentration so as to quantitatively confirm the amount of the antigen. As shown in FIG. 8, in (a), the fluorescence intensity at 330 nm decreases as the concentration of the antigen increases, and thus it is difficult to quantitatively measure the fluorescence intensity. However, as shown in FIG. 9 (b), the fluorescence intensity at 440 nm shows an increase in intensity as the concentration of the antigen increases. As shown in FIG. 9, the fluorescence intensity at 440 nm The fluorescence intensity ratio is more effective for the quantitative measurement by improving the sensitivity of the measured value according to the amount of the antigen.

In addition, since the present invention employs a chromatographic method using a membrane diagnostic apparatus, the presence of OTA was detected using a coffee as a sample which is difficult to be measured by the method using fluorescence resonance energy transfer phenomenon in a solution mode. OTA was added to the coffee liquid to prepare a sample, and otherwise, the same procedure as described above was performed. 10 is a graph showing the results of spectrum measurement of a coffee sample to which OTA is added according to the antigen detection method according to the present invention. As shown in Fig. 10, the change in the fluorescence intensity with respect to OTA was confirmed even in a dark coffee sample.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand.

It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

Claims (11)

(a) contacting a sample containing an antigen having an absorption wavelength at 300 to 400 nm, which is a detection target, with the other end of a diagnostic unit formed of a membrane containing an antibody against the antigen to be detected, and reacting the antibody with the antibody;
(b) irradiating the antigen-antibody complex formed by the step (a) with light to cause fluorescence resonance energy transfer and obtaining a spectrum therefrom; And
(c) determining the presence or absence of the antigen to be detected by analyzing the spectrum obtained in the step (b) and measuring the concentration,
Wherein the diagnostic device comprises: a reaction membrane including a sample contacting portion capable of contacting the sample and capable of injecting the sample, a test line containing the antibody, and a control line for confirming whether or not the sample is input; And an absorbing pad formed to absorb the contacted sample,
The reaction membrane is separated from one sample contacting portion and branched into two or more branches. Two or more antibodies capable of reacting with each of the separate antigens are positioned so that the presence of various antigens can be detected through a single diagnosis Can,
Wherein the detection is performed using the ratio of the fluorescence intensity at 440 nm to the fluorescence intensity at 330 nm satisfying the following condition (a).
(a) the fluorescence intensity emitted from the 440 nm wavelength band satisfies the following relational expression 1
[Relation 1]
I ₁ ? I ₁₀
(I ₁ : fluorescence intensity when the concentration of the antigen is 1 ng / ml, I ₁₀ : fluorescence intensity when the concentration of the antigen is 10 ng / ml)
delete delete The method according to claim 1,
Wherein the antigen is a fungal toxin, a biomarker or a polycyclic aromatic carbon.
The method of claim 4,
Wherein said mycotoxin is Aflatoxin, Ochratoxin (OTA) or gellulenone.
The method of claim 4,
Wherein the biomarker is neopterin, biopterin, nicotinamide adenine dinucleotide (NADH), or nicotinamide adenine dinucleotide phosphate (NADPH).
The method of claim 4,
Wherein the polycyclic aromatic carbon is benzopyrene, dibenzoanthracene, pyrene or benzofluorene.
The method according to claim 1,
The method of (a) further comprises a step of drying the diagnostic reagent after the antigen-antibody reaction.
The method according to claim 1,
Wherein the step (a) further comprises washing the buffer solution after the antigen-antibody reaction, followed by washing.
The method according to claim 1,
Wherein the spectral analysis in the step (c) is performed by comparing and analyzing the fluorescence intensity at the wavelength showing the maximum fluorescence intensity and the fluorescence intensity among the fluorescence spectrum of the antibody among the fluorescence spectra of the antigen.
The method of claim 10,
The ratio of the spectral analysis is fluorescence intensity (I _antigen) at the wavelength that indicates the maximum fluorescence intensity of the fluorescent spectrum of the antigen to the fluorescent group (I _antibody) at a wavelength showing the maximum fluorescence intensity of the fluorescent spectrum of the antibody I _Lt ; RTI ID = 0.0 &_gt; I / I _< / RTI &_gt; _antibody .

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