KR102185866B1 - Kit for simultaneously detecting multiple biomarkers and detection method using the same - Google Patents

Abstract

It includes a substrate, two or more detection antibody tags labeled with a fluorescent molecule, and two or more detection antibodies combined with the detection antibody tag, and simultaneously discriminates two or more types of biomarkers using FRET efficiency by the fluorescent molecule. It provides a kit for simultaneous detection of multiple biomarkers and a detection method using the same.

Description

Kit for simultaneously detecting multiple biomarkers and detection method using the same}

It includes a substrate, two or more detection antibody tags labeled with a fluorescent molecule, and two or more detection antibodies combined with the detection antibody tag, and simultaneously discriminates two or more types of biomarkers using the FRET efficiency by the fluorescent molecule. , A kit for simultaneous detection of multiple biomarkers and a detection method using the same are disclosed.

Biomarkers are biological indicators that can detect changes in the body using proteins, DNA, RNA, and metabolites. As a method of detecting a biomarker included in a specific sample derived from a living organism, it is possible to check the normal or pathological state of the living organism and objectively measure the degree of reaction to a drug. Recently, it has been in the spotlight of the medical community because it is an effective method for diagnosing various incurable diseases such as cancer, heart disease, stroke, and dementia.

A commonly used biomarker detection technique is mainly an ELISA-based technique, and is known to have a sensitivity of up to several tens of pg/ml. Therefore, in order to detect a biomarker, a biological sample of a certain amount or more in proportion to the sensitivity is required.

When using a biomarker to determine the presence or absence of a tumor, in most cases, one biomarker is identified in one test and the test is repeated several times. However, when a tumor occurs, not only one biomarker changes, but two or more biomarkers change. Therefore, in order to determine whether a tumor has occurred, detecting multiple biomarkers at the same time can increase the accuracy of determining whether a tumor has occurred.

In order to detect multiple biomarkers with the current technology, a kit and an amount of blood proportional to the number of biomarkers to be detected are required. This not only causes the psychological and physical burden of the subject due to a large amount of blood collection, and economic burden due to the use of multiple detection kits, but may also lead to difficulties in early diagnosis of the disease, so it is necessary to simultaneously diagnose two or more biomarkers. .

In order to simultaneously detect two or more biomarkers using a small amount of biological samples, there is a need for a technology capable of high sensitivity and multiple diagnosis.

Korean Patent Registration No. 1431062, "A multi-biomarker set for breast cancer diagnosis, a detection method thereof, and a breast cancer diagnostic kit including antibodies thereto" (Published: 2013.09.23) Korean Patent Application Publication No. 20170096619, “Method and Kit for Simultaneous Detection of Multiple Diseases Based on 3D Protein Nanoparticle Probe” (Published: 2017.08.24)

An object of the present invention is to include a substrate, two or more kinds of detection antibody tags labeled with a fluorescent molecule, and two or more kinds of detection antibodies to which the detection antibody tags are bound, and use two or more kinds of biomarkers by using the FRET efficiency of the fluorescent molecule. It is to provide a kit for simultaneous detection of multiple biomarkers that simultaneously discriminates the type.

Another object of the present invention relates to a method for simultaneously detecting multiple biomarkers, the step of attaching two or more biomarkers to a substrate, and two or more detection antibodies with a fluorescently labeled detection antibody tag attached to the two or more biomarkers. And measuring the FRET efficiency from the fluorescent signal generated by the binding, respectively, and after simultaneously determining the type of the biomarker by using the FRET efficiency by the fluorescent molecule, the biomarker per unit area To provide a method for simultaneous detection of multiple biomarkers for measuring biomarker concentration from the number of markers.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, a substrate; Two or more detection antibody tags labeled with fluorescent molecules; And two or more detection antibodies to which the detection antibody tag is bound, and simultaneously discriminating two or more types of biomarkers using FRET efficiency by the fluorescent molecule. A kit for simultaneous detection of multiple biomarkers is provided.

According to one side, the fluorescent molecule is a donor or acceptor on a specific base, 3'- or 5'-end, or a backbone of the detection antibody tag. It may be to act as.

According to one side, the detection antibody tag may be labeled with two or more different fluorescent molecules.

According to one side, the kit may further include a capture antibody.

According to one side, the capture antibody may be a combination of the capture antibody tag.

According to one side, the kit for simultaneous detection of multiple biomarkers may further include a detection probe.

According to one side, the detection probe may be complementarily combined with a detection antibody tag and a capture antibody tag.

According to one side, the detection probe may be a donor or acceptor labeled on a specific base, 3'- or 5'-end, or a backbone.

According to another embodiment of the present invention, there is provided a method for simultaneously detecting multiple biomarkers, comprising: attaching two or more biomarkers to a substrate; Binding two or more kinds of detection antibodies to which a fluorescently labeled detection antibody tag is attached to each of the two or more biomarkers; And measuring FRET efficiency from the fluorescent signal generated by the coupling. Including, and after simultaneously determining the type of the biomarker using the FRET efficiency of the fluorescent molecule, there is provided a method for simultaneously detecting multiple biomarkers to measure the biomarker concentration from the number of biomarkers per unit area.

According to one side, the fluorescent molecule may act as a donor or acceptor on a specific base, 3'- or 5'-end, or a backbone of the detection antibody tag. .

According to one side, the detection method may further include attaching a capture antibody to the substrate.

According to one side, the capture antibody may be a combination of the capture antibody tag.

According to one side, the detection method comprises the steps of combining the capture antibody tag and a detection probe; And combining the detection antibody tag with the detection probe.

The present invention can simultaneously detect two or more types of biomarkers using FRET efficiency, and can distinguish types of biomarkers with low concentration contained in a small amount of a sample.

Specifically, the present invention has an effect of simultaneously detecting up to 1000 biomarkers, including biomarkers present in an extremely small amount of 1fg/ml, and has the advantage that the time required for detection and analysis is very short.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 schematically shows a kit for simultaneous detection of multiple biomarkers according to an embodiment of the present invention.
2 schematically shows a donor and an acceptor labeled on a detection antibody tag according to an embodiment of the present invention.
3 shows a process of imaging using a single molecule microscope according to an embodiment of the present invention.
Figure 4 shows the measurement of the FRET efficiency of the donor and acceptor according to an embodiment of the present invention.
5 shows a detection antibody tag labeled with two or more donor-acceptor FRET pairs according to an embodiment of the present invention.
6 shows a case of a combination of a donor and an acceptor of two or more donor-acceptor FRET pairs according to an embodiment of the present invention.
7 schematically shows the detection of the type of biomarker according to an embodiment of the present invention.
8 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.
9 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.
10 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the rights of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes to the embodiments are included in the scope of the rights.

The terms used in the examples are used for illustrative purposes only and should not be interpreted as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that a detailed description of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed description thereof will be omitted.

The terms used in this specification will be briefly described.

In the present specification, “biomarker” includes all biomaterials capable of confirming the normal or pathological state of an organism.

In the present specification, “antibody” is used in a broad sense including “detection antibody”, specifically, monoclonal antibodies (including monoclonal antibodies and full-length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies) , Multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable regions and other portions of antibodies that exhibit a desired biological activity). In addition, the antibody of the present invention includes both monoclonal antibodies and polyclonal antibodies, and includes all chimeric antibodies, humanized antibodies, and human antibodies. Preferably, it may include Fab, F(ab)2, Fab', F(ab')2, Fv, diabody, nanobody, or scFv, more preferably scFv, Fab, It may be a nanobody and an immunoglobulin molecule.

In the present specification, the "detection antibody tag" refers to a nucleic acid in a state capable of attaching a fluorescent molecule onto a specific base or a single-stranded or double-stranded nucleic acid in which a fluorescent molecule is already attached.

In the present specification, “fluorescent molecule” refers to a detectable fluorescent dye compound bound to the detection antibody tag.

In the present specification, “FRET efficiency” refers to a phenomenon in which the energy of a fluorescent molecule excited by light is transferred to another adjacent fluorescent molecule, and indicates different energy transfer efficiencies according to the distance between the fluorescent molecules. And, it may include all the meanings of typical FRET efficiency recognized by those skilled in the art.

In the present specification, “donor” refers to a fluorescent molecule that transmits energy when excited by receiving light, and refers to a fluorescent molecule that absorbs or emits light of a relatively short wavelength when two or more fluorescent molecules are adjacent.

In the present specification, “acceptor” refers to a fluorescent molecule that receives energy from a donor in an excited state, and when two or more fluorescent molecules are adjacent, it refers to a fluorescent molecule that absorbs or emits light of a relatively long wavelength.

In the present specification, “capturing antibody” may be any one included in the above-described term “antibody”, and specifically refers to an antibody that performs a role of immobilizing a biomarker on a substrate.

In the present specification, "capturing antibody tag" refers to a nucleic acid strand bound to a capture antibody according to an embodiment of the present invention, and may have the same or similar configuration as the aforementioned "detection antibody tag".

In the present specification, the “detection probe” may have the same or similar configuration as the “detection antibody tag” described above. Specifically, it refers to a single-stranded nucleic acid that binds to a capture antibody tag and a detection antibody tag.

The present invention is a substrate; Two or more detection antibody tags labeled with fluorescent molecules; And it relates to a kit for simultaneous detection of multiple biomarkers comprising two or more detection antibodies to which the detection antibody tag is bound, and for simultaneously discriminating two or more types of biomarkers using FRET efficiency by the fluorescent molecule.

According to one side, the fluorescent molecule may act as a donor or acceptor on a specific base, 3'- or 5'-end, or a backbone of the detection antibody tag. .

The biomarker of the present invention may be a polypeptide, peptide, nucleic acid, protein, or metabolite detectable in biological fluids such as blood, saliva, urine, etc., but is not limited thereto, but is not limited to AFP (alpha fetoprotein), CA15-3, CA27-29, CA19-9, CA-125, calcitonin, calretinin, carcinoembryonic antigen (CEA), CD34, CD99, MIC-2, CD117, chromogranin, cytokeratin ( cytokeratin, various types: TPA, TPS, Cyfra21-1), desmin, EMA (epithelial membrane antigen), Factor VIII, 　CD31, 　FL1, GFAP (glial fibrillary acidic protein), GCDFP-15 (gross cystic disease fluid protein), HMB-45, hCG (human chorionic gonadotropin), immunoglobulin, inhibin, keratin, various types of keratin, leukocyte marker, MART-1 (Melan-A) ), Myo D1, MSA (muscle-specific actin), neurofilament (neurofilament), NSE (neuron-specific enolase), PLAP (placental alkaline phosphatase), PSA (prostate-specific antigen), PTPRC (CD45), S100 protein, SMA (smooth muscle actin), synaptophysin, TK (thymidine kinase), Tg (thyroglobulin), TTF-1 (thyroid transcription factor-1), M2-PK, vimentin, interleukin , CD24, CD40, integrin, cystatin, interferon, TNF (tumor necrosis factor), MCP, VEGF, GLP, ICA, HLA-DR, ICAM, EGFR, FGF, BRAF, GFEB, FRS, LZTS, CCN, mucine, lectin, apolipoprotein, tyrosine, nerve cell adhesion molecule-like protein, fibronectin, glucose, uric acid, carbonic anhydrase, or cholesterol.

The biomarker to be detected in the present invention can be used without limitation, as long as it is used in conventional science or medical fields such as biological treatment, pathogenic processes, and measurement or evaluation of pharmacological processes for treatment. It is not. Specifically, the present invention is to simultaneously detect two or more biomarkers by using a measurement value of FRET efficiency that is measured differently depending on the distance between a donor and an acceptor labeled on a detection antibody tag.

The detection antibody is for detecting a biomarker using an antigen-antibody reaction, and it is preferable to use as many different kinds of biomarkers as the type of biomarker to be detected. In addition, in order to measure the FRET efficiency during detection, it is preferable to use a detection antibody tag bound with a fluorescent molecule for each detection antibody. In the embodiments of the present invention, DNA was used as the detection antibody tag, but it is not necessarily limited thereto, and preferably, nucleic acids such as DNA, RNA, PNA, LNA, MNA, GNA, TNA, or analogs thereof may be used, Any one selected from the group consisting of polypeptides may be used.

The “FRET efficiency” is a phenomenon in which the energy of a fluorescent molecule excited by light is transferred to another adjacent fluorescent molecule, and represents the energy transfer efficiency different according to the distance between the fluorescent molecules. Specifically, it is calculated as follows. Means value.

FRET efficiency = (the intensity of light emitted by the acceptor)/(sum of the intensity of light emitted by the donor and acceptor)

In this case, the light intensity of the donor or acceptor may be affected by the distance between the donor and the acceptor, but the donor and acceptor are marked on the desired specific base, 3'- or 5'-end, or the basic skeleton. Thus, by controlling the distance between the donor and the acceptor, the FRET efficiency can be controlled.

At this time, the FRET efficiency is that the acceptor is excited by the light to excite the donor and the light emitted by the donor and the donor that is not completely separated by the dichroic mirror used to separate the light from the donor and the acceptor. It may be a value obtained by correcting a difference in detection efficiency of the image sensor according to a part of light emitted by the acceptor or a difference in wavelength of light emitted by the donor and the acceptor.

For example, if the number of bases present between the donor and the acceptor is 1, and the gap is 1, the gap between the donor and acceptor is 1, 2, 3,… Produce two or more detection antibody tags of n. The FRET efficiency for each interval is measured and the FRET efficiency for the interval is databased. The same type of detection antibody tag is attached to the same type of detection antibody, and a different type of detection antibody tag is attached to different types of detection antibody. Thereafter, when the reaction with the sample containing the biomarker proceeds, the specific biomarker binds to the specific detection antibody. At this time, the FRET efficiency is calculated by measuring the intensity of light emitted by the donor and acceptor of each molecule. The type of detection antibody used for detection can be confirmed by comparing the calculated value with the database value, so the type of biomarker can be finally confirmed.

As an embodiment, assuming that the interval at which the FRET efficiency can be classified as described above is an integer of 4 or more and 20 or less, there are 17 detection antibody tags that can be produced with one donor and one acceptor. If there are 2 pairs of donors and acceptors under the same conditions, there are 289 types, and when 3 pairs are present, there are 4,913 types. Therefore, various types of biomarkers can be detected using a minimum number of types of fluorescent molecules.

According to one side, the detection antibody tag may be labeled with two or more different fluorescent molecules.

1 schematically shows a kit for simultaneous detection of multiple biomarkers according to an embodiment of the present invention.

Referring to FIG. 1, a biomarker is fixed to a substrate, and a detection antibody with a detection antibody tag is bound to the biomarker. FIG. 1 shows the configuration of a kit including N types of detection antibodies that specifically bind to N types of biomarkers and N types of detection antibody tags attached thereto.

The reason why the kit of the present invention is constructed in this way is that if the same FRET efficiency is measured from the detection antibody tag of the detection antibody combined with each biomarker, the type of the detected biomarker cannot be specified. Therefore, in order to obtain a measured value of FRET efficiency that can be distinguished, it is preferable to use different detection antibody tags as many as the number of biomarkers to be detected. Specifically, two or more types of detection antibody tags may have different numbers of bases present between the labeled donor and acceptor. More specifically, the detection antibody tag may have different FRET efficiencies depending on the labeled positions of the donor and the acceptor. In this case, the detection antibody tag may be labeled with a plurality of donors or acceptors. In this case, the two or more donors may be different types of fluorescent molecules from the acceptor, and the two or more donors may be different types of fluorescent molecules. In another case, the two or more acceptors may be different types of fluorescent molecules from the donor, and the two or more acceptors may be different types of fluorescent molecules. As an example, as shown in FIG. 2, the detection antibody tag of the present invention may have one acceptor and a plurality of donors labeled on the detection antibody tag. In this case, it is preferable that the donor and the acceptor are different types of fluorescent molecules, and the plurality of donors are all different types of fluorescent molecules. For example, assuming that the interval at which the FRET efficiency can be classified as described above is an integer of 4 or more and 20 or less, theoretically, there are 17 detection antibody tags that can be produced with one FRET efficiency. In other words, in the case of manufacturing a detection antibody tag consisting of 4 donors and 1 acceptor, a total of 4 FRETs between donor 1-donor 2, donor 2-donor 3, donor 3-donor 4, donor 4-acceptor In theory, there are 17x17x17x17=83,521 types of detection antibody tags that can be produced efficiently.

FIG. 3 shows a process of measuring the FRET efficiency from fluorescent molecules labeled on the detection antibody tag constructed as shown in FIG. 2, and schematically shows the principle of imaging fluorescent molecules using a single molecule microscope.

First, after fixing a detection antibody tag labeled with a fluorescent molecule on a substrate, the brightness of light is observed using a single molecule fluorescence microscope as shown in FIG. 3. In this case, molecules located within about 300 nm due to light diffraction are not distinguished from each other and can be observed as a single point. Therefore, even if several fluorescent molecules are labeled on one detection antibody tag, fluorescent molecules arranged so that the total distance is less than 300 nm are seen as one dot. On the other hand, since different fluorescent molecules emit light of different wavelengths, the brightness of specific fluorescent molecules can be measured by separating the emitted light according to wavelength using an optical component (dichroic mirror) that reflects or passes only light in a specific wavelength range. I can.

The left image of FIG. 3 schematically shows a method of outputting and confirming four types of fluorescent molecules (Cy2, Cy3, Cy5, Cy7) on one camera, and the image on the right of FIG. 3 is a It schematically shows how to image through different cameras depending on the type. In the present invention, as a method of imaging the FRET efficiency generated by the donor and the acceptor, the above-described contents may be used, and there is no particular limitation, but specifically, the donor and the acceptor through the donor camera and the acceptor camera It is preferable in terms of analysis time to image the intensity of light of the septer.

Figure 4 shows the results of imaging by the above-described method using a detection antibody tag labeled with a donor of Cy3 and Cy5 and an acceptor of Cy7. Referring to FIG. 4, as a result of measuring brightness in the same manner as in the image on the left of FIG. 3, the results of imaging in order from the leftmost to Cy3, Cy5, and Cy7 were confirmed through one monitor. When irradiating a red laser on the detection antibody tag produced as shown in FIG. 4, Cy5 is first excited, and energy is transferred to Cy7 by FRET so that Cy7 can also emit light, so nothing appears in the Cy3 area, Bright spots are observed only in the areas of Cy5 and Cy7. At this time, the distance can be checked by measuring the FRET efficiency between Cy5 and Cy7 from the brightness ratio of each point. Additionally, when irradiating a green laser, Cy3 is excited, and energy is transferred to Cy5 and Cy7 by FRET, so that both light is emitted. In this case, the distance between Cy3 and Cy5 can be confirmed by using the FRET efficiency between Cy5 and Cy7, which was confirmed through the previous case.

According to another embodiment of the present invention, the kit for simultaneous detection of multiple biomarkers of the present invention may have a configuration in which two or more FRET pairs composed of donor-acceptors are labeled as shown in FIGS. 5 and 6.

When two or more FRET pairs consisting of a donor and an acceptor are labeled on one detection antibody tag, it is preferable to label each FRET pair at a certain distance in order to minimize the effect on each other's FRET efficiency. Specifically, it is more preferable that each FRET pair is labeled with a distance of 5 nm or more.

The detection antibody tag may include two or more FRET pairs consisting of a donor and an acceptor.

In this case, the two or more FRET pairs may have different intervals between the donor and the acceptor, and preferably, in consideration of the error range and noise that may occur during measurement, a sufficiently distinguishable FRET efficiency is measured. The spacing may be different.

As another example, in the case of using the same kind of donor for the two or more FRET pairs, each of the labeled acceptors may use the same or different types, but it is preferable that they are different types from the donors.

As another example, in the case of using the same type of acceptor for the two or more FRET pairs, each of the labeled donors may be of the same or different types, but it is preferable that they are of different types from the acceptors.

Specifically, when two pairs of FRET pairs are labeled on one detection antibody tag, the donors and acceptors of the FRET pair may be combined in the following four cases.

i) The donors of the FRET pair may be the same fluorescent molecules, and the acceptors may be the same fluorescent molecules.

ii) The donors of the FRET pair may be different fluorescent molecules, and the acceptors may be the same fluorescent molecules.

iii) The donors of the FRET pair may be the same fluorescent molecules, and the acceptors may be different fluorescent molecules.

iv) The donors of the FRET pair may be different fluorescent molecules, and the acceptors may be different fluorescent molecules.

5 shows the case of i) as an example. 5, donors 1 and 2 are labeled with a fluorescent molecule A, and acceptors 1 and 2 are labeled with a fluorescent molecule B. Since each donor and acceptor are labeled with the same fluorescent molecule, in this case, if all four donors and acceptors are adjacent to each other, the FRET efficiency of the FRET pair composed of each donor-acceptor is affected. Therefore, as shown in FIG. 5, when two or more of the same donors and acceptors are labeled on one detection antibody tag, it is preferable to label each FRET pair at least 5 nm apart.

6A to 6C exemplarily show combinations of FRET pairs of ii) to iv) described above.

6A shows the case of ii). In FIG. 6A, donor 1 is labeled with a fluorescent molecule A and donor 2 is labeled with a fluorescent molecule B. At this time, both the acceptor 1 and the acceptor 2 are labeled with the same fluorescent molecule C, and this is a diagram explaining a case where the same type of acceptor is labeled on two pairs of FRETs labeled with one detection antibody tag.

In addition, FIG. 6B shows the case of iii) above, where both donors 1 and 2 in FIG. 6B are labeled with the same fluorescent molecule A, acceptor 1 is fluorescent molecule B, and acceptor 2 is fluorescent molecule C. It is labeled. In addition, FIG. 6C shows the case of iv), in which donor 1 is labeled with a fluorescent molecule A, donor 2 is a fluorescent molecule B, acceptor 1 is a fluorescent molecule C, and acceptor 2 is labeled with a fluorescent molecule D. The case of a detection antibody tag labeled with a fluorescent molecule of is described.

6A to 6C, it can be seen that both the 1st FRET pair of the donor 1-acceptor 1 and the 2nd FRET pair of the donor 2-acceptor 2 are labeled with a predetermined distance apart. This is to minimize the influence of adjacent FRET pairs, as described above with reference to FIG. 5. In addition, the spacing between the donors and acceptors of the first FRET pair and the second FRET pair may be the same or different. However, if the types of donors and acceptors used in the 1st and 2nd FRET pairs are the same, the FRET efficiency of the 1st and 2nd FRET pairs is i) a, b, respectively, and ii) b, a. If there is a detection antibody tag, since the detection antibody tags of i) and ii) cannot be distinguished from each other, only one of the detection antibody tags i) or ii) can be used.

The detection antibody tag according to an embodiment of the present invention may include two or more FRET pairs, and the configuration may be the same as described above. That is, the detection antibody tag may be implemented similarly to that shown in FIG. 5, and may have the same configuration as that of FIGS. 6A, 6B, or 6C. However, since the drawings are merely schematic representations for the purpose of understanding one embodiment of the present invention, it is preferable to comprehensively judge the contents described in the specification of the present invention upon implementation.

In measuring the FRET efficiency using the kit configured as described above, after a certain time elapses after the donor is excited, the fluorescent molecule becomes a photobleached state in which no more light is emitted. Once photobleached, the intensity of light changes. Even in this case, the FRET efficiency on each donor-acceptor can be measured by measuring and comparing the brightness of light before and after photobleaching.

The basic principle of measuring FRET efficiency by comparing the brightness before and after photobleaching is as follows.

When the donor is photobleached, the donor no longer emits light or transfers energy to the acceptor due to photobleaching, and the acceptor cannot receive energy from the donor. Therefore, the light intensity of both the donor and the acceptor is reduced. Decrease. On the other hand, when the acceptor is photobleached, the donor cannot transmit energy to the acceptor, so all the energy is used to emit light, so the brightness of the donor is further increased, and the brightness of the acceptor is decreased. A more specific principle will be described through Example 3.

According to one side, the kit for simultaneous detection of multiple biomarkers may further include a capture antibody.

8 schematically shows a kit of the present invention including a capture antibody. In this case, the capture antibody may be attached to a substrate in advance, or may be attached to a detection process.

According to one side, the capture antibody may be a combination of the capture antibody tag.

At this time, the capture antibody tag may be made of the same or similar basic skeleton as the above-described detection antibody tag, and specifically, the capture antibody tag is preferably DNA, RNA, PNA, LNA, MNA, GNA, TNA and The same nucleic acid or an analog thereof may be used, and may be any one selected from the group consisting of polypeptides. For example, it may be configured as shown in FIG. 9, but is not particularly limited.

However, this is according to an embodiment of the present invention, and the biomarker may be directly attached to the substrate depending on the situation. This is for the speed, economy, and convenience of the measurement process, and if necessary, biomarkers may be detected through antigen-antibody reaction with the capture antibody attached to the substrate in order to increase the accuracy of the measurement result.

Accordingly, the substrate included in the kit of the present invention may have a surface treated so that the biomarker can be directly attached to the surface of the substrate or a capture antibody capable of capturing the biomarker can be attached when detecting the biomarker. . Alternatively, the capture antibody may be previously attached to the surface of the substrate. In this case, the substrate may be any one selected from the group consisting of slide glass, coverslip, quartz, plastic, and the like, but is not limited thereto.

According to one side, the kit for simultaneous detection of multiple biomarkers may further include a detection probe.

The detection probe of the present invention preferably uses the same or similar basic skeleton as the above-described detection antibody tag and capture antibody tag, and more preferably, a single-stranded nucleic acid having a base sequence is used.

According to one side, the detection probe may be complementarily combined with a detection antibody tag and a capture antibody tag.

When a single-stranded nucleic acid is used as the detection probe, some nucleotide sequences of the detection probe are complementary to some nucleotide sequences of the capture antibody tag, and other parts are complementary to some nucleotide sequences of the detection antibody tag, Part of the detection probe is complementarily combined with the detection antibody tag and the capture antibody tag, respectively, to form two different double-stranded nucleic acids. This structure can be represented as shown in FIG. 10.

According to a preferred embodiment, some of the total FRET pairs required to distinguish biomarkers are formed only when the detection antibody tag and the detection probe are complementarily combined, and the remaining FRET pairs are complementarily combined with the capture antibody tag and detection probe. It must be formed. Therefore, the fact that the entire FRET pair was observed in the biomarker detection process means that the detection probe is complementary to both the detection antibody tag and the capture antibody tag, which means that the biomarker is binding to the capture antibody and the detection antibody. . On the other hand, when only some of the plurality of FRET pairs are observed, it means that the detection antibody is non-specifically bound to a substrate other than a biomarker, which is a false positive signal and is a measurement error. . That is, by using a detection probe that complementarily binds the detection antibody tag and the capture antibody tag, the false positive signal can be removed, thereby improving the accuracy of the analysis result.

According to one side, the detection probe may be a donor or acceptor labeled on a specific base, 3'- or 5'-end, or a backbone. Preferably, it may be to measure the FRET efficiency by labeling a fluorescent molecule acting as a donor or an acceptor on a specific base of a detection probe.

In order to measure FRET efficiency by combining the capture antibody tag, detection antibody tag, and detection probe, a donor or acceptor may be labeled as follows.

1) When the detection antibody tag and the detection probe are complementarily combined to form one or more FRET pairs

1-1) When only the donor is labeled on the detection antibody tag and the acceptor is labeled on the detection probe

1-2) When only the acceptor is labeled on the detection antibody tag and the donor is labeled on the detection probe

1-3) When at least one of the donor and acceptor is labeled on the detection antibody tag, and at least one of the donor and acceptor is labeled on the detection probe

2) When the capture antibody tag and the detection probe are complementarily combined to form one or more FRET pairs

2-1) When only the donor is labeled on the capture antibody tag and the acceptor is labeled on the detection probe

2-2) When only the acceptor is labeled on the capture antibody tag and the donor is labeled on the detection probe

2-3) When at least one of the donor and acceptor is labeled on the capture antibody tag and at least one of the donor and acceptor is labeled on the detection probe

Also in the cases 1) and 2) above, the above-described principle may be applied. In this case, in the case of the FRET pair labeled with the same species, the FRET efficiencies of the 1st FRET pair and the 2nd FRET pair are i) a, b, and ii) b, a (a, b are respectively the FRET efficiency. Meaning) cannot be distinguished from each other, so it is preferable to manufacture a capture antibody tag, a detection antibody tag, and a detection probe so that only one of i) or ii) above can be used, and specifically, the degree to which the measured value of FRET efficiency can be distinguished. It is preferable to label each capture antibody tag, detection antibody tag, and detection probe at intervals of.

In addition, in the case of 1) and 2), it is preferable that the donor and the acceptor are labeled with different fluorescent molecules. In this case, when a plurality of donors or acceptors are labeled, the types of donors labeled on the capture antibody tag, detection antibody tag, or detection probe may be the same or different, and label on the capture antibody tag, detection antibody tag, or detection probe. The types of acceptors used may also be the same or different.

For example, in the case of a kit for detecting biomarker 1, capture antibody 1, detection antibody 1, and detection probe 1 are used to detect biomarker 1, and tags that bind to detection probe 1 are capture antibody tag 1 and Assume that it is the detection antibody tag 1. In this case, when the donor 1 is labeled on any one base of the capture antibody tag 1, acceptor 1 is labeled on any one of the sites that bind to the capture antibody tag 1 of detection probe 1 to achieve the desired FRET efficiency. . At the same time, donor 1 and acceptor 1 are labeled or completely different fluorescent materials, donor 2 and acceptor 2, so that the binding site of detection antibody tag 1 and detection probe 1 with detection antibody tag 1 can achieve the desired FRET efficiency in the same manner. May be a sign. Alternatively, donor 1 and acceptor 2 may be labeled, or donor 2 and acceptor 1 may be labeled, but donor 1 and acceptor 1 are different fluorescent molecules, and donor 2 and acceptor 2 must also be different fluorescent molecules. do.

The fluorescent molecules are rhodamine, Alexa, fluorescein, FITC (fluorescein isothiocyanate), FAM (5-carboxy fluorescein), Atto, BODIPY, CF, CY, DyLight Fluor, and Texas Red. (Texas Red) is preferably selected from the group consisting of, but is not necessarily limited thereto.

In addition, the kit of the present invention may be a RUO (research use only), an IUO (investigational use only) kit, or an IVD (in vitro diagnostic).

According to another embodiment of the present invention, there is provided a method for simultaneously detecting multiple biomarkers, comprising: attaching two or more biomarkers to a substrate; Binding two or more types of detection antibodies to which a fluorescent molecule-labeled detection antibody tag is bound to the two or more biomarkers, respectively; And measuring FRET efficiency from the fluorescent signal generated by the coupling. Including, and measuring the biomarker concentration from the number of biomarkers per unit area after simultaneously determining the type of the biomarker using the FRET efficiency by the fluorescent molecule, a method for simultaneously detecting multiple biomarkers is provided.

According to one side, the fluorescent molecule may act as a donor or acceptor on a specific base, 3'- or 5'-end, or a backbone of the detection antibody tag. . Preferably, it may be to measure the FRET efficiency by labeling a fluorescent molecule acting as a donor or an acceptor on a specific base of a detection antibody tag.

The simultaneous detection method of multiple biomarkers of the present invention may be a detection method using the aforementioned kit for simultaneous detection of multiple biomarkers, and may include a method described below.

In using the method of the present invention, the step of creating a FRET efficiency database to be described later is preceded. By measuring the distinguishable FRET efficiency reference value in advance and making a database, and comparing the FRET efficiency measurement value measured after the reaction based on this reference, the type of biomarker can be identified.

The FRET efficiency database is produced through the following process.

Using different fluorescent molecules A and B as donors and acceptors, respectively, A and B are labeled on a specific base on the DNA skeleton, and heterogeneous DNA is produced as many as necessary.

For example, if the required type on the database is 50 types, 50 types of DNA labeled with different distances between labeled A and B are prepared. At this time, at one end of each DNA, a material applied to the substrate and a binding-friendly material are attached to the end of each DNA so that it can be fixed to the substrate. If necessary, when the donor and acceptor to be used are commercially available fluorescent molecular materials, such a manufacturing process may be omitted and commercially available DNA with the donor and acceptor attached may be used.

However, in determining the fluorescent molecule used as a donor and an acceptor, it is preferable that the wavelength of the maximum value of the emission spectrum of the fluorescent molecule used as the donor is shorter than the wavelength of the maximum value of the absorption spectrum of the fluorescent molecule used as the acceptor. Do.

When two or more types of DNA are prepared, DNA with a distance of 1 is fixed to the substrate and the FRET efficiency of the distance is measured. Thereafter, starting from the DNA having a distance of 2, the same process as described above is sequentially repeated to obtain a reference value of FRET efficiency, and this is converted into a database for each distance.

Thereafter, by comparing the FRET efficiency measurement value measured in the biomarker detection process with the FRET efficiency reference value, the distance between the donor and the acceptor of the detection antibody tag used for detection can be known, which is the type of the detected biomarker. Can be seen.

However, since there may be an error due to noise in the measurement of FRET efficiency, the FRET efficiency of all distances may not be distinguishable. For example, if the FRET efficiency when the distance is 1 is 0.95 (average) ± 0.05 (error) and the FRET efficiency when the distance is 2 is 0.94 ± 0.05, the FRET efficiency cannot distinguish between distances 1 and 2. . Therefore, when detecting an actual biomarker, it is preferable to use only the distance from which the FRET efficiency is clearly distinguished among the values of the database.

When the FRET efficiency reference value is secured, in order to detect the biomarker, a sample containing a biomarker is injected and a step of attaching the biomarker to the substrate is performed.

According to one side, the simultaneous detection method of multiple biomarkers may further include attaching a capture antibody to a substrate.

According to one side, the capture antibody may be a combination of the capture antibody tag.

The method of adsorbing the capture antibody to the substrate is, for example, by diluting the capture antibody with a 0.06M carbonate buffer solution or bicarbonate buffer solution with a pH of 9.5, and contacting the diluted solution to the substrate for a predetermined period of time. It may be to let you do. Alternatively, the surface of the substrate is coated with biotin-conjugated PEG (polyethylene glycol) or BSA (bovine serum albumin), and streptavidin is bonded thereto. Can be. Thereafter, the capture antibody adsorbed on the substrate forms a biomarker and a conjugate contained in the sample when the sample or the processed sample is processed on the substrate. Additionally, after the formation of the conjugate, it may be desirable to wash with a washing buffer containing TWEEN 20, Triton X-100, etc. for the purpose of removing non-specifically bound antibodies or removing contaminants.

According to one side, the detection method comprises the steps of combining the capture antibody tag and a detection probe; And combining the detection antibody tag with the detection probe.

In this case, the detection probe may include a base sequence complementary to the base sequence of the detection antibody tag and the capture antibody tag. When the detection probe is a single-stranded nucleic acid, some nucleotide sequences may include a nucleotide sequence complementary to the nucleotide sequence of the detection antibody tag, and other parts may include a nucleotide sequence complementary to the capture antibody tag. The scope of the complementary bond is not particularly limited.

The capture antibody tag and the detection antibody tag used in the present invention can use nucleic acids such as DNA, RNA, PNA, LNA, MNA, GNA, TNA, or analogs thereof, respectively, and any one selected from the group consisting of polypeptides It may be included. In addition, the fluorescent molecules used in the present invention are rhodamine, Alexa, fluorescein, FITC (fluorescein isothiocyanate), FAM (5-carboxy fluorescein), Atto, BODIPY, CF, CY , DyLight Fluor and Texas Red (Texas Red) is preferably selected from the group consisting of, but is not limited thereto.

In addition, the sample used in the present invention may be selected from the group consisting of blood, plasma, serum, saliva, tissue fluid, and urine, but only those containing biomarkers that need to be detected according to the type or characteristic of diagnosis. It is not limited.

Thereafter, the detection of two or more types of biomarkers using the detection method of the present invention will be described later in more detail through examples.

In addition, all descriptions related to the above-described detection antibody tag can be applied equally to the capture antibody tag, and the same or similarly can be applied to kits and detection methods including the capture antibody.

Hereinafter, the present invention will be described in detail through examples.

Example 1. FRET efficiency database production

In order to construct the FRET efficiency database by the method described above, Cy3 as a donor and Cy5 as an acceptor were labeled on the DNA strand to produce a detection antibody tag. Biotin was attached to one end of the DNA, a donor and an acceptor were attached to a specific base, and a total of 50 detection antibody tags were prepared by labeling so that the distance between the donor and the acceptor was 1 to 50.

At this time, the reason for attaching biotin is to immobilize DNA on the substrate using antigen-antibody binding between streptavidin and biotin, and to perform a similar role, digoxigenin and its antibody or SNAP It is also possible to use a method of fixing to the substrate by using a bond between a tag such as -tag, CLIP-tag, FLAG-tag, His-tag, and each ligand.

After the substrate was coated with streptavidin, DNA having a distance of 1 between the donor and the acceptor was fixed, and the FRET efficiency when the distance was 1 was measured to obtain a reference value. In this case, the FRET efficiency is completely separated by 1) the light that the acceptor is excited and emitted by the light to excite the donor, and 2) the dichroic mirror used to separate the light from the donor and the acceptor. It may be a value obtained by correcting a difference in detection efficiency of an image sensor according to a difference in wavelength of light emitted by a non-donor and an acceptor and 3) a wavelength difference between the donor and the acceptor.

In the case of the remaining distances 2 to 50, each reference value was secured by measuring by the same method, and by calculating an error due to noise generated during measurement, only the distance of the FRET efficiency that is actually clearly distinguished was used. Hereinafter, in an embodiment to be described later, it is assumed that 10 clearly distinguishable values among the FRET efficiency reference values of distances 1 to 50 are described, and a detection example will be described.

Example 2. Preparation of detection antibody tag

The detection antibody tag should be produced as many as the number of biomarkers to be detected. The same DNA used in Example 1 can be used, but instead of biotin attached to one end of the DNA used in Example 1, a functional group attached to the detection antibody was used. In this case, the method of attaching may be the same as attaching a fluorescent molecule to DNA.

In Example 1, it has been assumed that 10 FRET efficiencies that can be clearly distinguished by one donor-acceptor pair are assumed, so if there are 10 types of biomarkers to be detected, only one donor-acceptor pair can be sufficiently detected. .

If the number of biomarkers to be detected exceeds 10, two or more pairs of donor-acceptors may be used. If two pairs of donor-acceptors are used, the maximum FRET efficiency that can be clearly distinguished will be 55, and if three pairs are used, the maximum will be 205. If calculated theoretically, if the maximum number of FRET efficiencies that can be divided into one pair of donor-acceptors is 10, it can be calculated that the number of FRET efficiencies that can be divided into two pairs is 100. However, if the FRET efficiency of FRET pair 1 and FRET pair 2 is i) a, b and ii) b and a, respectively, the cases of i) and ii) cannot be distinguished from each other, so i) Alternatively, if only one of ii) is used as a detection antibody tag, there may be a total of 55 cases in which the FRET efficiency can be substantially distinguished. If the types of the donors or acceptors of FRET pair 1 and FRET pair 2, or both of the fluorescent molecules are different, there are 100 FRET efficiencies that can be classified into two pairs, and 1,000 types of three pairs.

Example 3. Changes in FRET efficiency before and after photobleaching

Using the DNA labeled with the donor 1-acceptor 1 and donor 2-acceptor 2 separated by a certain distance, the brightness of each of the donors and acceptors before photobleaching and the FRET efficiency of the donor-acceptor pair were measured (Table 1). In this case, the FRET efficiency is completely separated by 1) the light that the acceptor is excited and emitted by the light to excite the donor, and 2) the dichroic mirror used to separate the light from the donor and the acceptor. It may be a value obtained by correcting a difference in detection efficiency of an image sensor according to a difference in wavelength of light emitted by a non-donor and an acceptor and 3) a wavelength difference between the donor and the acceptor.

For example, the FRET efficiency of FRET pair 1 is 0.5 and the FRET efficiency of FRET pair 2 is 0.7, but the observed FRET efficiency in this state is 0.6, which is the average of the two FRET pairs. FRET efficiency is unknown.

Donor 1-Acceptor 1 Donor 2-Acceptor 2 Doner 1 Acceptor 1 Doner 2 Acceptor 2 50 50 30 70 FRET efficiency 0.5 FRET efficiency 0.7

From the configuration shown in Table 1, four cases of photobleaching can occur, and in the case of the corrected FRET efficiency, the sum of the brightness of the donor and acceptor forming the FRET pair before and after photobleaching is constant, and in this embodiment, the sum is 100 to be. The FRET efficiency in each case can be measured as follows.

1) When donor 1 is photobleached

Since both donor 1 and acceptor 1 do not emit light, the sum of the brightness of the donor is 80 to 30, and the sum of the brightness of the acceptor is 120 to 70, so the amount of change in brightness before and after photobleaching is 50 for the donor and for the acceptor. Becomes 50. That is, it can be seen that the FRET efficiency measured from the difference in brightness before and after photobleaching is 0.5, and the FRET efficiency measured from the brightness observed after photobleaching is 0.7. Therefore, it can be seen that the FRET efficiency of the detection antibody tag is composed of 0.5 and 0.7.

2) When donor 2 is photobleached

Since both donor 2 and acceptor 2 do not emit light, the sum of the brightness of the donor is 80 to 50, and the sum of the brightness of the acceptor is 120 to 50, so the amount of change in brightness before and after photobleaching is 30 for the donor, and for the acceptor. Becomes 70. That is, it can be seen that the FRET efficiency measured from the difference in brightness before and after photobleaching is 0.7, and the FRET efficiency measured from the brightness observed after photobleaching is 0.5. Therefore, it can be seen that the FRET efficiency of the detection antibody tag is composed of 0.5 and 0.7.

3) When acceptor 1 is photobleached

Since donor 1 emits the light emitted by acceptor 1, the sum of the brightness of the donor is 80 to 130, and the sum of the brightness of the acceptor is 120 to 70, so the amount of change in brightness before and after photobleaching is 50 for the donor, and for the acceptor. Becomes 50. Since the sum of the brightness of the donor and acceptor forming the FRET pair before and after photobleaching is constant at 100, it can be seen that the FRET efficiency is 0.5 from the change amount of the acceptor 50. After photobleaching, it can be seen that the FRET efficiency measured from the brightness excluding 100 emitted by the donor 1 out of the sum 130 of the brightness of the donor is 0.7. Therefore, it can be seen that the FRET efficiency of the detection antibody tag is composed of 0.5 and 0.7.

4) When acceptor 2 is photobleached

Since donor 2 emits the light emitted by acceptor 2, the sum of the brightness of the donor becomes 80 to 150, and the sum of the brightness of the acceptor becomes 120 to 50, so the amount of change in brightness before and after photobleaching is 70 for the donor, and for the acceptor. Becomes 70. Since the sum of the brightness of the donor and acceptor forming the FRET pair before and after photobleaching is constant at 100, it can be seen that the FRET efficiency is 0.7 from the change amount of the acceptor 70. It can be seen that the FRET efficiency measured from the brightness excluding 100 emitted by the donor 2 out of the sum 150 of the brightness of the donor after photobleaching is 0.5. Therefore, it can be seen that the FRET efficiency of the detection antibody tag is composed of 0.5 and 0.7.

As above, by measuring the brightness until one fluorescent molecule is photobleached, and repeatedly measuring each fluorescent molecule by the same method, it can be confirmed that the FRET efficiency of the detection antibody tag is composed of 0.5 and 0.7.

Example 4. Measurement of FRET efficiency and identification of biomarker types

In order to detect biomarkers 1 or 2, a kit was prepared as follows.

Detection antibody tag 1 was attached to detection antibody 1 that binds to biomarker 1, and detection antibody tag 2 was attached to detection antibody 2 that binds to biomarker 2. The detection antibody tags 1 and 2 were labeled with 2 pairs of donor-acceptor FRET pairs, respectively. The reference values of FRET efficiency of detection antibody 1 were FRET pair 1 = 0.1, FRET pair 2 = 0.5, and FRET efficiency reference values of detection antibody 2 were FRET pair 1 = 0.2 and FRET pair 2 = 0.4.

Thereafter, as in Example 3, the FRET efficiency was analyzed by measuring the difference in brightness before and after photobleaching. Molecules 1 and 2 appear on the imaging camera observing the brightness, and the FRET efficiency of Molecule A was measured as 0.1 and 0.5, indicating that Molecule A is a biomarker 1. According to the same principle, the FRET efficiency of molecule B was measured as 0.2 and 0.4, and it was found that molecule B was a biomarker 2 (FIG. 7).

The biomarkers included in the sample have a very wide concentration range from fg/ml to mg/ml, depending on their type. If an excessively high concentration of biomarkers is fixed on the substrate, there are too many dots appearing on one screen, overlapping occurs between the dots, which makes it impossible to measure the FRET efficiency of individual molecules, so an appropriate number of biomarkers on one screen. Samples can be diluted in various ratios so that molecules are detected.

If biomarkers having a similar concentration range are detected, the analysis can be performed after diluting the biomarkers in the same ratio. For example, if biomarkers having a concentration of about 1 ng/ml are usually diluted 1:100, if an appropriate number of biomarker molecules are detected on one screen, biomarkers having a concentration of about 100 ng/ml are 1: After dilution to 10000, the analysis can be performed.

As described above, although the embodiments have been described by the limited drawings, a person of ordinary skill in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a form different from the described method, or are replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments and claims and equivalents fall within the scope of the following claims.

Claims (14)

Board;
Two or more detection antibody tags labeled with fluorescent molecules; And
It includes two or more detection antibodies to which the detection antibody tag is bound,
Using the FRET efficiency by the fluorescent molecule, two or more types of biomarkers are simultaneously identified,
The fluorescent molecule acts as a donor or acceptor on a specific base, 3'- or 5'-end, or backbone of the detection antibody tag,
The detection antibody tag is a kit for simultaneous detection of multiple biomarkers in which two or more different fluorescent molecules are labeled. delete delete The method of claim 1,
The kit is a kit for simultaneous detection of multiple biomarkers further comprising a capture antibody. delete delete delete delete It relates to a method of simultaneously detecting multiple biomarkers in a sample,
Attaching two or more kinds of biomarkers to the substrate;
Binding two or more kinds of detection antibodies to which a detection antibody tag labeled with a fluorescent molecule is attached, respectively, with the two or more biomarkers; And
Measuring FRET efficiency from the fluorescence signal generated by the coupling;
Including,
Simultaneously discriminating the type of the biomarker using the FRET efficiency by the fluorescent molecule,
The fluorescent molecule acts as a donor or acceptor on a specific base, 3'- or 5'-end, or backbone of the detection antibody tag,
The detection antibody tag is a method of simultaneously detecting multiple biomarkers in which two or more different fluorescent molecules are labeled. delete The method of claim 9,
The detection method further comprises attaching a capture antibody to the substrate. delete delete The method of claim 9,
Simultaneous multiple biomarker detection method further comprising the step of measuring the concentration of the biomarker in the sample from the number of biomarkers per unit area.

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