TW201815819A - Novel peptide, antibody thereof and method for evaluating risk of oral cancer wherein the peptide can be used as a biomarker for diseases related to HPV virus infection - Google Patents

Abstract

Disclosed is a novel peptide, an antibody thereof and a method for evaluating risk of oral cancer. The peptide disclosed in the invention is a THLW peptide, wherein the sequence of its amino acid is SEQ ID No.1. The THLW peptide can be used as a biomarker for diseases related to HPV virus infection, such as oral cancer, cervical cancer, and so on. Therefore, the disclosed method is able to use the THLW peptide as an antibody or prepare an antibody with the THLW peptide, so as to detect the risk of being stricken with cancer by screening a specimen provided by a sample provider.

Description

Novel peptides, antibodies and methods for assessing oral cancer risk

The present invention relates to a peptide and its use, and particularly to a novel peptide, its antibody, and a method for assessing the risk of oral cancer.

According to data from the Taiwan Ministry of Health and Welfare, the number of males suffering from oral cancer has increased year by year, and oral cancer was the fifth of the top ten causes of cancer death in Taiwan in 2013. According to statistics from the World Health Organization in 2008, about 260,000 people in the world have switched to oral cancer, and at least 120,000 deaths are related to oral cancer. For Taiwan, the high incidence of oral cancer is presumed to be related to chewing betel nut, smoking, drinking and other carcinogens in the diet. Among them, betel nut and smoking are the main causes of oral cancer. Specifically, areca nut, arecoline and added sassafras in areca are carcinogenic substances.

The initial symptoms of oral cancer are not obvious. The initial symptoms such as white spots and ulcers are often ignored by patients, so most patients delay the initial treatment time. As oral lesions continue to progress, more obvious symptoms begin to appear, such as pain, lumps, difficulty swallowing, bleeding, etc. Patients will seek medical treatment. However, most patients have advanced oral cancer when they seek medical treatment, resulting in increased difficulty in treatment. . Generally speaking, patients with oral cancer are found and treated in stages 1 to 2. The three-year survival rate is 72% and the five-year survival rate is 60%. The annual survival rate dropped to 61%, and the five-year survival rate was 30%. At present, in addition to the visual inspection and palpation by doctors, oral cancer in the early stage of clinical examination must be accompanied by pathological section and X-ray examination to confirm the diagnosis. Although oral cancer screening is actively promoted for high-risk groups suffering from oral cancer, due to the invasive screening process and the need for specialists to assist in screening, the public cannot actively participate in the screening, let alone the high-risk groups. Screening is performed regularly, so that the prevention or early treatment of oral cancer is still ineffective.

Therefore, a kit or method for predicting oral cancer risk in vitro is highly lacking in the market.

The main object of the present invention is to provide a THLW peptide, whose amino acid sequence is SEQ ID No.1. The peptide can be used as a biomarker for diseases related to HPV virus infection, such as oral cancer, cervical cancer, and the like.

Furthermore, by using the THLW peptide disclosed in the present invention as an antigen, a THLW antibody capable of specifically binding to the HPV virus E6 oncoprotein can be prepared by using the general knowledge in the technical field to which the present invention belongs.

A second object of the present invention is to provide a method for detecting the risk of suffering from oral cancer, which can be achieved in a non-invasive manner by using THLW peptide or / and anti-THLW peptide antibody as a detection molecule, thereby achieving Effectively and quickly detect whether the specimen provider is a high-risk group of patients with oral cancer.

In one embodiment of the present invention, a method for detecting the risk of oral cancer is disclosed, which uses THLW peptide to react with a specimen. When there is a reaction between the specimen and the peptide, it means that the specimen is in the specimen. Contains E6 oncoproteins against HPV virus.

In another embodiment of the present invention is a method for detecting the risk of oral cancer, comprising the following steps: (a) preparing an antibody against THLW peptide; (b) reacting a specimen with the antibody of step a, When the amount of force change between the specimen and the antibody is higher than a first predetermined value, it is shown that the specimen contains HPV virus, and the specimen provides a high risk of suffering from oral cancer; and when the specimen and When the amount of change in the acting force between the antibodies is higher than a second predetermined value, it is shown that the specimen provides a low risk of suffering from oral cancer.

The results obtained by the detection method disclosed in the embodiments of the present invention can be used to determine whether the specimen provider is a high-risk group suffering from oral cancer, and can also be used to regularly track the risk of oral cancer or detect pre-oral cancer.

And because HPV-positive oral cancer patients have a more limited range of canceration and are more sensitive to chemotherapy, clinically considered HPV-positive oral cancer patients have better treatment effects and prognosis than HPV-negative oral cancer patients. . Therefore, another object of the present invention is to provide a method for detecting and assessing the prognosis or treatment strategy of oral cancer, which uses THLW peptide or / and anti-THLW peptide antibody as a detection molecule to provide clinical Cancer patients develop treatment guidelines or auxiliary work to assess prognosis to achieve the effect of reducing medical costs and increasing cure rates.

Furthermore, the first predetermined value in step b is 100RU, and the second value is 40RU.

An embodiment of the present invention discloses a method for evaluating the prognosis or treatment policy of oral cancer, which uses THLW peptide to react with a specimen. When there is a reaction between the specimen and the peptide, it means that the specimen is in the specimen. Contains anti-HPV virus substances.

In another embodiment of the present invention is a method for assessing the prognosis or treatment strategy of oral cancer, which comprises the following steps: (a) preparing an antibody against THLW peptide; (b) performing the antibody from step a with a specimen In response, when the amount of change in the acting force between the specimen and the antibody is higher than a first predetermined value, it indicates that the specimen contains HPV virus, and the specimen provider has a poor prognosis or the treatment policy used is invalid And when the amount of change in the acting force between the specimen and the antibody is higher than a second predetermined value, it is shown that the specimen provider has a better prognosis or the treatment policy used is effective.

Based on the above test results, clinically, it is possible to simply and quickly determine whether a patient is HPV-positive and to formulate a suitable treatment plan; or the above evaluation method may be used to evaluate patients during or completing treatment to assess their prognosis or Effectiveness of treatment.

The first predetermined value in step b is 100RU, and the second value is 40RU.

In addition, the THLW peptides and / or anti-THLW peptides disclosed in the present invention can be combined with a detection tool or substrate, such as binding the peptide or antibody to a biochip or a biosensor to achieve more Quickly interpret the efficacy of test results.

Moreover, preferably, the detection system of the methods disclosed in the embodiments of the present invention can be saliva, oral tissue, and blood.

Unless otherwise defined, the meanings of the technical and scientific terms used in the description and patent application scope of the present invention are the same as those commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs. In case of conflict, the content of the present invention shall prevail.

The invention designs a novel peptide fragment: THLW peptide, the amino acid sequence of which is shown in SEQ ID No. 1 (EVYDFAFRDLCIVYRDGNP-amide), and its molecular weight is about 2290-2295Da. By using the peptide shown in SEQ ID No. 1 as an antigen and using the technique of immunological manipulation, antibodies can be prepared for detecting oral cancer. THLW peptides can be prepared through artificial synthesis or recombinant organism production platforms.

The so-called "synthetic method" refers to the well-known technology of ordinary technical persons in the technical field to which the present invention belongs, and the amino acids are sequentially connected by artificial methods to form a polypeptide. The artificial synthesis method includes chemical synthesis. Method and peptide synthesizer. The artificial synthesis method usually has the following advantages: it can conveniently change the primary structure of the peptide, add special amino acids, and modify the end of the peptide in the synthesis process. Generally speaking, chemical synthesis methods can be divided into solid phase peptide synthesis method and liquid phase peptide synthesis method. Among them, the liquid phase synthesis method must perform an extraction operation after completing the connection of each amino acid. However, since the polypeptide intermediate obtained by extraction is usually a mixture, a chromatographic purification step is still required. Therefore, the synthesis of polypeptide by liquid phase synthesis must involve tedious extraction and chromatographic purification steps. Only high-purity products can be obtained. The solid-phase synthesis method is performed in a solvent, and a peptide binding reaction is performed on solid polymer particles (or polymeric supports). In this method, the N-terminal amino acid of the desired polypeptide is first covalently bonded to the polymer particles, and then the subsequent amino acids are sequentially connected by means of specific bonding, and finally The polypeptide was synthesized. Since the polymer particles are not soluble in the solvent, the polymer particles (and the desired peptides attached to the polymer particles) and the reaction reagent need only be passed through the washing and filtering operations at the end of the reaction. And byproducts. Therefore, because solid phase peptide synthesis method does not require purification of intermediate products, it not only has better yield, but also can greatly reduce the reaction time. It also has advantages in the synthesis of long-chain peptides, which is widely used at present. The peptide synthesis method used.

The so-called "recombinant organism production platform" refers to the construction of a nucleic acid used to express a specific protein on a expression vector through biotechnology, and then transforming the recombinant expression vector into a host cell, such as E. coli, yeast Bacteria, lactic acid bacteria, etc., so that the recombinant expression vector can express the nucleic acid in the host cell to obtain the specific protein.

The so-called "technology of immune operation" refers to the antibody obtained by inducing an immune response through an antigen. Among them, the anti-system can be obtained as a multiple antibody or a single antibody. At present, there are many known techniques for preparing antibodies. For example, antibodies can be produced by driving antigens into individuals, such as mice, to induce immune reactions to occur. After confirming that antibodies have been produced, take spleen cells and myeloma After the cells undergo cell fusion, isolated antibodies can be obtained. There are also antigens that are used to infect poultry so that the yolk contains the antibodies, and then the antibodies are obtained by purification techniques.

The so-called "biosensor" refers to the use of immobilized biorecognition molecules, such as antibodies, antigens, sugars, proteins, etc., combined with signal conversion elements. After the biorecognition molecules react with the analyte, such as mass, optical, Changes in physical quantities, such as heat and charge, cause the signal conversion element to generate a detectable signal after receiving a signal that the biometric molecule has changed. Biosensors differ according to their energy conversion technology and equipment. For example, electrochemical biosensors use specific electrodes as signal conversion elements. The biosensor of the CM5 chip used in the examples of the present invention is based on the surface plasma resonance technology. It converts the change in the refractive index after the reaction between the biometric molecule and the test object into the change in the resonance angle to achieve Detect the effectiveness of the reaction between the two.

In the following, several examples are given to illustrate the efficacy of the present invention.

First of all, since HPV type 16 and 18 viruses are currently known to be associated with oropharyngeal cancer and cervical cancer, in which HPV type 16 infection of the host will cause the host cell to produce protein E6, the amino acid sequence of which is SEQ ID No. 2 is shown. E6 protein shutdown inhibits the function of tumor protein p53, causing abnormal cell proliferation and cancer formation. Therefore, in the following examples, E6 protein and its antibody related to HPV type 16 virus are used as a control group.

Example 1: Preparation of THLW peptides

To prepare THLW peptide by solid phase peptide synthesis, the steps are as follows:

Add Fomc-AM resin (0.125 mmole, 0.169 mg) to the PD-10 tube. Add 5 mL of dichloromethane to make the resin swell and repeat the reaction for 5 minutes. Add 5 mL of dimethylformamide to moisten the resin and react for 5 minutes. Repeat twice.

Add 5 mL of 30% piperidine / dimethylformamide solution and mix for 15 minutes to remove the Nα-Fmoc protecting group on the resin. Add 5 mL of dimethylformamide and mix for 5 minutes to wash away the piperidine / dimethylformamide solution remaining in the PD-10 tube. Take amino acid 0.25 mmole and react with coupling (HOBT: 0.25 mmole, 33.775 mg; HBTU: 0.25 mmole, 82.325 mg; DIEA: 87 μL) for 5 minutes, then mix and react with AM resin in the reaction tube for 2 hours. After the reaction was completed, 5 mL of dimethylformamide was added for 5 minutes, and the Ninhydrin test was used to determine whether the coupling was successful after each coupling reaction. By repeating the above steps until the amino acids of the THLW peptide are connected to the resin.

After the THLW peptide sequence is completed, add 5 mL of 30% piperidine / dimethylformamide solution to the reaction, and then add 5 mL of dimethylformamide solution to remove the N-terminus on the last amino acid of the sequence. Fmoc protecting group. Finally, the peptide was excised from the resin by chemical cleavage, the peptide was cleaved from the AM resin with a cleavage reagent (95% TFA in DDW), and the peptide side chain protecting group was excised. After filtering under reduced pressure and centrifugation, a crude peptide product was obtained.

The predetermined matrix and the crude peptide product were mixed with an equal volume. After co-crystallization, the molecular weight was analyzed by a MALDI-TOF mass spectrometer. The results are shown in the first figure. THLW The crude peptide contains the target product: THLW peptide (molecular weight: 2293.9Da). The amino acid sequence is shown in SEQ ID No.1.

Example 2: Purified crude peptide

The reverse peptide high-performance liquid chromatography was used to analyze the crude peptide obtained in Example 1. Among them, the chromatographic column used was a C18 column with a pore diameter of 10 μm, the detection wavelength was 225 nm, and the fixed flow rate was 4 mL / Min, the mobile phase solvent composition is as follows: solvent A: 4 L DDwater + 0.05% trifluoroacetic acid (TFA), and solvent B: 4 L Acetonitrile + 0.05% trifluoroacetic acid (TFA), and a small amount of TFA is added; solvent A / solvent B Content ratio: It is extracted in the manner of 90: 10 ~ 10: 90 in 30 minutes, then maintains the ratio of 10:90 for 7 minutes, and then converts to the ratio of 90:10 for 10 minutes. The results detected by the computer are shown in the second figure.

It can be seen from the second figure that the retention time of THLW peptide is about 14.02 minutes. After the solution of collecting the absorption peaks is subjected to freeze-drying treatment, the molecular weight is identified by a MALDI-TOF mass spectrometer. Among them, the operation flow is as described in Example 1, so it will not be repeated here. The molecular weight identification result is shown in the third figure, showing the purity of the purified THLW peptide and confirming its identity. Please refer to the fourth figure. The purified THLW peptide is freeze-dried, re-dissolved and analyzed by reverse-phase high performance liquid chromatography. It can be seen that there is only a signal of THLW peptide.

From the above results, it can be seen that the THLW peptide system disclosed in the present invention can be successfully synthesized and purified.

Example 3: Preparation of multiple antibodies

Let's start with the description. This example was performed according to the regulations of the Animal Care and Use Committee.

Take full-time Lai Hen chicken, and use purified THLW peptide as the antigen. Mix 150 μg of antigen with 150 μL of complete adjuvant at the ratio of 1: 1 for the first injection and shake it vigorously to make it emulsified. The mixed solution was dispersedly injected into different positions of chicken legs, and a second injection was performed after 14 days, and eggs were collected after 4 injections.

Take the yolk from the egg, add the same volume of deionized water, and mix. Add 15 mL of chloroform and mix. After centrifugation at 4000 rpm for 5 minutes, remove the supernatant, which contains THLW antibody, which can be stored at -20 ℃, or can be extracted into powder by a freeze dryer. The samples are dried and stored for subsequent examples.

Example 4: Detection of antibody titer of THLW peptide and its multiple antibodies

The purified THLW peptide (0.2 mg / mL) was fixed on a well plate, and the unbound THLW peptide was washed away. The THLW antibody prepared in Example 3 was diluted at the highest concentration of 0.2 mg / mL to 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, respectively, and the reaction between THLW peptide and antibody was detected by indirect ELASA. The results are shown in the fifth figure. From the fifth figure, it can be seen that the THLW antibody has a dose correlation with the immune response of the THLW peptide at a concentration of 0.0625 mg / mL to 0.25 mg / mL. Therefore, the THLW antibody was further serially diluted from 0.25 mg / mL to: 125, 62.5, The concentrations of 31.2515.625, 738125, 3.90625, and 1.953125 μg / mL were detected by ELISA, and the results are shown in the sixth figure.

The results of the sixth figure clearly show that the THLW antibody has a good linear relationship with the THLW peptide in the above concentration range.

Example 5: Collection of saliva samples

Saliva samples were collected from healthy subjects and oral cancer patients. Among them, any diet should be prohibited within one hour before saliva samples are collected. An enzyme inhibitor was added to the collected saliva samples, and the heart was taken at 4 ° C and 3000 rpm, and the supernatant was taken for subsequent examples.

Example 6: Determination of protein concentration

This example uses BCA analysis to determine the protein concentration in a saliva specimen.

First, a standard curve for protein concentration was established using BSA standards with final concentrations of 2, 1.5, 1, 0.5, and 0.25 mg / mL, as shown in Figure 7.

The saliva samples provided by patients diagnosed with oral cancer were diluted 1/2, and the total protein concentration in each sample was quantified by BCA analysis, as shown in Figure 8.

Example 7: Biosensor

Take a commercially available CM5 sensor chip, first activate the surface of the wafer with EDC / NHS, and then covalently bond the biometrics to the surface of the wafer at a predetermined concentration. After the attachment of the biometrics is completed, the remaining activated sites are shielded with EA . Thereby, a sensing chip is obtained.

In this example, a THLW peptide, a THLW antibody, an E6 protein, and an anti-E6 protein antibody are used as bioidentifiers to prepare a sensing chip. Before preparing the sensor wafer, test the bonding conditions with sodium acetate solutions with pH values of 3.5, 4.0, 4.5, 5.0, and 5.5. It is determined that the maximum bond amount can be obtained with a pH value of 3.5, that is, under these conditions. Perform biometric binding. In addition, in this example, the concentration of THLW peptide is 3.3 μM, the concentration of THLW antibody is 0.2 μg / mL; the concentration of E6 protein is 0.03 μg / mL; the concentration of anti-E6 protein antibody is 10 μg / mL.

Example 8: Detection with a sensor chip (1)

Saliva samples were taken from 12 subjects with oral cancer and 10 healthy subjects. Each saliva sample was diluted twice and eight times, respectively, and then reacted with the E6 protein sensor chip. The results are shown in Figures 9 and 10.

The results from the ninth figure show that substances in oral cancer saliva specimens will have a greater interaction with E6 protein, about 2300RU (Response unit), that is, the presence of saliva specimens in oral cancer subjects will interact with E6 protein interaction protein. It can be inferred that patients with oral cancer should be infected with HPV 16 virus, and antibodies against E6 protein have been produced in the body.

Furthermore, the results from the tenth graph show that although the saliva samples of healthy subjects and oral cancer subjects will increase with time, the saliva samples based on oral cancer subjects It has a substance that interacts with the E6 protein, so its increase is significantly higher than that of healthy subjects.

From the above, when a subject is infected with HPV type 16 virus and an E6 protein is detected in a saliva sample, the subject is a high-risk group suffering from oral cancer. In other words, by detecting the response of the saliva specimen, it can be inferred whether the specimen provider is at risk of developing oral cancer.

Example 9: Detection with a sensor chip (2)

Saliva samples from 24 patients with stage 4 oral cavity cancer were diluted twice, and then reacted with the THLW peptide sensor chip and the E6 protein sensor chip, respectively, and the sensor patterns were detected respectively. Figure 1 and Figure 12.

Please refer to the eleventh figure, because the substances in some saliva specimens have greater interaction with the E6 protein, it shows that these specimens have antibodies corresponding to the E6 protein. Based on this, it can be inferred that oral cancer patients who provided these specimens have a high possibility of contracting HPV type 16 virus, causing the E6 protein to generate an immune response in the body, and the presence of antibodies corresponding to the E6 protein will be detected in the specimen. .

Comparing Figure 11 and Figure 12, the interaction force between the substance in most saliva samples and THLW peptide in Figure 12 is similar to Figure 11, showing the THLW peptide disclosed in the present invention It should be able to replace E6 protein and become a biomarker for predicting the risk of oral cancer.

Example 10: Detection with sensing wafer (3)

The THLW was first mixed with the saliva sample diluted 3 times for 1 hour, and then reacted with the THLW peptide sensor chip respectively. The results are shown in FIG. 13, which shows that the THLW peptide containing the present invention was detected. The signal from the chip is stable.

It can be seen that the THLW peptides disclosed in the present invention can be effectively used as a biomarker for detecting the risk of oral cancer.

Example 11: Detection with a sensing chip (4)

The saliva samples from 24 patients were diluted 1/2, and then reacted with the THLW antibody sensor chip respectively. The detection results are shown in Figure 14.

As can be seen from the results in Figure 14, the saliva provided by patients infected with HPV type 16 virus has substances that can react with THLW antibodies, and therefore a larger interaction force with THLW antibodies will be detected.

In other words, the THLW anti-system prepared by using the THLW antigen disclosed in this case can be used to predict whether the sample provider is a high-risk population with oral cancer.

Example 12: Competitive ELISA

The purified THLW peptide (0.2mg / mL) was fixed on a well plate, and the serially diluted THLW peptide was used as an antigen, and preliminarily mixed with THLW antibody at a concentration of 125 μg / mL to compete and establish a standard competition curve. As shown in Figure 15.

From the results of the fifteenth figure, it can be seen that there is a clear concentration correlation in the concentration range of 15.625 to 62.5 μg / mL, which shows that this standard curve should be applicable to quantify the target protein in this concentration range. In other words, it can be used to convert samples Correct antigen content.

Example 13: Affinity analysis of THLW antibodies

The THLW antibody and anti-E6 monoclonal antibody diluted in sequence concentration were used as analytes, the highest concentration was 10.00 μg / mL, and the sequence was diluted to 5.00 μg / mL, 3.33 mg / mL, 2.50 mg / mL, 2.00 mg / mL and After the concentration of 1.66 μg / mL, it interacted with the E6 protein fixed on the wafer and detected it. The results are shown in Figure 17 and Figure 16. Then, a "1: 1 binding" kinetic analysis was performed, and the results are shown in Tables 1 and 2 below.

Table 1: Kinetic analysis of E6 protein-immobilized sensor chip and different concentrations of anti-E6 monoclonal antibodies

Table 2: Kinetic analysis of E6 protein-immobilized sensor chip and THLW antibodies at different concentrations

From the results of the sixteenth and seventeenth graphs, it can be known that both the anti-E6 monoclonal antibody and THLW antibody will change the RU, and as the concentration of the antibody increases, ΔRU also tends to increase.

Furthermore, the highest ΔRU and its concentration reached when the wafer was passed through the anti-E6 monoclonal antibody or THLW antibody at various concentrations were analyzed, and the results are shown in Figures 18 and 19, and the results of the affinity analysis were as follows: Table III shows.

Table 3: Affinity analysis between sensor chip with E6 protein immobilized and antibodies with different concentrations

From the above results, it can be seen that the anti-THLW peptide system disclosed in the present invention has good affinity with HPV virus, and the effect is similar to that of the anti-E6 monoclonal antibody. Therefore, the THLW anti-system disclosed in the present invention can be used to detect oral cancer Risk tool.

Example 14: THLW antibody knot and force analysis

Samples from non-oral cancer patients (11) and oral cancer patients (5) were collected and grouped according to the following conditions: the first group was a blank group with only phosphate buffer solution; the second group was The non-oral cancer patients 'specimens were diluted 1/2 with phosphate buffer; the third group was the oral cancer patients' specimens were diluted 1/2 with phosphate buffer; the fourth group was the oral cancer patients. The specimens were added with an equal volume of 0.01 μg / mLE6 protein; the fifth group was the specimens of oral cancer patients were added with an equal volume of 0.16 μg / mLE6 protein; the sixth group was the specimens with oral cancer patients were added with an equal volume of 0.5 μg / mLE6 protein.

The THLW antibody and the anti-E6 single antibody were fixed on the wafer, respectively, and used to test the RU value change and induction results of each group of samples. The results are shown in Figures 20 to 23.

From the results of the twentieth graph to the twenty-third graph, it can be known that when the anti-E6 monoclonal antibody or THLW antibody is used for detection, the RU change value of the second group is significantly lower than the RU change value of the third group, that is, The THLW antibody disclosed by the present invention can indeed determine the risk of oral cancer of a subject provider through the change of the RU value. Furthermore, from the results of the fourth group to the sixth group of the above results, it can be known that when the anti-E6 protein antibody is designed on the wafer, the change in the HPV virus content in the specimen is less detectable, and the detected RU The value change value is similar to the second group, so it is easy to cause misjudgment of the risk assessment of oral cancer. Relatively speaking, the THLW antibody disclosed by the present invention still has a detection effect on the change in the amount of subtle virus in the specimen, that is, the present invention The THLW antibody has higher sensitivity and specificity than the anti-E6 monoclonal antibody system.

In addition, from the above results, it can be known that when the detected RU change value is less than 40, the risk of oral cancer of the sample provider is low, and when the detected RU change value is higher than 100, the sample provider is infected with HPV virus. The chance of infection is high and therefore carries a high risk of developing oral cancer.

From the results of the above examples, it can be seen that the THLW peptide shown in SEQ ID No. 1 disclosed in the present invention can be used as a biomarker for detecting diseases related to HPV virus infection. Furthermore, the THLW peptide and anti-THLW peptide The resistance system can be used as a tool to detect the risk of oral cancer in vitro. In addition, by combining the THLW peptides and / or THLW antibodies disclosed in the present invention with detection tools such as biosensors, biochips, or techniques such as immunoenzyme analysis, it can be clinically simple and rapid to determine the risk of oral cancer. The efficacy of cancer risk.

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The first figure is the mass spectrum of the crude THLW peptide. The second figure is the RP-HPLC chromatogram of the crude THLW peptide. The third figure is the mass spectrum of the purified THLW peptide. The fourth figure is a RP-HPLC chromatogram of the purified THLW peptide. The fifth graph is the result of detecting the reaction between THLW peptide and THLW antibody. The sixth graph is the result of analyzing the reaction between THLW peptide and THLW antibody. The seventh graph is a standard curve of protein concentration established with BSA standards. The eighth graph is the result of measuring the total protein concentration in each saliva sample. The ninth figure is the result of detecting the response of each saliva sample by a two-fold dilution using an E6 protein sensor chip. The tenth figure is the result of detecting the response of each saliva sample by 8-fold dilution using an E6 protein sensor chip. The eleventh figure is the result of the two-fold dilution of each saliva sample with the E6 protein sensor chip. The twelfth figure is the result of the two-fold dilution of each saliva sample with the THLW peptide sensor wafer. The thirteenth figure is the result of reaction of each saliva sample with THLW peptide sensor wafer after 2 times dilution. The fourteenth figure is the result of the two-fold dilution of each saliva sample with the THLW antibody sensing wafer. The fifteenth figure is a standard competition curve established using THLW peptide as an antigen. The sixteenth figure is the SPR induction spectrum of the E6 protein-immobilized wafer and different concentrations of anti-E6 monoclonal antibodies. The seventeenth figure is the SPR induction spectrum of E6 protein-immobilized wafers and THLW antibodies at different concentrations. The eighteenth figure is the result of the reaction of the E6 protein-immobilized wafer with different concentrations of anti-E6 monoclonal antibodies. The nineteenth figure is the result of reacting the E6 protein-immobilized wafer with THLW antibodies at different concentrations. The twentieth graph is the analysis of the binding force between the anti-E6 monoclonal antibody and the saliva specimen. The twenty-first graph is the change of RU value after the reaction between the anti-E6 monoclonal antibody and different saliva samples. The twenty-second figure is an analysis of the binding force between the THLW antibody and the saliva specimen. The twenty-third figure is the change of RU value after the reaction between THLW antibody and different saliva samples.

<110> Taichung Rongmin General Hospital, Tokai University <120> Novel peptides, antibodies, and methods for assessing oral cancer risk <130> 1 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 19 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial Sequence ＜ 220 ＞ ＜ 223 ＞ Artificial Design ＜ 400 ＞ 1 Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp 1 5 10 15 Gly Asn Pro

Claims (19)

A THLW peptide, whose amino acid coding is SEQ ID No.1. According to the THLW peptide described in item 1 of the scope of the patent application, it is a biomarker of HPV virus-related diseases. The THLW peptide according to item 2 of the scope of the patent application, wherein the HPV virus-related disease is oral cancer. A method for detecting the risk of suffering from oral cancer, which uses the THLW peptide described in the first patent application to react with a specimen. When there is a reaction between the specimen and the peptide, it means that the specimen contains anti- HPV virus substance. The method according to item 4 of the scope of patent application, wherein the THLW peptide is bonded to a biochip. The method according to item 4 of the scope of the patent application, wherein the detection system is selected from the group consisting of saliva, oral tissue and blood. A method for detecting the risk of suffering from oral cancer, comprising the following steps: Step a: preparing an antibody against THLW peptide as described in item 1 of the scope of patent application; step b: reacting the antibody of step a with a specimen, when When the amount of change in the acting force between the specimen and the antibody is higher than a first predetermined value, it is shown that the specimen contains HPV virus, and the specimen provides a high risk of suffering from oral cancer; and when the specimen and the When the amount of change in the acting force between the antibodies is higher than a second predetermined value, it is shown that the specimen provides a low risk of suffering from oral cancer. The method according to item 7 of the patent application scope, wherein the antibody system is bonded to a biochip. The method according to item 7 of the scope of patent application, wherein step b is the analysis of the reaction between the antibody and the specimen by immunoenzyme analysis. The method according to item 7 of the scope of patent application, wherein the first predetermined value in step b is 100RU and the second value is 40RU. The method according to item 7 of the scope of patent application, wherein the detection system is selected from the group consisting of saliva, oral tissue and blood. A method for assessing the prognosis or treatment strategy of oral cancer, which uses the THLW peptide described in the first patent application scope to react with a specimen. When there is a reaction between the specimen and the peptide, it means that the specimen is in the specimen. Contains anti-HPV virus substances. The method according to item 12 of the patent application, wherein the THLW peptide is bonded to a biochip. The method according to item 12 of the scope of the patent application, wherein the detection system is selected from the group consisting of saliva, oral tissue and blood. A method for assessing the prognosis or treatment strategy of oral cancer, comprising the following steps: Step a: preparing an antibody against the THLW peptide described in item 1 of the scope of patent application; Step b: reacting the antibody of Step a with a specimen When the amount of change in the acting force between the specimen and the antibody is higher than a first predetermined value, it is shown that the specimen contains HPV virus, and the specimen provider has a poor prognosis or the treatment policy used is invalid; And when the amount of change in the acting force between the specimen and the antibody is higher than a second predetermined value, it indicates that the specimen provider has a better prognosis or the treatment policy used is effective. The method according to item 15 of the patent application, wherein the antibody system is bonded to a biochip. The method according to item 15 of the scope of patent application, wherein step b is the analysis of the reaction between the antibody and the specimen by immunoenzyme analysis. The method according to item 15 of the scope of patent application, wherein the first predetermined value in step b is 100RU and the second value is 40RU. The method according to item 15 of the scope of patent application, wherein the detection system is selected from the group consisting of saliva, oral tissue and blood.