Nasopharynx cancer antibody marker and application thereof

Abstract

The invention provides a nasopharynx cancer antibody marker and application thereof. Specifically, the invention provides application of the nasopharyngeal carcinoma antibody marker or the detection reagent thereof, the nasopharyngeal carcinoma antibody marker or the detection reagent thereof is used for preparing a diagnostic reagent or a diagnostic kit, and the diagnostic reagent or the diagnostic kit is used for judging the occurrence risk of nasopharyngeal carcinoma. The nasopharyngeal carcinoma antibody marker comprises the following antibody combination: (B) an EBV antibody; (B1) BKRF1-IgA; (B2) BMRF1 (BMRF1-IgA); and (B3) a BALF2-IgA, and (B4) a And (A) an autoantibody comprising any one of the autoantibodies selected from the group consisting of: (A1) anti-TPI1, or a combination thereof; (A2), anti-PRKACA (Pseudomonas And (A3) an anti-TRIM21 (Triglyceride- The nasopharyngeal carcinoma antibody marker can be used as a marker for judging nasopharyngeal carcinoma, and has high sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to nasopharyngeal carcinoma antibody markers and their applications. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor originating from the mucosa of the nasopharynx and prone to occur in the pharyngeal recess. The disease has a narrow onset location, insidious onset, and atypical early symptoms. Most patients are diagnosed at the middle and late stages when they seek medical treatment, missing the best treatment opportunity.

[0003] The clinical manifestations of nasopharyngeal carcinoma are not obvious, including otological diseases, neck masses, nasal problems, and headaches. Its anatomical isolation leads to delayed diagnosis and treatment, and patients start seeking medical treatment 6 weeks after the symptoms appear.

[0004] It is reported that the misdiagnosis rate of nasopharyngeal carcinoma patients is as high as 43.4%. Nasopharyngoscopy combined with biopsy pathological analysis is still the current gold standard for clinical nasopharyngeal carcinoma detection. However, this method is invasive and not suitable for asymptomatic individuals. Therefore, it is necessary to develop biomarkers for screening asymptomatic populations.

[0005] Currently, the pathogenesis of nasopharyngeal carcinoma is still unclear. Reports have pointed out that it may be related to multiple factors, such as genetic susceptibility, diet, environment, and Epstein - Barr virus (EBV) infection, etc. In 2011, EBV double - antibodies were included in the "Technical Plan for Cancer Early Diagnosis and Early Treatment Project". The results of large - scale randomized controlled cohort studies have shown that screening with double - antibodies can significantly improve the early diagnosis rate and survival rate of nasopharyngeal carcinoma patients and reduce the death risk of nasopharyngeal carcinoma.

[0006] However, the positive diagnosis rate of the double - antibody program is only about 5%. In addition, in high - incidence areas of nasopharyngeal carcinoma, 95% of nasopharyngeal carcinoma incidences are related to EBV infection. However, in low - incidence areas, only 20% of nasopharyngeal carcinoma incidences are related to EBV infection. Therefore, if only EBV antibodies are detected, nasopharyngeal carcinoma will be missed.

[0007] Therefore, it is necessary to develop more specific markers to improve the accuracy of screening and the economic benefits of nasopharyngeal carcinoma screening. Summary of the Invention

[0008] The present invention provides early - stage self - antibody markers for nasopharyngeal carcinoma with high sensitivity and high specificity.

[0009] One object of the present invention is to provide a biomarker for nasopharyngeal carcinoma, which is an antibody combination (including self - antibodies).

[0010] Another object of the present invention is to provide a reagent for detecting the antibody combination; and to provide the use of the antibody combination or the detection reagent in the preparation of products for predicting the risk of suffering from nasopharyngeal carcinoma, screening, prognostic evaluation, monitoring of treatment effect or recurrence monitoring, etc. The antibody combination is a combination of tumor-related autoantibodies and anti-EBV antibodies.

[0011] Another object of the present invention is to provide a kit and a method accordingly for predicting the risk of suffering from nasopharyngeal carcinoma, screening, prognostic evaluation, monitoring of treatment effect or recurrence monitoring, etc.

[0012] In the first aspect of the present invention, there is provided the use of a nasopharyngeal carcinoma antibody marker or its detection reagent for preparing a diagnostic reagent or a diagnostic kit for judging the occurrence risk of nasopharyngeal carcinoma; wherein, the nasopharyngeal carcinoma antibody marker comprises the following antibody combination: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, the autoantibodies comprising any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21, or a combination thereof.

[0013] In another preferred embodiment, the autoantibodies further comprise any one autoantibody selected from the group consisting of: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3, or a combination thereof.

[0014] In another preferred embodiment, the nasopharyngeal carcinoma antibody marker is an antibody combination selected from the group consisting of: (Z1) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A1) anti-TPI1; (Z2) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A2) anti-PRKACA; (Z3) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A3) anti-TRIM21; (Z4) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; and (A3) anti-TRIM21; (Z5) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; and (A2) anti-PRKACA; (Z6) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A2) anti-PRKACA; and (A3) anti-TRIM21; (Z7) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; and (A3) anti-TRIM21; (Z8) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; and (A5) anti-BMI; (Z9) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A3) anti-TRIM21; and (A6) anti-PDE4DIP; (Z10) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A7) anti-GATA3; (Z11) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A4) anti-SOX2; (Z12) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A5) anti-BMI; (Z13) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A6) anti-PDE4DIP; (Z14) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A7) anti-GATA3.

[0015] In another preferred embodiment, the autoantibody is IgA (such as IgA1, IgA2), IgM or IgG (such as IgG1, IgG2, IgG3, IgG4).

[0016] In another preferred embodiment, the diagnostic reagent or diagnostic kit is used to detect the level of the nasopharyngeal carcinoma antibody marker in a sample to be tested.

[0017] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

[0018] In another preferred embodiment, the tissue or cells are nasopharyngeal carcinoma tissue or cells, or adjacent tissue or cells of nasopharyngeal carcinoma.

[0019] In another preferred embodiment, the sample to be tested is derived from the following subjects to be tested: mammals, preferably primate mammals, more preferably humans.

[0020] In another preferred embodiment, the nasopharyngeal carcinoma includes: EBV-positive (EBV+) nasopharyngeal carcinoma and EBV-negative (EBV-) nasopharyngeal carcinoma.

[0021] In another preferred embodiment, the nasopharyngeal carcinoma includes: nasopharyngeal non-keratinizing carcinoma, nasopharyngeal keratinizing squamous cell carcinoma, and nasopharyngeal adenocarcinoma.

[0022] In another preferred embodiment, the nasopharyngeal carcinoma includes nasopharyngeal carcinoma at the following pathological stages: stage I, stage II, stage III, and stage IV; preferably stage I and stage II.

[0023] In a second aspect of the present invention, a kit is provided, the kit contains a detection reagent, and the detection reagent is used to detect a nasopharyngeal carcinoma antibody marker; wherein, the nasopharyngeal carcinoma antibody marker includes the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, said autoantibodies comprising any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21, or a combination thereof.

[0024] In another preferred embodiment, said autoantibodies further comprise any one autoantibody selected from the group consisting of: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3, or a combination thereof.

[0025] In another preferred embodiment, said detection reagent comprises: a specific antigen or specific binding molecule for said nasopharyngeal carcinoma antibody marker.

[0026] In another preferred embodiment, said detection reagent may be a reagent for enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, protein / peptide chip assay, immunoblotting, bead immunoassay or microfluidic immunoassay.

[0027] In another preferred embodiment, said detection reagent is used to detect said nasopharyngeal carcinoma antibody marker through an antigen-antibody reaction (e.g., by ELISA or fluorescence or chemiluminescent immunoassay).

[0028] In another preferred embodiment, said detection reagent comprises: (b) EBV antigens: BKRF1, BMRF1 and BALF2; and (a) antigens selected from the group consisting of: TPI1, PRKACA, TRIM21, SOX2, BMI, PDE4DIP, GATA3, or a combination thereof.

[0029] In another preferred embodiment, said detection reagent comprises the following antigens: BKRF1, BMRF1, BALF2, TPI1, PRKACA and TRIM21.

[0030] In another preferred embodiment, said detection is for an in vitro sample.

[0031] In another preferred embodiment, said in vitro sample is selected from the group consisting of: whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

[0032] In another preferred embodiment, said tissue or cells are nasopharyngeal carcinoma tissue or cells, or paraneoplastic tissue or cells of nasopharyngeal carcinoma.

[0033] In another preferred embodiment, the detection reagent is conjugated with or carries a detectable label.

[0034] In another preferred embodiment, the detectable label is selected from the group consisting of: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

[0035] In another preferred embodiment, the kit contains a nasopharyngeal carcinoma risk marker as a reference or a quality control product.

[0036] In another preferred embodiment, the kit further includes a label or an instruction manual, and the label or the instruction manual indicates that the kit is used for (a) determining the risk of nasopharyngeal carcinoma, and / or (b) evaluating the therapeutic effect of nasopharyngeal carcinoma.

[0037] In another preferred embodiment, the detection of the nasopharyngeal carcinoma risk marker can be quantitatively detected.

[0038] In the third aspect of the present invention, a set of nasopharyngeal carcinoma antibody markers is provided, and the set of nasopharyngeal carcinoma antibody markers includes the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, and the autoantibodies include any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21, or a combination thereof.

[0039] In another preferred embodiment, the autoantibodies further include any one autoantibody selected from the group consisting of: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3, or a combination thereof.

[0040] In the fourth aspect of the present invention, a nasopharyngeal carcinoma risk assessment device is provided, and the device includes: (a) An input module, which is configured to input data of nasopharyngeal carcinoma antibody markers in a sample to be tested; the data of nasopharyngeal carcinoma antibody markers is the level of nasopharyngeal carcinoma antibody markers in the sample to be tested; the set of nasopharyngeal carcinoma antibody markers includes the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, and the autoantibodies include any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; (b) A risk assessment module configured to assess the input nasopharyngeal carcinoma antibody marker data to obtain a risk assessment result; the assessment includes: Comparing the level of the nasopharyngeal carcinoma antibody marker with a control reference value (cut-off); when the level of the nasopharyngeal carcinoma antibody marker ≥ the control reference value, it indicates a high risk of nasopharyngeal carcinoma in the subject to be tested; otherwise, it indicates a low risk of nasopharyngeal carcinoma in the subject to be tested; (c) An output module configured to output the assessment result.

[0041] In another preferred embodiment, the risk assessment is for early screening or auxiliary diagnosis.

[0042] In another preferred embodiment, the device further includes a detection module configured to detect the level of the nasopharyngeal carcinoma antibody marker.

[0043] In another preferred embodiment, the autoantibody further includes any one autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0044] In another preferred embodiment, the subject to be tested is a mammal, preferably a primate mammal, more preferably a human.

[0045] In the fifth aspect of the present invention, a detection method is provided, including the steps of: (a) Providing a test sample; (b) Detecting the expression level of the nasopharyngeal carcinoma antibody marker in the test sample, denoted as C1; and (c) Comparing the concentration C1 of the nasopharyngeal carcinoma antibody marker with a control reference value C0; Wherein, the nasopharyngeal carcinoma antibody marker includes the following antibody combination: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, the autoantibodies include any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; If the detection result of the nasopharyngeal carcinoma risk of the test subject meets the following conditions, it indicates a high risk of nasopharyngeal carcinoma in the subject: the level of the nasopharyngeal carcinoma antibody marker ≥ the control reference value (cut-off value).

[0046] In another preferred embodiment, the autoantibody further comprises any one autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0047] In another preferred embodiment, the test sample is selected from the following group: whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

[0048] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0049] In another preferred embodiment, the method is an in vitro method.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0051] Figure 1 Shows the distribution of individual antibody levels in the enzyme-linked immunosorbent assay.

[0052] Figure 2 Shows a scatter plot of the distribution of individual antibody levels in the chemiluminescence method.

[0053] Figure 3 Shows the receiver operating characteristic curve (ROC) analysis chart. Detailed Description of the Invention

[0054] Through extensive and in-depth research, the present inventors have first developed an antibody marker for nasopharyngeal carcinoma. The antibody marker is an antibody combination, specifically including the following antibodies: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) autoantibodies, the autoantibodies including any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3. The antibody combination of the present invention can not only effectively detect EBV-positive nasopharyngeal carcinoma, but also effectively detect EBV-negative nasopharyngeal carcinoma. Experiments of the present invention show that the antibody combination of the present invention has high sensitivity and high specificity and can be used for the auxiliary diagnosis or early screening of nasopharyngeal carcinoma. On this basis, the present invention has been completed.

[0055] Terms To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meanings given below. Other definitions are set forth throughout the application.

[0056] The term "about" can refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined.

[0057] As used herein, the terms "comprising" or "including (containing)" can be open-ended, semi-closed, and closed. In other words, the terms also include "consisting essentially of...", or "consisting of...".

[0058] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range and, where appropriate, fractional values thereof (such as one-tenth and one-hundredth of an integer).

[0059] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0060] The term "sample" or "specimen" as used herein refers to a material specifically associated with a subject from which specific information related to the subject can be determined, calculated, or inferred. The sample can be composed in whole or in part of biological material from the subject.

[0061] As used herein, the "presence" or "absence" of an antibody can be used interchangeably with "positive" or "negative"; making such a determination is routine in the art. For example, it can be detected by an antigen-antibody specific reaction between the antigen that causes the appearance of any antibody in the combination and the antibody.

[0062] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically related to a specific result when compared with the analysis result. In a preferred embodiment, the reference value is determined by comparing the levels of nasopharyngeal carcinoma antibody markers and performing statistical analysis. Some such studies are shown in the example section herein. However, studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. The reference value can also be determined by considering circumstances and results specifically related to the ethnicity, medical history, genetics, age, and other factors of the patient.

[0063] Nasopharyngeal Carcinoma Antibody Marker As used herein, the terms "antibody", "antibody marker", "nasopharyngeal carcinoma antibody marker", and "nasopharyngeal carcinoma risk marker" have the same meaning and can be used interchangeably.

[0064] The nasopharyngeal carcinoma antibody markers of the present invention are a combination of the following antibodies: a combination of anti-Epstein-Barr virus antibodies and tumor-related autoantibodies.

[0065] In another preferred embodiment, the anti-Epstein-Barr virus antibodies include: BKRF1-IgA, BMRF1-IgA, BALF2-IgA; the tumor-related autoantibodies are selected from the group consisting of: anti-TPI1, anti-PRKACA, anti-TRIM21, anti-SOX2, anti-BMI, anti-PDE4DIP, anti-GATA3, or a combination thereof.

[0066] The antigens of the above-mentioned tumor autoantibodies are TPI1, PRKACA, TRIM21, SOX2, BMI, PDE4DIP, GATA3, respectively.

[0067] The corresponding antigens in the above-mentioned Epstein-Barr virus antibodies are: BKRF1, BMRF1, BALF2, respectively.

[0068] The following Table A is a sorted list of the antigen names of tumor autoantibodies and their UniProt sequence numbers.

[0069] Table A

[0070] The following Table B is a sorted list of the antigen names of Epstein-Barr virus antibodies and their UniProt sequence numbers.

[0071] Table B

[0072] In another preferred embodiment, the nasopharyngeal carcinoma includes nasopharyngeal non-keratinizing carcinoma, nasopharyngeal keratinizing squamous cell carcinoma, and nasopharyngeal adenocarcinoma. In another preferred embodiment, the nasopharyngeal carcinoma includes EBV-positive nasopharyngeal carcinoma and EBV-negative nasopharyngeal carcinoma.

[0073] Detection method Based on the significant differences in the levels of nasopharyngeal carcinoma antibody markers in tissue samples or blood samples, the present invention also provides a corresponding method for judging the risk of nasopharyngeal carcinoma.

[0074] The present invention relates to an experimental method for quantitatively detecting the levels of nasopharyngeal carcinoma antibody markers. These tests are well known in the art. The levels of human nasopharyngeal carcinoma antibody markers detected in the tests can be used to judge (including assisting in judging) whether there is a risk of nasopharyngeal carcinoma.

[0075] In another preferred embodiment, the present invention provides a method for predicting the risk of nasopharyngeal carcinoma, screening, prognostic evaluation, monitoring the treatment effect, or recurrence monitoring, which includes the following steps: (1) Detect the level (quantity) of the nasopharyngeal carcinoma antibody markers (antibodies) provided by the present invention in a sample from a subject. (2) Compare the level of the nasopharyngeal carcinoma antibody marker (or the quantity of the antibody) with a reference threshold (hereinafter referred to as the CUT - OFF value). When it is greater than or equal to the CUT - OFF value, it is determined that the subject has a risk of nasopharyngeal carcinoma, has nasopharyngeal carcinoma, has a poor prognosis of nasopharyngeal carcinoma, or has a poor treatment effect.

[0076] In step (1), the quantification includes detecting each antibody in the antibody combination using the reagent provided by the present invention or a kit containing the reagent.

[0077] In step (2), the CUT - OFF value can be a reference level from healthy individuals or a healthy population; for example, it can be defined as the average value of the antibody detection results of a population confirmed to have no cancer through physical examination plus 2 standard deviations.

[0078] Kit Based on the correlation between the nasopharyngeal carcinoma antibody marker and the risk of nasopharyngeal carcinoma, the nasopharyngeal carcinoma antibody marker can thus be used as a marker for judging the risk of nasopharyngeal carcinoma.

[0079] The present invention also provides a kit for judging or evaluating the risk of nasopharyngeal carcinoma. The kit contains a detection reagent for detecting the level of the nasopharyngeal carcinoma antibody marker. Preferably, the kit contains the antigen or immunoconjugate of the nasopharyngeal carcinoma antibody marker (antibody) of the present invention, or an active fragment thereof.

[0080] In another preferred embodiment, the kit is an enzyme - linked immunosorbent assay (ELISA) detection kit. That is, using this kit, whether the antibody and / or antigen in the sample of the subject is positive is detected by enzyme - linked immunosorbent assay. Correspondingly, the kit may further include other components required for ELISA detection of the antibody and / or antigen, which are all well - known in the art. For detection purposes, for example, the antigen protein and / or antibody in the kit may be linked with a tag peptide, such as His tag, streptavidin tag, Myc tag; or, the kit may include a solid - phase carrier, such as a carrier with micropores for immobilizing the antigen protein and / or antibody, such as an enzyme - linked immunosorbent assay plate; or a microbead or magnetic bead solid - phase carrier. It may also include an adsorption protein for immobilizing the antigen protein and / or antibody on the solid - phase carrier, a diluent for blood such as serum, a washing solution, a secondary antibody with an enzyme label or a secondary antibody with fluorescence or chemiluminescence, a chromogenic solution, a termination solution, etc. The content of the corresponding antibody in the body fluid is detected based on the principle that the antigen protein and / or antibody indirectly or directly coated on the surface of the solid - phase carrier reacts with the antibody and / or antigen in serum / plasma / tissue fluid to form an antigen - antibody complex.

[0081] In another preferred example, the kit is a chemiluminescence immunoassay kit with magnetic microparticles. Antibodies in the sample are mixed with magnetic beads coated with corresponding antigens, incubated to form a complex, and unreacted substances are removed by washing with a washing solution. Then, it is mixed with a secondary antibody labeled with a fluorescent or chemiluminescent substance to form a complex, and unreacted substances are removed by washing with a washing solution. Finally, a substrate solution is added to cause the complex to generate a chemiluminescence signal, and the chemiluminescence signal is collected by a chemiluminescence analyzer. The instrument calculates the test result through a calibration curve. Correspondingly, the kit may further include other components required for ELISA detection of the antibody and / or antigen, which are all well-known in the art. For detection purposes, for example, the antigen protein and / or antibody in the kit may be linked to a tag peptide, such as His tag, streptavidin tag, Myc tag; the secondary antibody of the fluorescent or chemiluminescent substance may be acridinium ester.

[0082] In another preferred example, the kit further includes a label or an instruction manual.

[0083] The main advantages of the present invention include: (a) The antibody combination described in the present invention can achieve a sensitivity of 99.2% in the screening of nasopharyngeal carcinoma.

[0084] (b) The diagnostic performance of the tumor autoantibody and EBV antibody combination of the present invention is significantly superior to the EBV antibody combination (EA-IgA + VCA-IgA + Rta-IgA) currently used for clinical symptoms.

[0085] (c) The sensitivity of the antibody combination described in the present invention in nasopharyngeal carcinoma patients with negative EBV antibodies can reach 97.18% or more.

[0086] (d) The sensitivity of the antibody combination described in the present invention in early nasopharyngeal carcinoma patients can reach 97.22% or more.

[0087] (e) Among different subtypes of nasopharyngeal carcinoma, the sensitivity of the non-keratinizing carcinoma of the antibody combination described in the present invention can reach 98.33% or more, the sensitivity of the keratinizing squamous cell carcinoma can reach 97.50% or more, and the sensitivity of the adenocarcinoma can reach 96.67% or more.

[0088] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0089] General Method (I) Preparation of Recombinant Antigen Protein The cDNA fragment of the tumor antigen was cloned into the PET28(a) expression vector containing a 6XHis tag. At the N-terminus or C-terminus of the antigen, streptavidin protein or an analog (a biotin-binding tag protein) was introduced. The obtained recombinant expression vector was transformed into Escherichia coli for expression. The protein expressed in the supernatant was purified by Ni-NTA affinity column and ion column. When the protein was expressed in inclusion bodies, the protein was denatured with 6M guanidine hydrochloride and refolded in vitro according to standard methods, and then purified by Ni-NTA affinity column through the 6XHis tag to obtain the antigen protein.

[0090] (II) Preparation and Preservation of Serum or Plasma Serum or plasma of nasopharyngeal carcinoma patients was collected when the patients were initially diagnosed with nasopharyngeal carcinoma and had not received any radiotherapy, chemotherapy, or surgical treatment. Plasma or serum was prepared according to standard clinical procedures and stored in a -80 °C refrigerator for a long time.

[0091] (III) ELISA Detection The content of antibody markers in the sample was quantified by sandwich enzyme-linked immunosorbent assay (ELISA). The purified tumor antigen and Epstein-Barr virus antigen were immobilized on the microplate surface through their tag streptavidin or analog. The microplate was pre-coated with biotin-labeled bovine serum albumin (BSA). Serum or plasma samples were diluted 1:110 times with phosphate buffer and added to the microplate for reaction (50 μL / well). After washing away the unbound serum or plasma components with the washing solution, horseradish peroxidase (HRP)-conjugated anti-human IgG was added to each well for reaction. Then the reaction substrate TMB (3,3',5,5'-tetramethylbenzidine) was added for color development. The termination solution (1N HCl) was added, and the absorbance at 450 nm was read on a microplate reader (OD). At this time, the amount of enzyme carried on the solid phase carrier was positively correlated with the amount of the substance to be detected in the specimen, and the enzyme catalyzed the substrate to become a colored product. Qualitative or quantitative determination of the antibody was carried out according to the degree of the color reaction.

[0092] Magnetic Particle Chemiluminescence Assay The magnetic particle chemiluminescence method is used to detect antibodies in the sample to be tested by an automatic chemiluminescence immunoassay analyzer. The detection process for each sample to be tested is as follows: The sample to be tested (10 μL), the magnetic bead working solution coated with antigen markers (40 μL), and the sample diluent (50 μL) are incubated together for 18 min, washed for 5 min, and then reacted with the acridinium ester-labeled anti-human IgG antibody working solution (secondary antibody) (50 μL) to form a magnetic particle-antigen-secondary antibody-chemiluminescence marker complex. The immune complex is separated by a magnetic bead separator, the free components are washed away, and the chemiluminescence marker is excited by a chemiluminescence excitation solution to emit light. The automatic chemiluminescence immunoassay analyzer automatically completes the detection and records the relative luminescence intensity (RLU).

[0093] Example 1 Training cohort study on the sensitivity and specificity of tumor antibodies and Epstein-Barr virus antibodies 1.1 Sample information of the subjects in the training cohort This example includes 184 cases of healthy physical examination population (age range: 18 - 88, average age 54) and 130 cases of nasopharyngeal carcinoma patients. The healthy physical examination population comes from no less than 3 different physical examination centers. Serum samples of all nasopharyngeal carcinoma patients were collected when the patients were diagnosed with nasopharyngeal carcinoma and had not received any radiotherapy, chemotherapy, or surgical treatment, and were stored in a -80°C refrigerator. The information of nasopharyngeal carcinoma patients in this example is shown in Table 1.

[0094] Table 1 Sample information table of the subjects in the training cohort

[0095] 1.2 Detection method and analysis of the sensitivity and specificity of individual antibodies in the subjects Nasopharyngeal carcinoma-related antigen and Epstein-Barr virus antigen were expressed and purified and then coated on the surface of a 96-well plate. After blocking, it was reacted with nasopharyngeal carcinoma serum or physical examination control population serum diluted 1:110 times, then reacted with anti-human IgG antibody-HRP horseradish peroxidase, and then a color reaction was carried out, and the absorbance was detected at a wavelength of 450 nm by an enzyme-linked immunosorbent assay reader. Tables 2 and 3 show the detection sensitivity and specificity.

[0096] Table 2 Sensitivity and specificity of individual tumor autoantibodies as nasopharyngeal carcinoma antibody markers

[0097] Note: Both IgG and IgA types of tumor autoantibodies have good sensitivity and specificity, and IgA is superior to IgG. This invention elaborates on tumor autoantibodies.

[0098] Table 3 Sensitivity and specificity of individual EBV antibodies as nasopharyngeal carcinoma antibody markers

[0099] Note: In current clinical applications, the performance of Epstein-Barr virus IgA antibody is superior to that of IgG antibody. Therefore, this invention elaborates on Epstein-Barr virus IgA antibody.

[0100] Scatter plots of the antibody levels of tumor autoantibodies anti-TPI1, anti-PRKACA, anti-TRIM21, anti-SOX2, anti-BMI, anti-PDE4DIP, anti-GATA3 and Epstein-Barr virus antibodies BKRF1-IgA, BMRF1-IgA, BALF2-IgA in the tumor group and the control group are shown in Figure 1 and Figure 2 and Table 4-7.

[0101] Table 4 Distribution of individual antibodies in enzyme-linked immunosorbent assay (OD value, mean ± standard deviation)

[0102] Table 5 Distribution of individual antibodies in enzyme-linked immunosorbent assay in the training cohort (number of subjects) (For each autoantibody, number of subjects with ≥ cut-off value)

[0103] Table 6 Distribution of individual antibodies in chemiluminescence immunoassay (RLU value, mean ± standard deviation)

[0104] Table 7 Distribution of individual antibodies in chemiluminescence immunoassay in the training cohort (number of subjects) (For each autoantibody, number of subjects with ≥ cut-off value)

[0105] Due to the different strengths of the immune systems of tumor patients and the diversity of tumor generation mechanisms, the distribution sensitivity of a single tumor antibody in tumor patients is low. Using the Mann-Whitney U test for statistical analysis of the level distribution of antibodies in the tumor group and the control group, it was found that the level distributions of (anti-TPI1, anti-PRKACA, anti-TRIM21, anti-SOX2, anti-BMI, anti-PDE4DIP, anti-GATA3, BKRF1-IgA, BMRF1-IgA, BALF2-IgA) antibodies in the tumor group and the control group were significantly different (p < 0.05).

[0106] 1.3 Screening of antibody combinations Based on the detection of a single candidate antibody in the population, while ensuring the high specificity of a single antibody, combined with the individual positive contribution of the antibody (i.e., excluding candidate molecules with high overlapping positive detection rates), the detection model was maximally made to cover more nasopharyngeal carcinoma patients, forming different antibody combinations, and corresponding detection reagents were used to detect them. The results are shown in Table 8.

[0107] Table 8 Sensitivity and Specificity of Antibody Combinations in the Training Cohort

[0108] Note: EA(P54 - BMRF1, P138 - BALF2), VCA (p18 - BFRF3, P23 - BLRF2), RTA.

[0109] It can be seen from the performance of the antibody combinations in Table 5 that the diagnostic performance of the combination of tumor autoantibodies and Epstein - Barr virus antibodies of the present invention is significantly better than that of the Epstein - Barr virus antibody combination (EA - IgA + VCA - IgA + Rta - IgA) currently used in clinical symptoms. And the performance of the antibody combination is better than that of a single antibody, increasing the detection rate in the nasopharyngeal carcinoma patient population while ensuring the specificity of the healthy population.

[0110] As can be seen in Table 5, the antibody combinations (anti - TPI1, anti - PRKACA, anti - TRIM21 and BKRF1 - IgA, BMRF1 - IgA, BALF2 - IgA) have the best sensitivity and specificity. Therefore, further performance studies were carried out on this antibody combination. The performance study is as shown in 1.4 below.

[0111] 1.4 Receiver Operating Characteristic Curve (ROC) Analysis of the Combined Detection Model of Tumor Autoantibodies and Epstein - Barr Virus Antibodies of the Present Invention in the Population The present invention further uses ROC curve analysis to analyze the screening ability of the antibody combination (anti - TPI1, anti - PRKACA, anti - TRIM21 and BKRF1 - IgA, BMRF1 - IgA, BALF2 - IgA) of the present invention for nasopharyngeal carcinoma patients in the population.

[0112] As Figure 3 shown, using the healthy physical examination population as a control, when the Youden index is at its maximum, the sensitivity of the antibody combination of the present invention reaches 98.46%, the specificity is 94.02% at this time, and the area under the curve is 0.993.

[0113] 1.5 The Combined Detection Model of Tumor Autoantibodies and Epstein - Barr Virus Antibodies of the Present Invention Has Ideal Detection Ability for Early Nasopharyngeal Carcinoma The inventors of the present invention classified the pathological stages of nasopharyngeal carcinoma in the test patients, including 72 patients with early-stage (stage 0, I, and II) nasopharyngeal carcinoma and 58 patients with late-stage (stage III and IV) nasopharyngeal carcinoma, a total of 130 nasopharyngeal carcinoma patients, among which 59 nasopharyngeal carcinoma patients were co-infected with Epstein-Barr virus. Using the antibody combination detection model of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, BALF2-IgA), the serum was detected by enzyme-linked immunosorbent assay, and the detection ability of the antibody combination in nasopharyngeal carcinoma at different stages was analyzed for the obtained detection data. It was evaluated according to the reference values in Table 9 below.

[0114] Table 9

[0115] Positive and negative judgment rules: If the OD450 value detected by any one or more indicators is greater than or equal to its corresponding CUT-OFF, it is recorded as positive; If the OD450 values detected by all indicators are less than their corresponding CUTOFF, it is recorded as negative; The total detection sensitivity of the detection model of the present invention for nasopharyngeal carcinoma is 98.46%, and the specificity is 94.02% at this time. The detection sensitivity for early-stage nasopharyngeal carcinoma is 98.61%, and the detection sensitivity for late-stage nasopharyngeal carcinoma is 98.28%. The detection model of the present invention has an ideal detection ability for early-stage nasopharyngeal carcinoma.

[0116] 1.6 The combined detection model of tumor autoantibodies and Epstein-Barr virus antibodies of the present invention has an ideal detection ability for different subtypes of nasopharyngeal carcinoma The inventors of the present invention classified the detection results of different pathological subtypes of nasopharyngeal carcinoma, including 60 cases of nasopharyngeal non-keratinizing squamous cell carcinoma, 40 cases of nasopharyngeal keratinizing squamous cell carcinoma, and 30 cases of nasopharyngeal adenocarcinoma patients, a total of 130 nasopharyngeal carcinoma patients, among which 59 nasopharyngeal carcinoma patients were co-infected with Epstein-Barr virus and 71 nasopharyngeal carcinoma patients were not co-infected with Epstein-Barr virus.

[0117] Using the antibody combination detection model of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, BALF2-IgA), the serum was detected by enzyme-linked immunosorbent assay, and the detection ability of the antibody combination in nasopharyngeal carcinoma of different histopathological subtypes was analyzed regardless of whether the nasopharyngeal carcinoma patients were infected with Epstein-Barr virus. It was evaluated according to the reference values in Table 8.

[0118] Positive and negative judgment rules: If the OD450 value detected by any one or more indicators is greater than or equal to its corresponding CUT-OFF, it is recorded as positive; The OD450 value detected for all indicators is less than its corresponding CUT-OFF, which is recorded as negative; The detection sensitivity of the detection model of the present invention for nasopharyngeal non-keratinizing carcinoma is 100%, the detection sensitivity for nasopharyngeal keratinizing squamous cell carcinoma is 97.50%, and the detection sensitivity for nasopharyngeal adenocarcinoma is 96.67%. The detection sensitivity for nasopharyngeal carcinoma patients co-infected with Epstein-Barr virus is 100%, and the detection sensitivity for nasopharyngeal carcinoma patients not co-infected with Epstein-Barr virus is 97.18%. The detection model of the present invention has good sensitivity for various subtypes of nasopharyngeal carcinoma, regardless of whether nasopharyngeal carcinoma patients are infected with Epstein-Barr virus.

[0119] 1.7 Using chemiluminescence immunoassay with magnetic microparticles to detect that the combined detection model of tumor autoantibodies and Epstein-Barr virus antibodies of the present invention has ideal detection ability for early nasopharyngeal carcinoma and different subtypes of nasopharyngeal carcinoma The inventors of the present invention classified and statistically analyzed the detection results of different stages of nasopharyngeal carcinoma and different pathological subtypes of nasopharyngeal carcinoma. Different stages of nasopharyngeal carcinoma included 72 patients with early-stage (stage 0, I, and II) nasopharyngeal carcinoma and 58 patients with advanced-stage (stage III and IV) nasopharyngeal carcinoma, for a total of 130 nasopharyngeal carcinoma patients, among which 59 nasopharyngeal carcinoma patients were co-infected with Epstein-Barr virus and 71 nasopharyngeal carcinoma patients were not co-infected with Epstein-Barr virus. Different histopathological subtypes included 60 cases of nasopharyngeal non-keratinizing carcinoma, 40 cases of nasopharyngeal keratinizing squamous cell carcinoma, and 30 cases of nasopharyngeal adenocarcinoma patients. Chemiluminescence immunoassay with magnetic microparticles was used for detection, and the combined detection model of antibody combinations of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, BALF2-IgA) was used to analyze its detection ability for nasopharyngeal carcinoma of different stages and different histopathological subtypes.

[0120] Evaluate according to the reference values in Table 10 below.

[0121] Table 10

[0122] Positive and negative judgment rules: If the RLU value detected for any one or more indicators is greater than or equal to its corresponding CUT-OFF, it is recorded as positive; If the RLU values detected for all indicators are less than their corresponding CUT-OFF, it is recorded as negative; The detection sensitivity of the detection model of the present invention for nasopharyngeal carcinoma patients in the early stage (stage 0, stage I, and stage II) is 97.22%, and for those in the advanced stage (stage III and stage IV) is 98.28%. The detection sensitivity for nasopharyngeal carcinoma patients co-infected with Epstein-Barr virus is 98.59%, and for those not co-infected with Epstein-Barr virus is 100%. The detection model of the present invention has good sensitivity for various stages of nasopharyngeal carcinoma, regardless of whether the nasopharyngeal carcinoma patients are infected with Epstein-Barr virus.

[0123] The detection sensitivity of the detection model of the present invention for nasopharyngeal non-keratinizing carcinoma is 98.33%, for nasopharyngeal keratinizing squamous cell carcinoma is 97.50%, and for nasopharyngeal adenocarcinoma is 96.67%. The detection sensitivity for nasopharyngeal carcinoma patients co-infected with Epstein-Barr virus is 98.59%, and for those not co-infected with Epstein-Barr virus is 100%. The detection model of the present invention has good sensitivity for various subtypes of nasopharyngeal carcinoma, regardless of whether the nasopharyngeal carcinoma patients are infected with Epstein-Barr virus.

[0124] Example 2 Verification cohort verifies the sensitivity and specificity of the antibody combination 2.1 Sample information of the subjects in the verification cohort This example includes 150 cases of healthy physical examination population (age range: 18 - 88, average age 56) and 150 cases of nasopharyngeal carcinoma patients. The healthy physical examination population comes from no less than 3 different physical examination centers. Serum of all nasopharyngeal carcinoma patients was collected when the patients were diagnosed with nasopharyngeal carcinoma and had not received any radiotherapy, chemotherapy, or surgical treatment, and was stored in a -80°C refrigerator. The information of nasopharyngeal carcinoma patients in this example is shown in Table 11.

[0125] Table 11 Sample information table of the subjects in the verification cohort

[0126] 2.2 Number of subjects meeting the cut-off value measured by enzyme-linked immunosorbent assay and chemiluminescence immunoassay In this example, in the verification cohort, for each autoantibody marker, the number of subjects with a value ≥ cut-off value measured by the two methods respectively is shown in Tables 12 - 13.

[0127] Table 12 Distribution of single antibodies in the enzyme-linked immunosorbent assay in the verification cohort (number of subjects) (For each autoantibody, number of subjects with a value ≥ cut-off value)

[0128] Table 13 Distribution of single antibodies in the chemiluminescence immunoassay in the verification cohort (number of subjects) (For each autoantibody, number of subjects with a value ≥ cut-off value)

[0129] 2.3 Detection and analysis of the sensitivity and specificity of the antibody combination by enzyme-linked immunosorbent assay According to the enzyme-linked immunosorbent assay and CUT-OFF value determined from the training cohort, 150 samples of nasopharyngeal carcinoma patients and 150 samples of healthy physical examination population included in the study were detected, and the sensitivity and specificity of the antibody combination were analyzed.

[0130] The sensitivity and specificity of the antibody combination in this example are shown in Table 14.

[0131] Table 14 Sensitivity and specificity of the enzyme-linked immunosorbent assay for detecting the validation cohort

[0132] Note: The detection sensitivity of early nasopharyngeal carcinoma, advanced nasopharyngeal carcinoma, nasopharyngeal non-keratinizing carcinoma, nasopharyngeal keratinizing squamous cell carcinoma, and nasopharyngeal adenocarcinoma is the analysis of the detection results of the antibody combination (anti-TPI1, anti-PRKACA, anti-TRIM21 and BKRF1-IgA, BMRF1-IgA, BALF2-IgA).

[0133] From the above results, it can be seen that the sensitivity and specificity of the detection results of the subjects in the validation cohort detected by enzyme-linked immunosorbent assay are consistent with those of the training cohort.

[0134] In this example, the enzyme-linked immunosorbent assay technology was used. The detection sensitivity for early-stage (stage 0, I, and II) nasopharyngeal carcinoma patients was 98.80%, and for late-stage (stage III and IV) nasopharyngeal carcinoma patients was 98.51%. The detection sensitivity for nasopharyngeal non-keratinizing carcinoma was 100%, for nasopharyngeal keratinizing squamous cell carcinoma was 97.78%, and for nasopharyngeal adenocarcinoma was 97.14%. The detection sensitivity for nasopharyngeal carcinoma patients co-infected with Epstein-Barr virus was 100%, and for nasopharyngeal carcinoma patients not co-infected with Epstein-Barr virus was 97.73%. The detection model of the present invention has good sensitivity for various nasopharyngeal carcinoma stages and histopathological subtypes, regardless of whether nasopharyngeal carcinoma patients are infected with Epstein-Barr virus.

[0135] 2.4 Detection and analysis of the sensitivity and specificity of the antibody combination by magnetic particle chemiluminescence immunoassay According to the magnetic particle chemiluminescence immunoassay and CUT-OFF value determined from the training cohort, 150 samples of nasopharyngeal carcinoma patients and 150 samples of healthy physical examination population included in the study were detected, and the sensitivity and specificity of the antibody combination were analyzed. The sensitivity and specificity of the antibody combination in this example are shown in Table 15.

[0136] Table 15 Sensitivity and specificity of the magnetic particle chemiluminescence immunoassay for detecting the validation cohort

[0137] Note: The detection sensitivities of early nasopharyngeal carcinoma, advanced nasopharyngeal carcinoma, nasopharyngeal non-keratinizing carcinoma, nasopharyngeal keratinizing squamous cell carcinoma, and nasopharyngeal adenocarcinoma are analyzed based on the detection results of the antibody combination (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, BALF2-IgA).

[0138] From the above results, it can be seen that the sensitivity and specificity of the detection results of the test population in the validation cohort detected by the magnetic particle chemiluminescence method are consistent with those of the training cohort.

[0139] In this embodiment, the magnetic particle chemiluminescence detection technology is adopted. The detection sensitivity for early-stage (stage 0, I, and II) nasopharyngeal carcinoma patients is 100%, and the detection sensitivity for late-stage (stage III and IV) nasopharyngeal carcinoma patients is 95.56%. The detection sensitivity for nasopharyngeal non-keratinizing carcinoma is 100%, the detection sensitivity for nasopharyngeal keratinizing squamous cell carcinoma is 97.78%, and the detection sensitivity for nasopharyngeal adenocarcinoma is 97.14%. The detection sensitivity for nasopharyngeal carcinoma patients co-infected with Epstein-Barr virus is 98.39%, and the detection sensitivity for nasopharyngeal carcinoma patients not co-infected with Epstein-Barr virus is 97.73%. The detection model of the present invention has good sensitivity for various nasopharyngeal carcinoma stages and histopathological subtypes, regardless of whether nasopharyngeal carcinoma patients are infected with Epstein-Barr virus.

[0140] All documents mentioned in the present invention are cited herein by reference as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. Use of a nasopharyngeal carcinoma antibody marker or its detection reagent, characterized in that, For preparing a diagnostic reagent or a diagnostic kit, which is used for judging the occurrence risk of nasopharyngeal carcinoma; Among them, the nasopharyngeal carcinoma antibody markers include the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, which include any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21.

2. The use according to claim 1, wherein The autoantibodies also include any one autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

3. The use according to claim 2, characterized in that, The nasopharyngeal carcinoma antibody markers are antibody combinations selected from the following group: (Z1) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A1) anti-TPI1; (Z2) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A2) anti-PRKACA; (Z3) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A3) anti-TRIM21; (Z4) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; and (A3) anti-TRIM21; (Z5) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; and (A2) anti-PRKACA; (Z6) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A2) anti-PRKACA; and (A3) anti-TRIM21; (Z7) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; and (A3) anti-TRIM21; (Z8) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; and (A5) anti-BMI; (Z9) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A3) anti-TRIM21; and (A6) anti-PDE4DIP; (Z10) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A7) anti-GATA3; (Z11) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A4) anti-SOX2; (Z12) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A5) anti-BMI; (Z13) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A6) anti-PDE4DIP; (Z14) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A7) anti-GATA3.

4. The use according to claim 1, characterized in that, The diagnostic reagent or diagnostic kit is used to detect the level of the nasopharyngeal carcinoma antibody marker in a sample to be tested.

5. The use according to claim 1, characterized in that, The sample to be tested is selected from the group consisting of: whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

6. The use according to claim 1, characterized in that, The nasopharyngeal carcinoma includes: EBV-positive nasopharyngeal carcinoma and EBV-negative nasopharyngeal carcinoma.

7. A kit, characterized in that, The kit contains a detection reagent for detecting a nasopharyngeal carcinoma antibody marker; wherein, the nasopharyngeal carcinoma antibody marker includes the following antibody combinations: (B) EBV antibody: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibody, the autoantibody includes any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21, or a combination thereof.

8. A set of nasopharyngeal carcinoma antibody markers, characterized in that, The set of the nasopharyngeal carcinoma antibody markers includes the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, said autoantibodies including any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21, or a combination thereof.

9. The set according to claim 8, wherein The autoantibodies further include any one autoantibody selected from the group consisting of: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3, or a combination thereof.

10. A nasopharyngeal carcinoma risk assessment device, characterized in that, The device includes: (a) An input module configured to input data of nasopharyngeal carcinoma antibody markers in a sample to be tested; the data of nasopharyngeal carcinoma antibody markers being the levels of nasopharyngeal carcinoma antibody markers in the sample to be tested; the set of nasopharyngeal carcinoma antibody markers including the following antibody combinations: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) Autoantibodies, said autoantibodies including any one autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; (b) A risk assessment module configured to assess the input data of nasopharyngeal carcinoma antibody markers to obtain a risk assessment result; the assessment including: comparing the levels of nasopharyngeal carcinoma antibody markers with a control reference value or a cut-off value, and when the levels of nasopharyngeal carcinoma antibody markers ≥ the control reference value or the cut-off value, it indicates a high risk of nasopharyngeal carcinoma in the object to be tested; otherwise, it indicates a low risk of nasopharyngeal carcinoma in the object to be tested; (c) An output module configured to output the assessment result.

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