WO2020046069A1 - Peptide specifically degraded by pepsin and reflux laryngopharyngitis diagnosing kit comprising same - Google Patents

Abstract

The present invention relates to a peptide specifically degraded by pepsin, a reflux laryngopharyngitis diagnosing kit comprising same, and a reflux laryngopharyngitis diagnosing method using same.

Description

Peptides specifically degraded by pepsin and reflux pharyngitis diagnostic kit comprising the same

The present invention relates to a peptide specifically cleaved by pepsin, a kit for diagnosing reflux pharyngitis comprising the same, and a method for diagnosing reflux pharyngitis using the same.

Reflux pharyngitis refers to chronic inflammation of the larynx and pharynx or damage to the mucous membrane caused by the contents of gastric acid that flow back into the larynx and pharynx through the esophagus. As the changes in dietary habits, industrialization, and urbanization of modern people have increased, the number of patients complaining of reflux pharyngitis is increasing.However, there is still no simple and accurate way to diagnose it. Most of the treatment method is only in the suppression of gastric acid secretion, the symptoms persist after treatment, such as difficult to treat effective conditions, accurate and rapid diagnosis is necessary for proper treatment.

The 24-hour esophageal acidity test, known as the gold standard for diagnosing reflux pharyngitis, is expensive, lengthy, inconvenient, difficult to use, and unpredictable in patients with atypical symptoms. Patients with reflux pharyngitis often complain of nonspecific symptoms such as sore throat, cough, and voice changes, rather than reflux symptoms, but it is difficult to diagnose correctly. The development of non-invasive and objective diagnostic methods is needed.

On the other hand, since pepsin is a protease produced only in the stomach, saliva pepsin detection technology may be useful as a sensitive and non-invasive method for reflux of the gastric contents. Saritas et al. Have shown that the detection of pepsin in saliva can be helpful in the diagnosis of reflux esophagitis using a rapid lateral flow device (Saritas Yuksel, E. et al., Laryngoscope, 2012, 122 (6). (1212-1316), Darija et al. Found that the amount of pepsin in saliva was significantly higher in the patients with reflux pharyngitis (Darija, B. et al., Collegium Antropologicum, 2012, Supp. 2, 36: 83-). 86). As a result, many countries, including the United States, are currently developing simpler, more reliable, and non-invasive diagnostic methods.

In general, immunochromatography, also known as rapid test, is a method that can qualitatively and quantitatively analyze trace amounts of analytes in a short time by using antigen-antibody reactions. Or it is used in various fields such as inspection, medicine, agriculture, animal husbandry, food, military, environment. In the immunochromatography analysis, an assay strip including a reactant capable of reacting with an analyte to be detected and displaying a change, or an analytical device in the form of a device equipped with the assay strip in a plastic case is generally used. . 1 is a cross-sectional view of an assay strip used in a conventional immunochromatographic assay. As shown in FIG. 1, a typical assay strip includes a conjugate obtained by conjugating a conjugate to a ligand such as an antigen, an antibody, or the like, which generates a signal that can be detected using a sample pad, a naked eye or a sensor containing a liquid sample. Conjugate pads containing, analyte in the sample and / or porous membrane pads immobilized with a binding agent (antibody or antigen) that specifically binds to the conjugate, and a hygroscopic pad that finally receives the liquid sample. In the order listed above they are connected in a partially overlapped form, attached on a solid support and arranged continuously. When the assay strip is mounted and used inside the plastic case, a sample inlet for dropping the sample at the sample pad is positioned on the upper part of the case, and a result for confirming the test result is confirmed at the location where the binder of the porous membrane pad is fixed. A window is formed. As described above, in the immunochromatographic analysis method using the assay strip, when a liquid sample is dropped onto the sample pad, the liquid sample moves through the conjugate pad and the porous membrane pad by capillary action, and finally is accommodated in the hygroscopic pad. At this time, the conjugate contained in the conjugate pad also moves with the liquid sample, and if the substance to be analyzed exists in the sample, the conjugate binds to the binder fixed to the porous membrane pad through the analyte (usually, "sandwich"). ("sandwich reaction"), whereby the conjugate and the analyte are competitively bound to the binder (commonly referred to as the "competition reaction") to determine the presence of the analyte in the sample, either visually or using a sensor. Can be detected. In order to use commonly used sandwich reactions, two kinds of antibodies specific to the analyte are required, and the antibodies must react with different antigen binding sites of the same antigen. In the case of using an antibody as described above, there is an advantage that selective and sensitive detection is possible using the high specificity and affinity of the antibody, but due to the manufacturing process using an animal or a cell, production is difficult, takes a long time, and is expensive. The activity may vary depending on the time, so it is not easy to obtain a high quality antibody having a high binding ability with a desired antigen. In addition, since the antibody is low in stability, it is difficult to store because it is affected by the environment such as pH and temperature.

The present inventors have made diligent research to provide a kit for diagnosing reflux pharyngitis using saliva, which is a noninvasive collectible sample that contains no antibody for economical and easy storage. By using the proteolysis of pepsin in saliva, which is an indicator, an amino acid sequence was devised that can be specifically degraded only by pepsin without being degraded by other enzymes. In addition, by using the appropriate combination of ligands, we can apply the principle of immunochromatography, but can also make a kit for diagnosing reflux pharyngitis based on this qualitative and quantitative analysis of pepsin, which does not contain antibodies, The present invention has been completed.

One object of the present invention is a peptide that is specifically degraded by pepsin, one end of the labeling material and the first ligand, the other end is bound to the second ligand; And a test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, including a third ligand specifically binding to the first ligand, and a fourth binding specifically to the second ligand. A membrane pad including a control line including a ligand and a control line formed in parallel with a predetermined distance apart from the test line, and an absorption pad for finally receiving a liquid sample, wherein the membrane A kit for diagnosing reflux pharyngitis comprising an immunochromatography strip in which a pad and a hygroscopic pad are connected in a partially overlapped form, wherein the first ligand and the second ligand, and the third and fourth ligands are different from each other. And a fourth ligand and a second ligand and a third ligand are selected so as not to bind to each other.

Another object of the present invention is a first step of deploying saliva collected from an individual suspected of developing reflux laryngitis into the membrane pad of the reflux laryngitis diagnostic kit in the direction of the hygroscopic pad to cross the test line and control line; A second step of confirming signals by the labeling substance in the inspection line and the control line; And a third step of determining positive for reflux pharyngitis when a signal is detected in a test line and a control line, and negative when a signal is detected only in a control line, and for providing reflux pharyngitis. .

Another object of the present invention is to continuously position two hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan, each of which is 1 to the sock end of the consecutively located hydrophobic amino acids A first step of preparing a series of peptides including a total of 6 or more amino acids to which 10 amino acids are added; And a second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming degradation by each enzyme. It is to provide a manufacturing method.

Another object of the present invention is to provide a peptide having an amino acid sequence of GDFLMGRDMR (SEQ ID NO: 1), GDFLGGRDAR (SEQ ID NO: 2), CGDFLMGRDMR (SEQ ID NO: 3), GDFLMGRD (SEQ ID NO: 4) or GDFLMG (SEQ ID NO: 5). It is.

Since the peptide of the novel amino acid sequence of the present invention is rarely degraded by other proteolytic enzymes or enzymes in saliva, but specifically degraded only by pepsin, it can be usefully used for diagnosing reflux pharyngitis. On the other hand, by using the specific degradation of the peptide by pepsin, by constructing a diagnostic kit using the peptide instead of the antibody can provide a kit for the diagnosis of reflux pharyngitis, which is economical and advantageous for long-term storage.

1 is a schematic cross-sectional view of an assay strip used for conventional immunochromatographic analysis.

Figure 2 is a schematic diagram showing the principle of operation of the peptide-based reflux laryngitis diagnostic strip according to the invention.

3 is a diagram showing peptides of an amino acid sequence specifically cleaved by pepsin according to the present invention, pepsin detection principle using the same, and degradation rate according to reaction time.

Figure 4 is a diagram showing the resolution according to the peptide length of the amino acid sequence that is specifically cleaved pepsin according to the present invention.

5 is a diagram showing the resolution according to the pepsin concentration for the pepsin-specific amino acid sequence of the present invention.

6 is a diagram showing pepsin specific degradation of other enzymes according to the present invention.

7 is a complex for detecting pepsin comprising an oligonucleotide for selective capture with a peptide of the amino acid sequence specifically cleaved pepsin according to the present invention, gold nanoparticles as a color marker, and a peptide fragment digested by pepsin And (B) shows pepsin specific resolution of the complex.

Figure 8 shows the structure of (A) a pepsin specific peptide functionalized with maleimide for selectively detecting a specific fragment when the peptide to be specifically degraded by pepsin according to the present invention, (B) of the peptide Figures show the specific binding force upon functionalization and (C) optimization of pH for the selective binding.

9 is a diagram showing pepsin detection by an assay strip using pepsin specific peptide bond degradation properties according to the present invention. Specifically, the strip was composed of a test line that selectively captures peptide fragments degraded by pepsin and a control line that captures undigested peptides. (A) and (B) show the results of applying the experimental group treated with pepsin and the comparative group not treated with pepsin to pepsin specific peptide complexes labeled with fluorescent dyes as colorimetric markers, respectively, on the assay strip. Furthermore, (C) shows the results of optimizing the peptide concentration for the preparation of gold nanoparticle-pepsin specific peptide complex, and (D) shows the resolution by pepsin of the gold nanoparticle-pepsin specific peptide complex. In addition, (E) and (F) are the results of applying the experimental group treated with pepsin and the comparative group not treated with pepsin to the gold nanoparticle-pepsin specific peptide complex containing gold nanoparticles as colorimetric markers on the assay strip, respectively. Indicates.

In order to achieve the above object, the first aspect of the present invention

A peptide specifically cleaved by pepsin, one end of which is labeled with a label and a first ligand, and the other end of which is bound to a second ligand; And

A test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, including a third ligand that specifically binds to the first ligand, and a fourth ligand that specifically binds to the second ligand. Membrane pad including a control line formed parallel to the test line spaced apart from the inspection line by a predetermined distance, and an absorption pad (absorption pad) for finally receiving a liquid sample, the membrane pad And a reflux pharyngitis diagnostic kit comprising an immunochromatography strip in which a hygroscopic pad is connected in a partially overlapping form.

The first and second ligands, and the third and fourth ligands are different from each other, and the first and fourth ligands and the second and third ligands are selected so as not to bind to each other.

For example, the immunochromatography strip included in the kit of the present invention may be a sample pad containing a liquid sample and / or a reaction pad for reacting the sample with a peptide specifically degraded by pepsin. ) May be further included. These pads may optionally be located at the front of the membrane pad, ie at the other side of the hygroscopic pad. These pads are located in the order of the sample pad, the reaction pad, the membrane pad, and the hygroscopic pad, and may be arranged so that the samples on the solution may sequentially move toward the hygroscopic pad by partially overlapping through the ends.

On the other hand, the peptide specifically degraded by pepsin included in the kit of the present invention is provided independently of the immunochromatography strip, it can be applied to the immunochromatography strip by reacting with the sample from the outside during the diagnosis using the same. . Alternatively, the immunochromatography strip may be provided as bound or adsorbed on the reaction pad included in the immunochromatography strip to react with pepsin in the sample on the reaction pad when the sample is developed. However, this is merely an example of how to implement a kit, and the scope of the present invention is not limited thereto.

The present invention applies the principle of immunochromatography, but can be configured to a diagnostic kit containing no antibody using the specific peptide resolution of pepsin, i) not degraded by other enzymes in saliva, specific by pepsin Ii) a peptide having a specific amino acid sequence of appropriate length that can be solubilized to be able to move through the assay strip by reaction and capillary action with the sample in solution. In addition, a labeling substance is introduced at one end of the amino acid sequence, and a different ligand is introduced into each of the two fragmented fragments, so that the fragment to which the labeling substance is bound is attached to the inspection line, the other fragments and the undecomposed peptides are added to the control line. By forming a test line and a control line with each corresponding ligand to be bound, it can be utilized for the diagnosis of reflux pharyngitis.

The immunochromatography used in the conventional rapid test is an analysis method that combines the principle of the immune response based on the antigen-antibody reaction and the principle of chromatography in which the sample and the reagent move along the medium by the mobile phase. The present invention utilizes the chromatographic principle that dual samples and reagents are moved along the medium by a mobile phase, but in order to avoid the use of antibodies that are difficult to produce and are prone to denaturation and have poor shelf-life, they are specific instead of antigen-antibody reactions. It is designed to utilize the binding between ligands. Specifically, the present invention utilizes the protein resolution of pepsin in detecting pepsin in saliva as a method for diagnosing reflux pharyngitis. For example, pepsin preferentially cleaves bonds between successive hydrophobic peptides rather than cleaving at random positions in the amino acid sequence. Accordingly, the inventors have discovered a peptide consisting of an amino acid sequence that is specifically degraded by pepsin compared to other proteolytic enzymes or enzymes in saliva. In addition, as described above, in order to detect such a phenomenon by specific binding between ligands, the present invention introduces different ligands into two fragments formed by cleaving the peptide, respectively, The kit is configured to identify whether the peptide is degraded by forming a test line and a control line using the ligands that bind to each other.

The term "labelled substance" of the present invention means a substance that generates a signal that can be detected by the naked eye or by using a sensor. The labeling material may include latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, or magnetic particles, but is not limited thereto. In addition, the signal may be generated by an intrinsic property of the labeling material such as light emission, or may be generated by external stimulation such as fluorescence.

The term "ligand" of the present invention means a substance that specifically binds to each other. For example, an antibody that specifically binds to an antigen, a ligand that specifically binds to a specific receptor, a DNA pair of complementary sequences that specifically hybridize with each other, and the like act as ligands. In addition, the ligand in the present invention may be used without limitation so long as it exhibits the above-defined properties. As used throughout this specification, a ligand means a substance that specifically binds to each other as defined above, unless specified as a ligand that specifically binds to a specific receptor. For example, the first ligand may be a maleimide group, and the third ligand may be a material including a thiol group specifically bound thereto, for example, biotin including a cysteine having a thiol group, but is not limited thereto. For example, the second ligand may be biotin, wherein the fourth ligand may be streptavidin specifically bound thereto, but is not limited thereto. As another example of the first ligand, the third ligand, and the second ligand and the fourth ligand pair, a material that enables selective binding of histidine and nickel may be used, but is not limited thereto. Another example of the first ligand and the third ligand pair specifically binding thereto may be, but is not limited to, a single stranded DNA pair capable of hybridizing by complementary binding to form a double stranded DNA.

On the other hand, the peptide specifically degraded by pepsin included in the reflux laryngitis diagnostic kit of the present invention is a site for introducing a ligand lysine (lysine; Lys; K) or cysteine (cysteine) having a reactive functional group at its terminal Cys; C), but is not limited thereto. In a specific embodiment of the present invention, lysine was included at the 3 'end of the peptide to introduce biotin as the second ligand.

The test line selectively captures the labeling substance formed by the decomposition of the peptide contained in the sample pad and the first ligand bound fragment, thereby confirming the presence or absence of a degradation reaction by pepsin, that is, whether pscine is present in the sample. It is formed to, and may be configured to include a third ligand specifically binding to the first ligand for such selective capture.

On the other hand, the control line is formed to capture the other fragment that does not contain a labeling substance and / or unreacted peptides, ie undecomposed peptides among the peptide fragments digested after the reaction of the peptide contained in the sample pad with pepsin For such capture, the other end of the peptide to which the labeling substance is bound is bound to a second ligand, and the control line may be configured to include a fourth ligand that can specifically bind thereto. The control line may be a reference for determining whether the kit operates normally. Specific working principle will be described later.

The peptide specifically degraded by pepsin may comprise two hydrophobic amino acids located consecutively, selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. The two amino acids may be the same or different from each other. By including a moiety consisting of the two hydrophobic amino acids, a cleavage site for pepsin can be provided. Furthermore, for development into the solution phase, it may further comprise polar and / or positive or negatively charged amino acids, the peptides being, for example, 6 to 20 amino acids in total, specifically 6 to 15 amino acids. It may consist of amino acids, but is not limited thereto. For example, the peptide specifically cleaved by pepsin may include -FL- as a cleavage site by pepsin, and may be a peptide having a sequence further comprising one or more amino acids in its sock end. For example, the peptide specifically cleaved by pepsin may include an amino acid sequence of SEQ ID NO: 1, ie, GD FL MGRDMR, an amino acid sequence of SEQ ID NO: 2, ie, GD FL GGRDAR, an amino acid sequence of SEQ ID NO: 3, ie, CGD FL MGRDMR, It may be a peptide of the amino acid sequence of SEQ ID NO: 4, ie GD FL MGRD, or the amino acid sequence of SEQ ID NO: 5, ie the sequence represented by GD FL MG.

In a specific embodiment of the present invention, as a cleavage site for pepsin, a peptide specifically cleaved by pepsin consisting of a total of 5 to 10 amino acids including -FL- is treated with pepsin and the degradation rate is determined according to amino acid length. The resolution was confirmed, and as a result, peptides consisting of five amino acids were hardly decomposed until 30 minutes, while other peptides were confirmed to increase in resolution as their length increased. If the length of the peptide is too short, it is hardly used for pepsin detection because it is hardly degraded by pepsin, whereas if the length of the peptide is too long, it is not economical due to excessive time, effort, and cost to synthesize it. Is disadvantageous.

For example, the diagnostic kit of the present invention may further include a sample pad and / or a reaction pad, as described above. In this case, when the reaction pad includes a peptide, a flow control membrane for delaying the flow of the sample so that the peptide can sufficiently react with the sample, a heating plate for improving the decomposition reaction rate by pepsin, or both, are added to the reaction pad. It may further include, but is not limited thereto.

Specifically, the flow control separation membrane is a typical example of adding a material such as vinyl between the membrane to thereby suppress or block the flow of the liquid sample, so that it can flow again through the membrane when removing it, in addition to hydrophobic or superhydrophobic The surface treatment may be implemented by interrupting or retarding the flow of the liquid sample on the membrane, or by additionally configuring a channel through which the fluid flows on the membrane to adjust the width or number of channels.

On the other hand, the heating plate can be configured in the heating plate using a chemical reaction in consideration of the manufacturing cost. At this time, it can be implemented by using a chemical plate, such as sodium acetate or sodium thiosulfate and a metal plate used in a hand stove. Another example may include an inexpensive electric heating film, which may be implemented by using a graphene heating film having an even heating property, but is not limited thereto.

Both of these flow control separators and heating plates may be provided at the bottom of the reaction pad to control the reaction time and / or the reaction temperature to promote peptide degradation by pepsin, thereby helping to achieve efficient reactions and / or sensitive detection.

The liquid sample used in the diagnostic kit of the present invention may contain saliva. For example, the liquid sample may use a solution obtained by diluting saliva in a buffer to adjust concentration and / or viscosity and to impart fluidity, but is not limited thereto.

As described above, the diagnostic kit of the present invention utilizes the principle of chromatography in which a mobile phase comprising an analyte moves along a medium. Therefore, the analysis using the same requires a mobile phase for moving the sample containing the analyte along the strip. The buffer not only acts as a mobile phase to move the sample along the assay strip, but may also serve as a diluent to dilute the sample, if necessary. For example, conventional buffers such as phosphate buffered solution (PBS), nonionic or amphoteric surfactants, methanol, or mixtures thereof may be used as the buffer without limitation. In addition, a pH adjuster or citric acid may be further included to provide suitable conditions for the degradation reaction of pepsin. Specifically, a solution of 5% by volume and 95% by volume of methanol and 0.1 M citric acid solution may be used, but is not limited thereto.

Thus, the diagnostic kit of the present invention may be provided with a buffer as a mobile phase for dilution of a sample and / or development on a strip, but is not limited thereto.

The diagnostic kit of the present invention includes a hygroscopic pad, and the sample is transferred from a sample pad into which a liquid sample is introduced, through a reaction pad including a peptide specifically degraded by pepsin, and then to a membrane pad provided with a test line and a control line. Driving force can be provided.

In order to develop such a specimen, the series of pads were formed by using a membrane pad to move by capillary action, and at one end thereof, a moisture absorbing pad was provided to provide a driving force for transporting the specimen. The moisture absorbing pad may be positioned to partially overlap the membrane pad. The membrane may be a nitrocellulose membrane, a glass fiber membrane, a polyethersulfone (PES) membrane, a cellulose membrane, a nylon membrane, or a combination thereof. This is not restrictive.

The diagnostic kit may be additionally prepared by including a solid support at the bottom. As the solid support, a plastic support can be used, and thus, by attaching a kit to the solid support, the durability can be increased, and handling and storage can be facilitated. In addition, it is possible to facilitate additional external case mounting. As a plastic material that may be used as the solid support, a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, or the like may be used, but is not limited thereto.

The diagnostic kit may additionally be fixed in the case. The lower case may be provided with a plurality of guides and / or kit supports for positioning and securing or crimping the diagnostic kit in an appropriate position. Optionally, the guide and kit support may be provided in the upper case at a position corresponding to the guide and kit support provided in the lower case. That is, the guide and / or kit support may be formed in the lower case or both the upper case and the lower case as necessary. In addition, the upper case may be provided with a result confirmation window for detecting a signal from the labeling material at a position corresponding to the sample inlet and the inspection line. The sample inlet may be formed in the form of a hole or a slit at one end of the membrane pad, that is, at a point sufficiently spaced apart from the test line and the sample along the membrane pad at the opposite end where the hygroscopic pad is positioned based on the test line. Can be. The result confirmation window may be formed to a size sufficient to be visible from the outside or visually through the sensor, including the control line where the inspection line is located on the membrane pad and / or additionally the control line in some cases. . The size and shape may be formed without limitation as long as the inspection line and / or control line can be identified.

The upper and lower cases may be manufactured using a conventional plastic material, for example, a material such as polycarbonate, acrylonitrile butadiene styrene (ABS) may be used, but is not limited thereto. The upper and lower cases may be manufactured separately and include coupling grooves, coupling protrusions, and the like, and may be combined by conventional means, and in some cases, may be integrally manufactured.

The second aspect of the present invention comprises a first step of injecting saliva collected from a subject suspected of developing reflux pharyngitis into the membrane pad of the kit of the first aspect to develop in the direction of the hygroscopic pad to cross the test and control lines; A second step of confirming signals by the labeling substance in the inspection line and the control line; And a third step of determining positive for reflux laryngitis when the signal is detected in the test line and the control line, and negative when the signal is detected only in the control line.

For example, in the diagnostic kit of the present invention, the control line is included in the reaction pad, and the labeling substance of the peptide, which is specifically degraded by pepsin that moves toward the hygroscopic pad according to the fluid flow when the liquid sample is added, is bound to the other end not bound. A fourth ligand that specifically binds to a second ligand and is formed parallel to the test line by a predetermined distance, wherein the peptide fragment and the undecomposed peptide are decomposed by pepsin to which the second ligand is bound. As long as the peptide contains a sufficient amount of peptide in the hygroscopic pad, the control line will release the signal of the labeling substance by the binding of the undigested peptide. Therefore, if no signal is detected from the control line in the third step, it may be determined that the diagnosis is invalid. This can occur if the kit itself is poor, for example, if the amount of peptide contained in the hygroscopic pad is insufficient, the flow of liquid sample is not smooth, or the pepsin concentration in the sample is too high.

A third aspect of the present invention provides two consecutive hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan in succession, each in the sock end of the successively located hydrophobic amino acids. A first step of preparing a series of peptides including a total of 6 or more amino acids, to which amino acids have been added; And a second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming degradation by each enzyme. It provides a method of manufacturing.

In order to facilitate the degradation by the enzyme in the second step, a labeling substance may be introduced at one end of the peptide. On the other hand, in order to facilitate the separation of the two fragments cut, the peptide may be bound to a predetermined particle, and the two fragments cut from each other by a simple method of centrifugation using the weight difference by the particles. Can be separated. In this case, by designing the labeling material and the particles to be located in different fragments, by measuring the signal of the labeling material for any one of the recovered fragments, and observes the change to determine whether the peptide is cleaved by each enzyme and / or You can check the degree. However, this is only one example for performing the second step, the scope of the present invention is not limited thereto, and the second step may be performed by applying various methods known in the art.

For example, the peptide may be a peptide having an amino acid sequence that is designed to be water soluble for development onto a solution. Specifically, the peptide may further comprise polar and / or positive or negatively charged amino acids at sites other than the two consecutively located hydrophobic amino acids providing cleavage sites by pepsin, wherein the peptides are, for example, a total of 6 To 20 amino acids, specifically, 6 to 15 amino acids, but is not limited thereto.

Furthermore, the peptide can be used in a kit for diagnosing reflux throat. To this end, appropriate labeling material and / or one or more ligands can be introduced at the end of the sock and the sequence of the peptide.

A fourth aspect of the invention is GD FL MGRDMR (SEQ ID NO: 1), GD FL GGRDAR (SEQ ID NO: 2), CGD FL MGRDMR (SEQ ID NO: 3), GD FL MGRD (SEQ ID NO: 4) or GD FL MG (SEQ ID NO: 5). Peptide having an amino acid sequence of ().

As described above, the peptide of the present invention has a sequence designed to be specifically degraded by pepsin, and is only degraded by pepsin, and by amylase, lysozyme or mucin, which is an enzyme present in saliva, or a protease trypsin. Does not decompose. This suggests that by using the peptide of the above sequence, it is possible to qualitatively and / or quantitatively detect pepsin without being influenced by other enzymes contained in saliva used as a sample.

Specifically, the peptide of the present invention is degraded by pepsin to GD (SEQ ID NO: 7) and MGRDMR (SEQ ID NO: 8), GD (SEQ ID NO: 4) and GGRDAR (SEQ ID NO: 9), CGD (SEQ ID NO: 10) and MGRDMR ( SEQ ID NO: 8), GD (SEQ ID NO: 7) and MGRD (SEQ ID NO: 11), or two fragments of GD (SEQ ID NO: 7) and MG (SEQ ID NO: 12).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1 Synthesis and Separation of Peptides

To synthesize peptides of any desired sequence, they were performed according to general peptide solid phase synthesis (Wang C. Chan and Peter D. White, Fmoc Solid phase peptide synthesis, Oxiford). Specifically, amino acid monomers were coupled one by one from the C-terminus by Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) using an autosynthesizer (ASP48S, Peptron, Inc.). All monomer raw materials used for peptide synthesis are amino acids protected with Fmoc at the N-terminus and residues protected with trityl (trt), t-butyloxycarbonyl (Boc), t-butyl (t-Bu), etc. It was. As a coupling reagent, HBTU (2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate / HOBt (hydroxybenzotriazole) / NMM (N-methylmorpholine) was used.

The protected amino acid (8 equivalents) and the coupling agent HBTU (8 equivalents) / NMM (16 equivalents) were dissolved in DMF (dimethyl formamide) and added, followed by reaction at room temperature for 2 hours. Then, in order to remove Fmoc, 20% (v / v) piperidine / DMF was added and reacted twice for 5 minutes at room temperature. The two reactions were repeated to prepare peptides of the desired sequence.

Subsequently, peptide separation in the resin and amino acid protecting groups was carried out by trifluoroacetic acid (TFA) / EDT (1,2-ethanedithol) / thioanisole / TIS (triisopropylsilane) / H ₂ O (90 / 2.5 / 2.5 / 2.5 by weight). /2.5 at the ratio). The mixed solution thus prepared was treated with an excess of refrigerated diethyl ether to form a precipitate. The precipitate was centrifuged to completely precipitate the precipitate, and excess TFA, EDT, thioanisole and TIS were first removed. . The same procedure was repeated twice to obtain a solid precipitate.

The precipitate obtained was water-containing 0.1% (v / v) TFA using a high performance liquid chromatography apparatus (Shimadzu Prominence HPLC, Japan) equipped with an Ace C18 column (250 mm × 22 mm, 10 μm, UK). Purification was carried out by separation by acetonitrile linear gradient (acetonitrile concentration: 10 to 75% (v / v)) method. The molecular weight of the purified peptide was confirmed by LC / MS (Shimadzu LC / Mass-2020, Japan), and the purified fractions were lyophilized to give a white powder in the form of a TFA salt.

Example 2: Validation of Pepsin Specific Peptides

In order to confirm experimentally pepsin specific degradation of the peptide having the amino acid sequence of SEQ ID NO: 1 according to the present invention, 6-aminohexanoic acid (6 -aminohexanoic acid) synthesized a peptide linked to the phosphor, FITC and confirmed by mass spectrometry.

FITC- (6-aminohexanoic acid) -GDFLGRDMRK (biotin)

Mass Analysis

Instrument: SHIMADZU LCMS-2020

MS Expected: 2053.5

MS Found: 2053

Prepared as described above, a peptide having an amino acid sequence of SEQ ID NO: 1 labeled with a fluorescent substance FITC at one end and a biotin at the other end was prepared at a concentration of 10 μM, and reacted with pepsin at a concentration of 100 ng / mL. The reaction was carried out at 42 ℃, immediately after the start of the reaction, 5 minutes, 10 minutes and 30 minutes, using agarose beads streptavidin-bound on the surface to recover the peptide fragments with biotin. Fluorescence of FITC was measured from the recovered peptide fragments to quantify the amount of peptide cleaved by pepsin. Upon digestion with pepsin, the fragment labeled with phosphor FITC was removed by washing, and thus the degree of degradation of the peptide by pepsin was confirmed from the reduction of fluorescence. The fluorescence intensity according to the reaction time measured accordingly is shown in FIG. 3. As shown in Figure 3, about 30% of the peptide was degraded within 5 minutes after the start of the reaction, after 30 minutes it was confirmed that more than 70% degradation.

Example  3: pepsin specific Peptide  Resolution according to length

In order to confirm the difference in resolution according to amino acid length of the specifically decomposed pepsin specifically decomposed peptide according to the present invention, in the same manner as in Examples 1 and 2, respectively, GDFLMGRD (SEQ ID NO: 4), GDFLMG ( 6-aminohexanoic acid comprising 5, 6 and 8 amino acids represented by SEQ ID NO: 5) and DFLMG (SEQ ID NO: 6), 3 'terminal K with biotin linked, and 5' terminal linked to Through the synthesis of the peptide FITC is connected to the phosphor was confirmed by mass spectrometry.

FITC- (6-aminohexanoic acid) -DFLMGK (biotin)

Mass Analysis

Instrument: SHIMADZU LCMS-2020

MS Expected: 1438.21

MS Found: 1438

FITC- (6-aminohexanoic acid) -GDFLMGK (biotin)

Mass Analysis

Instrument: SHIMADZU LCMS-2020

MS Expected: 1495.23

MS Found: 1495

FITC- (6-aminohexanoic acid) -GDFLMGRDK (biotin)

Mass Analysis

Instrument: SHIMADZU LCMS-2020

MS Expected: 1766.36

MS Found: 1766

A peptide comprising a total of 10 amino acid sequences of SEQ ID NO: 1 and peptides containing 5, 6, and 8 amino acid sequences represented by SEQ ID NOs: 4, 5, and 6, respectively, prepared as described above were prepared in a solution of 10 μM concentration, respectively. After reacting it with pepsin at a concentration of 100 ng / mL for 5 minutes, 10 minutes, and 30 minutes at 42 ° C, biotin-bound peptide fragments were recovered using agarose beads having streptavidin-bound on the surface. . The fluorescence of FITC was measured from the recovered peptide fragments to determine the resolution of peptides by pepsin, and the results are shown in FIG. 4. As shown in FIG. 4, as the length of the peptide to be specifically cleaved for pepsin increases, that is, as the number of amino acids constituting it increases, the degradation rate of the peptide increases. For example, the peptide containing the 5 amino acid sequence was hardly degraded by pepsin until 30 minutes, but the peptide of the sequence represented by SEQ ID NO: 1 containing 10 amino acids was about 30% within 5 minutes after the start of the reaction. It was decomposed to a degree, and the reaction rate approached 70% when reacted up to 30 minutes.

Example  4: according to pepsin concentration Peptide  Resolution

In order to confirm the possibility of quantitative analysis according to pepsin concentration, peptide degradation was measured by a method similar to Example 2 while varying the concentration of pepsin. Peptide solutions of 5, 10, 20 and 40 μM concentrations were prepared, respectively, and reacted with pepsin solutions at 250, 500, 1,000 and 2,000 ng / mL concentrations for 5 minutes at room temperature, and the remaining amount of peptide was calculated. Shown in 10 μM of the peptide, which showed a linear change according to the concentration of pepsin, was reacted with pepsin solution at concentrations of 0, 10, 50, 100, 500 and 1,000 ng / mL for 30 minutes, and then the amount of remaining peptide was calculated. 5 together. As shown in the right side of Figure 5, when the peptide was reacted with a concentration of 10 μM, it was confirmed that the linear degradation rate according to the pepsin concentration in the concentration range of 0 to 1,000 ng / mL. This indicates that quantitative analysis of pepsin concentration is possible by measuring fluorescence signal or color change in the corresponding range.

Example  5: according to the type of enzyme Peptide  Decomposition

In order to identify the possibility of cleavage of pepsin specific peptides specifically designed according to the invention by enzymes other than pepsin and other proteolytic enzymes, trypsin (1.5 units, instead of pepsin (1.5 units, 50 ng)) 1500 ng), amylase (1.5 units, 5000 ng), lysozyme (1.5 units, 150 ng) and mucin (1000 ng) were reacted for 5 minutes and 1 hour, respectively, and then the fluorescence intensity was changed in a similar manner to Example 1. Was measured and the result is shown in FIG. As shown in Figure 6, agarose beads themselves (resin) did not fluoresce at all, when treated with pepsin compared to the fluorescence signal of the peptide itself (PEP-Only) without any enzyme, within 5 minutes Although the fluorescence intensity decreased to less than 5%, the other enzyme treatment showed only a decrease of less than 15% within 5 minutes and a maximum reduction of 35% even after 1 hour. Since the peptide having the amino acid sequence of SEQ ID NO: 1 designed according to the present invention is a sequence that is specifically cleaved by pepsin, it is possible to qualitatively and quantitatively detect pepsin in a sample without interference of other sequences. To indicate.

Example  6: degraded by pepsin Peptide  For selective joining of fragments Oligonucleotides Preparation of Peptide Complexes and Evaluation of Specific Degradation Performance by Pepsin

Synthesis of pepsin specific peptide complexes having oligonucleotides bound at one end thereof in order to utilize hybridization of complementary DNA as a method for selectively detecting fragments digested by pepsin of a pepsin specific peptide designed according to the present invention It was prepared using a post-synthetic coupling method. As the pepsin specific peptide, a peptide having the amino acid sequence of SEQ ID NO: 3 was used.

Step 1 : First, considering the G / C content of the sequence and secondary structures such as hairpins and self-dimers, the pre-validation using the OligoAnalyzer 3.1 program (IDT, Inc.) and the result To reflect the oligonucleotide was prepared. The length of the oligonucleotide was set to 21mer, a thiol group was introduced at the 5 'end, and a maleimide group was introduced at the 3' end. Oligonucleotides having functional groups at both ends were purified by PAGE (polyacrylamide gel electrophoresis) and confirmed by MALDI-TOF QC. The prepared oligonucleotide sequence is 5 '-[thioC6] -AAg gAg ggg gAg AAg TgA ggT- [maleimide] -3' (SEQ ID NO: 13), which is a 5 'as a functional group for binding to the gold nanoparticles as colorimetric markers. It contains a thiol group at the terminal and is designed to bind C (cysteine) at the pepsin specific peptide 5 'terminal through a maleimide group at the 3' end.

Step 2 : Next, in order to bind the oligonucleotide having a functional group to the sock end obtained from Step 1 with the peptide having the amino acid sequence of SEQ ID NO: 3, the oligonucleotide and the peptide were neutralized at a concentration of 1: 2 ( 100 mM KH 2 PO 4, pH 7.2) for at least 4 hours at room temperature. The conjugate of the obtained oligonucleotide and peptide was electrophoresed on a 10% TBE-urea polyacrylamide gel and stained with oligonucleotide with methylene blue to confirm the location of the conjugation band on the gel. Based on this, the band was cut from the gel and the conjugate was extracted by the Crush and Soak technique. The concentration of oligonucleotide-peptide conjugate extracted with nanodrops was calculated.

Step 3 : Finally, tris (2-chloroethyl) phosphate (TCEP) reducing beads (-SS-) contained in the 5 'end of the oligonucleotide in the oligonucleotide-peptide conjugate obtained from step 2 are reduced. bead) was reduced to a thiol group (-SH) to bind the gold nanoparticles of the color marker (Fig. 7 (A)).

In order to determine whether the oligonucleotide-peptide complex specifically designed according to the present invention is specifically degraded by pepsin, the oligonucleotide-peptide complex containing gold nanoparticles prepared according to steps 1 to 3 was introduced in 2,000. Reacted with pepsin at ng / mL concentration. The reaction was performed at 42 ° C. for 30 minutes, and after completion of the reaction, biotin-bound peptide fragments were removed by centrifugation using agarose beads having streptavidin bound to the surface. Thereafter, the absorbance of the supernatant was measured to quantify gold nanoparticles to evaluate degradation performance by pepsin. Specifically, the oligonucleotide-peptide complex into which the gold nanoparticles are introduced includes gold nanoparticles at one end and biotin at the other end. After reacting with pepsin, streptavidin is bound to agarose beads. When contacted and centrifuged, undigested oligonucleotide-peptide complexes containing biotin at one end and other fragments containing no gold nanoparticles in the complexes decomposed by pepsin are bound to the beads and precipitated, and the supernatant Since only the fragments containing gold nanoparticles were included in the complex decomposed by pepsin, the degree of degradation of the peptides by pepsin was calculated by quantifying the gold nanoparticles by measuring the absorbance of the recovered supernatant. In other words, the high absorbance measured in the supernatant indicates that more peptide was degraded by pepsin.

Example  7: As ligand for the implementation of in situ diagnostic strips for pepsin detection Peptide  Experimental validation of functionalization and selective binding by the ligand

In order to implement a diagnostic strip that can be used for actual in situ diagnosis using a pepsin specific peptide designed according to the present invention, the amino acid of SEQ ID NO: 2 so that fragments containing colorimetric markers of peptides degraded by pepsin can be identified on a test line. Maleimide groups were introduced as ligands on the side to which the colorimetric marker of the peptide having the sequence was bound. In addition, TAMRA, which can confirm color change not only with fluorescence but also with the naked eye, was introduced as a color marker. The peptide complex thus synthesized was isolated and identified as follows:

TAMRA-MiniPEG2-K (4-maleimidobutyryl) -MiniPEG2-GDFLGGRDARK (biotin);

R _t = 5.40 min, various concentration gradients over 5 minutes from 5% (v / v) to 100% (v / v) DW / acetonitrile containing 0.01% (v / v) TFA;

MS (ESI) 2412.96 m / e, [M + H] ⁺ = 2413.

Using a peptide complex functionalized with maleimide synthesized and identified as described above, a cysteine having a thiol group is included to confirm whether the peptide fragment containing the same can be selectively detected by the maleimide upon cleavage by pepsin. Experiment using the prepared biotin (CK-Biotin) solution as follows. First, the peptide complex of 10 μM concentration was reacted with 2,000 ng / mL pepsin solution at 42 ° C. for 30 minutes, and then, the biotin-bound peptide fragment was removed. Then, to the supernatant (pH 3 to 4) containing the peptide fragment containing TAMRA and maleimide group, a biotin solution containing cysteine having a thiol group (2 mM CK-Biotin) was added and maintained at room temperature for 20 minutes. To react with CK-Biotin through the maleimide group. CK-Biotin bound to maleimide group was collected with streptavidin-bound agarose beads, and then quantitatively analyzed binding of maleimide and CK-Biotin by measuring the fluorescence intensity of the collected pellet and the remaining supernatant, respectively. The results are shown in FIG. 8 (B). At this time, the structure of the pepsin-specific peptide complex functionalized with the maleimide used was shown in Figure 8 (A) together. As shown in FIG. 8 (B), the fluorescence intensity was strong in the pellet collected from the CK-Biotin-bound peptide fragment with streptavidin-bound agarose beads (about fluorescence measured before CK-Biotin binding). 89% level). On the other hand, there was little fluorescence from the supernatant from which the peptide fragment bound to CK-Biotin was removed (about 5.6% of the fluorescence measured before CK-Biotin binding). This indicates that the peptide fragment degraded by pepsin effectively bound CK-Biotin through the functionalized maleimide group.

On the other hand, in order to minimize the measurement error caused by the false-positive signal due to the binding of the functionalized peptide complex with CK-Biotin, which is not degraded by pepsin, the functionalized peptide complex which is not degraded by pepsin while changing the reaction conditions The binding force of CK-Biotin was confirmed. First, as described above, the peptide complex functionalized with maleimide at a concentration of 10 μM was allowed to react with streptavidin-bound agarose beads for 10 minutes at room temperature. After the addition of the CK-Biotin solution capable of binding to the maleimide group was further reacted. At this time, the pH of the reaction solution was adjusted to 2 (0.1 M citric acid solution), 4 (1 × PBS) and 7 (1 × PBS), respectively, for 10 minutes at room temperature, and then streptavidin-coupled agar was added. After collecting the loss beads, the degree of binding was confirmed by measuring the fluorescence of the recovered beads, and the results are shown in Fig. 8C. As shown in FIG. 8 (C), the degree of binding of the maleimide group to the CK-Biotin was different depending on pH. The pH of the maleimide and CK-Biotin was increased from about 31% at pH 7 to about 7% at pH 2. The binding force was shown to decrease pH dependent. This indicates that only peptide fragments degraded by pepsin can be selectively detected by performing a reaction with CK-Biotin at low pH, and considering the use of citric acid solution at pH 2 in saliva collection as a sample for diagnosis, This suggests that the collected saliva can be used directly for diagnosis without further treatment.

Example  8: In situ diagnostic strips for pepsin detection

In order to check whether the presence of pepsin in the sample can be detected by using a diagnostic strip comprising pepsin-specific peptide complex functionalized at both ends functionalized according to Example 7 of the present invention, a thiol group on the nitrocellulose membrane The pepsin detection strip was briefly constructed by immobilizing cysteine-containing biotin or cysteine amino acid aggregates on the test line and streptavidin on the control line.

First, a peptide having an amino acid sequence of SEQ ID NO: 2, at which one end was labeled with TAMRA, which is a phosphor and visually identifiable red, and a maleimide group was introduced, and at which the other end was biotin bound, was prepared at a concentration of 2 mM. The experimental group reacted the peptide modified at both ends with pepsin at a concentration of 60 μg / mL, and then flowed into a new strip. In the comparative group, the non-reacted pepsin was allowed to flow into the strip. The strips of the experimental group and the comparative group were visually compared and observed, and the results are shown in FIGS. 9 (A) and (B), respectively. As shown in Figure 9 (A) and (B), the comparison group that did not react with pepsin turned red only in the control region (Fig. 9 (B)), the test line for the experimental group developed by reacting with pepsin And it was confirmed that both the control line turned red. This indicates that the pepsin specific peptide complex devised according to the present invention can be used to detect the presence of pepsin in the sample together with the strip of the above-described configuration.

Next, to determine whether pepsin was detected using a strip including gold nanoparticles and pepsin-specific peptide complexes as colorimetric markers, first, the concentration of pepsin-specific peptides to be bound to gold nanoparticles was optimized. Specifically, pepsin specific peptides having the amino acid sequence of SEQ ID NO: 2 designed according to the present invention were prepared at concentrations of 3.5, 7, 14, 28 and 56 μg / mL, and were prepared using gold nanoparticles (80 nm in diameter, NHS-activated). Gold nanoparticles, OD20), were covalently coupled to the gold nanoparticles-pepsin specific peptide complex. Gold nanoparticle-pepsin-specific peptide complex (OD 0.6) prepared as described above was shed on the streptidine-immobilized strep, visually observed and quantitatively displayed using the image J software. It is shown in FIG. 9 (C). As shown in FIG. 9 (C), the gold nanoparticle-pepsin specific peptide complex prepared by reacting with a pepsin specific peptide solution at a concentration of 14 μg / mL showed the largest color change. This indicates that pepsin specific peptides bind most efficiently with gold nanoparticles of 80 nm diameter at 14 μg / mL to form gold nanoparticle-pepsin specific peptide complexes.

In order to evaluate the degree of peptide degradation according to the pepsin concentration of the gold nanoparticles-pepsin specific peptide complex prepared as described above, it was reacted with pepsin at 0, 0.5 and 1 μg / mL concentration, respectively. The reaction was carried out at 42 ° C. for 30 minutes, and after the reaction was completed, the solution was flowed onto the streptavidin-fixed strip and the result is shown in FIG. 9 (D). As shown in FIG. 9 (D), as the pepsin concentration was increased, the degree of change to red decreased, which means that the gold nanoparticles bound to the streptavidin immobilized on the strip as the degradation of the peptide by pepsin increased. This is due to the decrease in the color change due to the decrease in the amount of the pepsin specific peptide complex, from which it can be primarily confirmed that the complex is effectively degraded by pepsin.

For the gold nanoparticle-peptin specific peptide complex prepared according to the present invention, specifically, a peptide complex having an amino acid sequence of SEQ ID NO: 2, in which gold nanoparticles are bonded at one end and biotin at the other end (OD). 0.6) and 50 μg / mL pepsin concentration of the experimental group and the non-pepsin reaction group was flown to the strip, respectively. Strips of the experimental group and the comparative group were visually compared and observed, and the results are shown in FIGS. 9 (E) and (F), respectively. As shown in Fig. 9 (E) and (F), it was confirmed that only the test line and the control line turned red in the case of the experimental group developed by reacting with pepsin. This indicates that the pepsin specific peptide complex devised according to the present invention can be applied to strips of the above-described configuration and used to detect the presence of pepsin in the sample.

Claims (14)

1. A peptide specifically cleaved by pepsin, one end of which is labeled with a label and a first ligand, and the other end of which is bound to a second ligand; And

   A test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, including a third ligand that specifically binds to the first ligand, and a fourth ligand that specifically binds to the second ligand. Membrane pad including a control line formed parallel to the test line spaced apart from the inspection line by a predetermined distance, and an absorption pad (absorption pad) for finally receiving a liquid sample, the membrane pad And a reflux pharyngitis diagnostic kit comprising an immunochromatography strip in which a hygroscopic pad is connected in a partially overlapping form.

   And wherein the first and second ligands, and the third and fourth ligands are different from each other, and the first and fourth ligands and the second and third ligands are selected so as not to bind to each other.
2. The method of claim 1,

   And wherein said peptide specifically cleaved by pepsin comprises two hydrophobic amino acids sequentially positioned, selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan.
3. The method of claim 1,

   The peptide that is specifically degraded by pepsin is a hydrophobic amino acid including phenylalanine and leucine, and a kit that will include a total of 6 or more amino acids, including 1 to 10 amino acids each in its sockdan.
4. The method of claim 1,

   The immunochromatography strip further comprises a sample pad, a reaction pad, or both.
5. The method of claim 4, wherein

   The reaction pad further comprises a flow control membrane, a heating plate or both.
6. The method of claim 1,

   The liquid sample is a kit containing saliva.
7. A first step in which saliva collected from an individual suspected of developing reflux pharyngitis is put into a membrane pad of the kit of any one of claims 1 to 6 and deployed in a direction of a hygroscopic pad to cross a test line and a control line;

   A second step of confirming signals by the labeling substance in the inspection line and the control line; And

   And a third step of determining positive for reflux laryngitis when a signal is detected in a test line and a control line, and negative when a signal is detected only in a control line.
8. The method of claim 7, wherein

   And determining that the diagnosis is invalid when no signal is detected from the control line in the third step.
9. Two hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan are consecutively located, with 1 to 10 amino acids added to the sock end of the consecutively located hydrophobic amino acids, respectively. A first step of preparing a series of peptides including six or more amino acids; And

   A second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming degradation by each enzyme, wherein the peptides are specifically degraded by pepsin. Manufacturing method.
10. The method of claim 9,

    Wherein said peptide is water soluble.
11. The method of claim 9,

    The peptide is a manufacturing method that is used in the reflux laryngitis diagnostic kit.
12. A peptide having the amino acid sequence of GDFLMGRDMR (SEQ ID NO: 1), GDFLGGRDAR (SEQ ID NO: 2), CGDFLMGRDMR (SEQ ID NO: 3), GDFLMGRD (SEQ ID NO: 4) or GDFLMG (SEQ ID NO: 5).
13. The method of claim 12,

    A peptide that is specifically degraded by pepsin without being degraded by trypsin, which is a protease, or amylase, lysozyme, or mucin, which are enzymes present in saliva.
14. The method of claim 12,

    By pepsin GD (SEQ ID NO: 7) and MGRDMR (SEQ ID NO: 8), GD (SEQ ID NO: 4) and GGRDAR (SEQ ID NO: 9), CGD (SEQ ID NO: 10) and MGRDMR (SEQ ID NO: 8), GD (SEQ ID NO: 8) 7) and MGRD (SEQ ID NO: 11), or GD (SEQ ID NO: 7) and MG (SEQ ID NO: 12) two fragments.

Images