Nasopharyngeal carcinoma antibody markers and their applications

Abstract

The present invention provides nasopharyngeal carcinoma antibody markers and their applications. Specifically, the present invention provides uses of nasopharyngeal carcinoma antibody markers or detection reagents thereof for preparing diagnostic reagents or diagnostic kits for determining the risk of nasopharyngeal carcinoma; wherein 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, the autoantibodies including any autoantibody selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21. The nasopharyngeal carcinoma antibody markers of the present invention can be used as markers for determining the occurrence of nasopharyngeal carcinoma with high sensitivity and specificity.

Description

Technical Field

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

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck that originates from the nasopharyngeal mucosa and is more common in the pharyngeal recess. The disease occurs in a narrow location, with insidious onset and atypical early symptoms. Most patients are diagnosed in the middle or late stages of the disease, missing the optimal time for treatment.

[0003] The clinical manifestations of NPC are subtle, including ear problems, neck masses, nasal problems, and headaches. Its anatomical isolation leads to delayed diagnosis and treatment, with patients not seeking medical attention until 6 weeks after symptom onset.

[0004] The misdiagnosis rate for nasopharyngeal carcinoma (NPC) is reported to be as high as 43.4%. Nasal endoscopy combined with biopsy and pathological analysis remains the gold standard for clinical NPC detection. However, this method is invasive and unsuitable for asymptomatic individuals, necessitating the development of biomarkers for screening in asymptomatic individuals.

[0005] The pathogenesis of nasopharyngeal carcinoma remains unclear, but reports suggest it may be related to a variety of factors, including genetic susceptibility, diet, environment, and Epstein-Barr virus (EBV) infection. In 2011, EBV dual antibody screening was included in the "Technical Plan for Early Diagnosis and Treatment of Cancer." Large-scale randomized controlled cohort studies have shown that dual antibody screening can significantly improve the early diagnosis and survival rates of nasopharyngeal carcinoma patients and reduce the risk of death from nasopharyngeal carcinoma.

[0006] However, the dual-antibody approach has a positive diagnosis rate of only approximately 5%. Furthermore, in areas with a high incidence of NPC, 95% of NPC cases are associated with EBV infection, whereas in areas with a low incidence, only 20% of NPC cases are associated with EBV infection. Therefore, testing only for EBV antibodies can lead to missed NPC diagnoses.

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

[0008] The present invention provides an early-stage autoantibody marker for nasopharyngeal carcinoma with high sensitivity and specificity.

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

[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 detection reagent in the preparation of products for nasopharyngeal carcinoma risk prediction, screening, prognosis assessment, treatment effect monitoring, or recurrence monitoring. The antibody combination is a combination of tumor-associated autoantibodies and anti-EBV antibodies.

[0011] Another object of the present invention is to provide a kit and corresponding methods for risk prediction, screening, prognosis assessment, treatment effect monitoring or recurrence monitoring of nasopharyngeal carcinoma.

[0012] In a first aspect of the present invention, there is provided a use of a nasopharyngeal carcinoma antibody marker or a detection reagent thereof for preparing a diagnostic reagent or a diagnostic kit, wherein the diagnostic reagent or the diagnostic kit is used to determine the risk of nasopharyngeal carcinoma;

[0013] The nasopharyngeal carcinoma antibody markers include the following antibody combinations:

[0014] (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and

[0015] (A) An autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21.

[0016] In another preferred embodiment, the autoantibody further comprises any autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0017] In another preferred embodiment, the nasopharyngeal carcinoma antibody marker is an antibody combination selected from the following group:

[0018] (Z1) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A1) anti-TPI1;

[0019] (Z2) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A2) anti-PRKACA;

[0020] (Z3) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; and (A3) anti-TRIM21; <h2 style=";text-align:left;direction:ltr">

[0021] <h2 style=";text-align:left;direction:ltr"> (Z4) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A3) anti-TRIM21;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0022] <h2 style=";text-align:left;direction:ltr"> (Z5) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A2) anti-PRKACA;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0023] <h2 style=";text-align:left;direction:ltr"> (Z6) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A2) anti-PRKACA;(A3) anti-TRIM21;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0024] <h2 style=";text-align:left;direction:ltr"> (Z7) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A2) anti-PRKACA;(A3) anti-TRIM21;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0025] <h2 style=";text-align:left;direction:ltr"> (Z8) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A2) anti-PRKACA;(A5) anti-BMI;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0026] <h2 style=";text-align:left;direction:ltr"> (Z9) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A3) anti-TRIM21;(A6) anti-PDE4DIP;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0027] <h2 style=";text-align:left;direction:ltr"> (Z10) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A2) anti-PRKACA;(A3) anti-TRIM21;(A7) anti-GATA3;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0028] <h2 style=";text-align:left;direction:ltr"> (Z11) (B1) BKRF1-IgA;(B2) BMRF1-IgA;(B3) BALF2-IgA;(A1) anti-TPI1;(A2) anti-PRKACA;(A3) anti-TRIM21;(A4) anti-SOX2;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0029] (Z12) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A5) anti-BMI;

[0030] (Z13) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A6) anti-PDE4DIP;

[0031] (Z14) (B1) BKRF1-IgA; (B2) BMRF1-IgA; (B3) BALF2-IgA; (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; and (A7) anti-GATA3.

[0032] In another preferred embodiment, the autoantibody is IgA (eg, IgA1, IgA2), IgM, or IgG (eg, IgG1, IgG2, IgG3, IgG4).

[0033] 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.

[0034] In another preferred embodiment, the sample to be tested is selected from the following group: whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

[0035] In another preferred embodiment, the tissue or cell is nasopharyngeal carcinoma tissue or cell, or para-cancerous tissue or cell of nasopharyngeal carcinoma.

[0036] In another preferred embodiment, the sample to be tested is derived from the following test subjects: mammals, preferably primates, and more preferably humans.

[0037] In another preferred embodiment, the nasopharyngeal carcinoma includes: EBV-positive (EBV+) nasopharyngeal carcinoma and EBV-negative (EBV-) nasopharyngeal carcinoma.

[0038] In another preferred embodiment, the nasopharyngeal carcinoma includes: nasopharyngeal non-keratinizing carcinoma, nasopharyngeal keratinizing squamous cell carcinoma and nasopharyngeal adenocarcinoma.

[0039] In another preferred embodiment, the nasopharyngeal carcinoma includes nasopharyngeal carcinoma of the following pathological stages: stage I, stage II, stage III and stage IV; preferably stage I and stage II.

[0040] In a second aspect of the present invention, a kit is provided, comprising a detection reagent for detecting nasopharyngeal carcinoma antibody markers; wherein the nasopharyngeal carcinoma antibody markers comprise the following antibody combinations:

[0041] (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and

[0042] (A) An autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21.

[0043] In another preferred embodiment, the autoantibody further comprises any 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 detection reagent includes: a specific antigen or specific binding molecule for the nasopharyngeal carcinoma antibody marker.

[0045] In another preferred embodiment, the detection reagent can be a reagent used for enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, protein / peptide chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

[0046] In another preferred embodiment, the detection reagent is used to detect the nasopharyngeal carcinoma antibody marker through antigen-antibody reaction (for example, through ELISA or fluorescence or chemiluminescence immunoassay).

[0047] In another preferred embodiment, the detection reagent includes:

[0048] (b) EBV antigens: BKRF1, BMRF1, and BALF2; and

[0049] (a) An antigen selected from the group consisting of TPI1, PRKACA, TRIM21, SOX2, BMI, PDE4DIP, GATA3, or a combination thereof.

[0050] In another preferred embodiment, the detection reagent includes the following antigens: BKRF1, BMRF1, BALF2, TPI1, PRKACA and TRIM21.

[0051] In another preferred embodiment, the detection is a detection of an in vitro sample.

[0052] In another preferred embodiment, the 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.

[0053] In another preferred embodiment, the tissue or cell is nasopharyngeal carcinoma tissue or cell, or para-cancerous tissue or cell of nasopharyngeal carcinoma.

[0054] In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.

[0055] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0056] In another preferred embodiment, the kit contains a nasopharyngeal carcinoma risk marker as a reference substance or quality control substance.

[0057] In another preferred embodiment, the kit further includes a label or instructions, wherein the label or instructions indicate that the kit is used for (a) determining the risk of nasopharyngeal carcinoma, and / or (b) evaluating the therapeutic effect of nasopharyngeal carcinoma.

[0058] In another preferred embodiment, the detection of the nasopharyngeal carcinoma risk marker can be quantitative.

[0059] In a third aspect of the present invention, a set of nasopharyngeal carcinoma antibody markers is provided, wherein the set of nasopharyngeal carcinoma antibody markers comprises the following antibody combinations:

[0060] (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and

[0061] (A) An autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21.

[0062] In another preferred embodiment, the autoantibody further comprises any autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0063] In a fourth aspect of the present invention, a nasopharyngeal carcinoma risk assessment device is provided, comprising:

[0064] (a) An input module configured to input nasopharyngeal carcinoma antibody marker data in a sample to be tested; the nasopharyngeal carcinoma antibody marker data is the level of the nasopharyngeal carcinoma antibody marker in the sample to be tested; the set of nasopharyngeal carcinoma antibody markers includes the following antibody combinations:

[0065] (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and

[0066] (A) an autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21;

[0067] (b) a risk assessment module, wherein the risk assessment module is configured to evaluate the input nasopharyngeal carcinoma antibody marker data to obtain a risk assessment result; the evaluation includes:

[0068] Comparing the nasopharyngeal carcinoma antibody marker level with a control reference value (cut-off); when the nasopharyngeal carcinoma antibody marker level is greater than or equal to the control reference value, it indicates that the subject has a high risk of nasopharyngeal carcinoma; otherwise, it indicates that the subject has a low risk of nasopharyngeal carcinoma;

[0069] (c) an output module, wherein the output module is configured to output the evaluation result.

[0070] In another preferred embodiment, the risk assessment is early screening or auxiliary diagnosis.

[0071] In another preferred embodiment, the device further comprises a detection module, and the detection module is configured to detect the level of the nasopharyngeal carcinoma antibody marker.

[0072] In another preferred embodiment, the autoantibody further comprises any autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0073] In another preferred embodiment, the subject to be tested is a mammal, preferably a primate mammal, and more preferably a human.

[0074] In a fifth aspect of the present invention, a detection method is provided, comprising the steps of:

[0075] (a) provide a test sample;

[0076] (b) detecting the expression level of nasopharyngeal carcinoma antibody markers in the test sample, denoted as C1; and

[0077] (c) comparing the nasopharyngeal carcinoma antibody marker concentration C1 with a control reference value C0;

[0078] The nasopharyngeal carcinoma antibody markers include the following antibody combinations:

[0079] (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and

[0080] (A) an autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21;

[0081] If the test result of the nasopharyngeal carcinoma risk of the test subject meets the following conditions, it indicates that the risk of nasopharyngeal carcinoma of the test subject is high: the level of the nasopharyngeal carcinoma antibody marker is ≥ the control reference value (cut-off value).

[0082] In another preferred embodiment, the autoantibody further comprises any autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

[0083] In another preferred embodiment, the sample to be tested is selected from the following group: whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid, urine, or a combination thereof.

[0084] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0085] In another preferred embodiment, the method is an in vitro method.

[0086] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The distribution of individual antibody levels in enzyme-linked immunosorbent assay is shown.

[0088] Figure 2 Scatter plots showing the distribution of individual antibody levels by chemiluminescence are shown.

[0089] Figure 3 Receiver operating characteristic (ROC) analysis plots are shown. DETAILED DESCRIPTION

[0090] After extensive and in-depth research, the present inventors have developed, for the first time, an antibody marker for nasopharyngeal carcinoma. This antibody marker is an antibody combination, specifically comprising the following antibodies: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) autoantibodies, including any one or a combination thereof selected from the group consisting of: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; and (A7) anti-GATA3. This antibody combination can effectively detect not only EBV-positive nasopharyngeal carcinoma but also EBV-negative nasopharyngeal carcinoma. Experiments conducted by the present inventors have demonstrated that this antibody combination has high sensitivity and specificity and can be used for auxiliary diagnosis or early screening of nasopharyngeal carcinoma. Based on this, the present invention was completed.

[0091] the term

[0092] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0093] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0094] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0095] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer).

[0096] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0097] As used herein, the term "sample" or "specimen" refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. A sample may consist entirely or in part of biological material from a subject.

[0098] As used herein, the "presence" or "absence" of an antibody is used interchangeably with "positive" or "negative"; determining this is a routine skill in the art. For example, detection can be performed by an antigen-antibody specific reaction between the antigen that causes the appearance of any antibody in the combination and the antibody.

[0099] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular result when compared to an analysis result. In a preferred embodiment, the reference value is based on comparing nasopharyngeal carcinoma antibody marker levels and performing statistical analysis to determine. Some such studies have been shown in the Examples section of this paper. However, user experience from research in the literature and the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering situations and results that are particularly relevant to the patient's ethnic group, medical history, genetics, age, and other factors.

[0100] Nasopharyngeal carcinoma antibody markers

[0101] 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.

[0102] The nasopharyngeal carcinoma antibody marker of the present invention is a combination of the following antibodies: a combination of an anti-Epstein-Barr virus antibody and a tumor-related autoantibody.

[0103] In another preferred embodiment, the anti-EB virus antibodies include: BKRF1-IgA, BMRF1-IgA, BALF2-IgA; the tumor-related autoantibodies are selected from the following group: anti-TPI1, anti-PRKACA, anti-TRIM21, anti-SOX2, anti-BMI, anti-PDE4DIP, anti-GATA3, or a combination thereof.

[0104] The antigens of the above-mentioned tumor autoantibodies are TPI1, PRKACA, TRIM21, SOX2, BMI, PDE4DIP, and GATA3.

[0105] The corresponding antigens in the above-mentioned EB virus antibodies are: BKRF1, BMRF1, and BALF2.

[0106] Table A below is a list of the antigen names and UniProt sequence numbers corresponding to tumor autoantibodies.

[0107] Table A

[0108]

[0109] Table B below is a collated list of antigen names and UniProt sequence numbers corresponding to EB virus antibodies.

[0110] Table B

[0111]

[0112] 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.

[0113] Detection method

[0114] 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 determining the risk of nasopharyngeal carcinoma.

[0115] The present invention relates to an experimental method for quantitatively detecting the levels of nasopharyngeal carcinoma antibody markers. These assays are well known in the art. The levels of nasopharyngeal carcinoma antibody markers detected in the assay can be used to determine (including assist in determining) whether a person has a risk of nasopharyngeal carcinoma.

[0116] In another preferred embodiment, the present invention provides a method for risk prediction, screening, prognosis assessment, treatment effect monitoring, or recurrence monitoring of nasopharyngeal carcinoma, comprising the following steps:

[0117] (1) Detecting the level (quantity) of the nasopharyngeal carcinoma antibody marker (antibody) provided by the present invention in a sample from a subject;

[0118] (2) comparing the NPC antibody marker level (or the amount of the antibody) with a reference threshold value (hereinafter referred to as the CUT-OFF value); and when the level is greater than or equal to the CUT-OFF value, determining that the subject has a risk of NPC, has NPC, has a poor prognosis for NPC, or has a poor treatment effect on NPC.

[0119] 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.

[0120] In step (2), the CUT-OFF value may be a reference level from a healthy person or a healthy population; for example, it may be defined as the mean value of the antibody test results of a population confirmed to be cancer-free by physical examination plus 2 standard deviations.

[0121] Reagent test kit

[0122] Based on the correlation between NPC antibody markers and NPC risk, NPC antibody markers can be used as markers for judging NPC risk.

[0123] The present invention also provides a kit for determining or assessing the risk of nasopharyngeal carcinoma, the kit comprising a detection reagent for detecting the level of a nasopharyngeal carcinoma antibody marker. Preferably, the kit comprises an antigen or immunoconjugate of a nasopharyngeal carcinoma antibody marker (antibody) of the present invention, or an active fragment thereof.

[0124] In another preferred embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) test kit. That is, the kit is used to detect whether a subject's sample is positive for antibodies and / or antigens using an ELISA assay. Accordingly, the kit may also include other components required for ELISA detection of the antibodies and / or antigens, all of which are 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 a His tag, a streptavidin tag, or a Myc tag. In another example, the kit may include a solid phase support, such as a support with micropores for immobilizing the antigen protein and / or antibody, such as an ELISA plate, or a microbead or magnetic bead solid phase support. It may also include an adsorbent protein for immobilizing the antigen protein and / or antibody on the solid phase support, a diluent for blood, such as serum, a wash solution, a secondary antibody labeled with an enzyme or a fluorescent or chemiluminescent substance, a color development solution, a stop solution, and the like. The content of corresponding antibodies in body fluids is detected by the principle that antigen proteins and / or antibodies indirectly or directly coated on the surface of a solid phase carrier react with antibodies and / or antigens in serum / plasma / tissue fluid to form antigen-antibody complexes.

[0125] In another preferred embodiment, the kit is a magnetic microparticle chemiluminescence detection kit. The antibody in the sample is mixed with the magnetic beads coated with the corresponding antigen, incubated to form a complex, washed with a cleaning solution to remove substances that have not participated in the reaction, and then mixed with a secondary antibody labeled with a fluorescent or chemiluminescent substance to form a complex, and washed with a cleaning solution to remove substances that have not participated in the reaction. Finally, a substrate solution is added to cause the complex to generate a chemiluminescent signal, and the luminescent signal is collected by a chemiluminometer, and the instrument calculates the detection result through a calibration curve. Accordingly, the kit may also 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 connected to a label peptide, such as a His tag, a streptavidin tag, or a Myc tag; the secondary antibody for the fluorescent or chemiluminescent substance may be an acridinium ester.

[0126] In another preferred embodiment, the kit further comprises a label or instructions.

[0127] The main advantages of the present invention include:

[0128] (a) The antibody combination of the present invention can achieve a sensitivity of 99.2% in nasopharyngeal carcinoma screening.

[0129] (b) The diagnostic performance of the combination of tumor autoantibodies and EB virus antibodies of the present invention is significantly better than the EB virus antibody combination (EA-IgA+VCA-IgA+Rta-IgA) currently used in clinical symptoms.

[0130] (c) The sensitivity of the antibody combination of the present invention in nasopharyngeal carcinoma patients with negative EBV antibodies can reach 97.18% or above.

[0131] (d) The sensitivity of the antibody combination of the present invention in patients with early-stage nasopharyngeal carcinoma can reach 97.22% or above.

[0132] (e) The antibody combination of the present invention can detect different subtypes of nasopharyngeal carcinoma, with a sensitivity of 98.33% or higher for non-keratinizing carcinoma, 97.50% or higher for keratinizing squamous cell carcinoma, and 96.67% or higher for adenocarcinoma.

[0133] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those 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 indicated, percentages and parts are by weight.

[0134] General Methods

[0135] (I) Preparation of recombinant antigen protein

[0136] The cDNA fragment of the tumor antigen is cloned into the PET28(a) expression vector containing a 6XHis tag. Streptavidin or a biotin-binding tag protein is introduced at the N-terminus or C-terminus of the antigen. The resulting recombinant expression vector is transformed into Escherichia coli for expression. The protein expressed in the supernatant is purified using a Ni-NTA affinity column and an ion column. Once the protein is expressed in inclusion bodies, it is denatured with 6M guanidine hydrochloride and folded in vitro according to standard methods. The antigen protein is then purified using a Ni-NTA affinity column via the 6XHis tag to obtain the antigen.

[0137] (2) Preparation and storage of serum or plasma

[0138] Serum or plasma from NPC patients was collected at the time of initial diagnosis and before any chemotherapy, radiotherapy, or surgery. The plasma or serum was prepared according to standard clinical procedures and stored long-term in a -80°C freezer.

[0139] (3) ELISA test

[0140] The amount of antibody markers in a sample is quantified using a sandwich enzyme-linked immunosorbent assay (ELISA). Purified tumor antigens and Epstein-Barr virus antigens are immobilized on the microwell surface via their tags, streptavidin or an analog. The microwells are pre-coated with biotinylated bovine serum albumin (BSA). Serum or plasma samples are diluted 1:110 with phosphate buffer and added to the microwells for reaction (50 ml / well). After washing away unbound serum or plasma components with a wash buffer, horseradish peroxidase (HRP)-conjugated anti-human IgG is added to each well for reaction. The reaction substrate, TMB (3,3',5,5'-tetramethylbenzidine), is then added for color development. Stop solution (1N HCl) is added, and the absorbance (OD) is read on a microplate reader at 450 nm. The amount of enzyme bound to the solid support is positively correlated with the amount of the analyte in the sample. The enzyme catalyzes the substrate to produce a colored product. The degree of color reaction is used to determine the qualitative or quantitative nature of the antibody.

[0141] Magnetic microparticle chemiluminescence detection

[0142] The magnetic microparticle chemiluminescence assay uses a fully automated chemiluminescence analyzer to detect antibodies in the sample. The testing process for each sample is as follows: the sample (10 μL) is incubated with a working solution of magnetic beads coated with an antigen marker (40 μL), and a sample diluent (50 μL) for 18 minutes. The sample is then washed for 5 minutes and reacted with a working solution of an acridinium ester-labeled anti-human IgG antibody (secondary antibody) (50 μL) to form a magnetic microparticle-antigen-secondary antibody-chemiluminescent marker complex. The immune complex is then separated using a magnetic bead separator, free components are washed away, and the chemiluminescent marker is stimulated with a chemiluminescence excitation solution. The fully automated chemiluminescence analyzer automatically completes the test and records the relative luminescence intensity (RLU).

[0143] Example 1: Training Cohort Study on Sensitivity and Specificity of Tumor Antibodies and EB Virus Antibodies

[0144] 1.1 Sample information of the training cohort subjects

[0145] This example includes 184 healthy individuals (age range: 18-88, mean age 54) and 130 NPC patients undergoing physical examinations. The healthy individuals were recruited from at least three different physical examination centers. All NPC patient serum was collected at the time of diagnosis and before receiving any chemotherapy, radiotherapy, or surgery and stored at -80°C. Information on the NPC patients in this example is shown in Table 1.

[0146] Table 1 Sample information of the training cohort subjects

[0147]

[0148] 1.2 Detection Method and Analysis of Sensitivity and Specificity of Individual Antibodies in the Test Population

[0149] Nasopharyngeal carcinoma-associated antigens and Epstein-Barr virus antigens were expressed, purified, and coated onto the surface of a 96-well plate. After blocking, the plate was reacted with nasopharyngeal carcinoma serum or serum from a control group diluted 1:110. The plate was then reacted with an anti-human IgG antibody-HRP horseradish peroxidase enzyme, followed by color development and detection using a microplate reader at OD 450 nm. Tables 2 and 3 show the sensitivity and specificity of the assay.

[0150] Table 2 Sensitivity and specificity of single tumor autoantibodies as antibody markers for nasopharyngeal carcinoma

[0151]

[0152] Note: Both IgG and IgA types of tumor autoantibodies have good sensitivity and specificity.

[0153] IgA is superior to IgG. The present invention describes tumor autoantibodies.

[0154] Table 3 Sensitivity and specificity of single EBV antibodies as antibody markers for nasopharyngeal carcinoma

[0155]

[0156] Note: In current clinical applications, the performance of EB virus IgA antibodies is better than that of IgG antibodies, so the present invention

[0157] Elucidation of EB virus IgA antibodies.

[0158] The scatter plot 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 is shown in the figure. Figure 1 and Figure 2 and Table 4-7.

[0159] Table 4 Distribution of individual antibodies in enzyme-linked immunosorbent assay (OD value, mean ± SD)

[0160]

[0161] Table 5 Distribution of individual antibodies in ELISA in the training cohort (number of subjects)

[0162] (For each autoantibody, number of subjects with ≥ cut-off value)

[0163]

[0164] Table 6 Distribution of individual antibodies in chemiluminescence assay (RLU values, mean ± SD)

[0165]

[0166] Table 7 Distribution of individual antibodies in chemiluminescence assay in the training cohort (number of subjects)

[0167] (For each autoantibody, number of subjects with ≥ cut-off value)

[0168]

[0169] Due to the varying strengths of cancer patients' immune systems and the diverse mechanisms of tumor development, the distribution sensitivity of individual tumor antibodies in cancer patients is low. Statistical analysis of antibody levels between the tumor and control groups using the Mann-Whitney U test revealed significant differences in the distribution of antibodies (anti-TPI1, anti-PRKACA, anti-TRIM21, anti-SOX2, anti-BMI, anti-PDE4DIP, anti-GATA3, BKRF1-IgA, BMRF1-IgA, and BALF2-IgA) between the tumor and control groups (p<0.05).

[0170] 1.3 Screening of Antibody Combinations

[0171] Based on the detection results of individual candidate antibodies in the population, while ensuring the high specificity of individual antibodies, the individual positive contributions of the antibodies were combined (i.e., candidate molecules with high overlapping positive detection rates were excluded) to maximize the coverage of the detection model for more nasopharyngeal carcinoma patients. Different antibody combinations were formed and tested using corresponding detection reagents. The results are shown in Table 8.

[0172] Table 8 Sensitivity and specificity of antibody combinations in the training cohort

[0173]

[0174] Note: EA (P54-BMRF1, P138-BALF2), VCA (p18- BFRF3, P23-BLRF2), RTA.

[0175] As shown in Table 5, the diagnostic performance of the combination of tumor autoantibodies and Epstein-Barr virus antibodies of the present invention is significantly superior to the Epstein-Barr virus antibody combination (EA-IgA + VCA-IgA + Rta-IgA) currently used in clinical settings. Furthermore, the performance of the antibody combination is superior to that of individual antibodies, increasing the detection rate in NPC patients while maintaining specificity in healthy subjects.

[0176] As shown in Table 5, the antibody combination (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, and BALF2-IgA) offers the best sensitivity and specificity. Therefore, further performance studies were conducted on this antibody combination. This performance study is shown in Section 1.4 below.

[0177] 1.4 Receiver Operating Characteristic (ROC) Analysis of the Combined Detection Model of Tumor Autoantibodies and EBV Antibodies in the Human Population

[0178] The present invention further uses ROC curve analysis to determine the screening ability of the antibody combination of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21 and BKRF1-IgA, BMRF1-IgA, BALF2-IgA) for nasopharyngeal carcinoma patients in the population.

[0179] like Figure 3 As shown, compared with the healthy population under physical examination, 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%, and the area under the curve is 0.993.

[0180] 1.5 The combined detection model of tumor autoantibodies and EB virus antibodies of the present invention has ideal detection capabilities for early nasopharyngeal carcinoma

[0181] The inventors classified the pathological stages of nasopharyngeal carcinoma (NPC) in a cohort of 72 patients with early-stage NPC (stages 0, I, and II) and 58 with advanced NPC (stages III and IV), for a total of 130 patients. Fifty-nine of these patients were also infected with Epstein-Barr virus (EBV). Using the antibody combination detection model of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, and BALF2-IgA), serum was tested using an enzyme-linked immunosorbent assay (ELISA). The data obtained were analyzed for the ability of the antibody combination to detect NPC at different stages. Evaluation was performed using the reference values ​​in Table 9 below.

[0182] Table 9

[0183]

[0184] Rules for judging yin and yang:

[0185] Any OD450 value detected by one or more indicators was greater than or equal to its corresponding CUT-OFF value and was recorded as positive;

[0186] All the indexes tested with OD450 values ​​less than their corresponding CUTOFF values ​​were recorded as negative;

[0187] The detection model of the present invention has an overall sensitivity of 98.46% for nasopharyngeal carcinoma and a specificity of 94.02%. It has a sensitivity of 98.61% for early-stage nasopharyngeal carcinoma and 98.28% for late-stage nasopharyngeal carcinoma, demonstrating its ideal detection capabilities for early-stage nasopharyngeal carcinoma.

[0188] 1.6 The combined detection model of tumor autoantibodies and EB virus antibodies of the present invention has ideal detection capabilities for different subtypes of nasopharyngeal carcinoma

[0189] The inventors of the present invention classified the test results of different nasopharyngeal carcinoma pathological subtypes, including 60 patients with nasopharyngeal non-keratinizing squamous cell carcinoma, 40 patients with nasopharyngeal keratinizing squamous cell carcinoma and 30 patients with nasopharyngeal adenocarcinoma, totaling 130 nasopharyngeal carcinoma patients, of which 59 patients with nasopharyngeal carcinoma were simultaneously infected with Epstein-Barr virus, and 71 patients with nasopharyngeal carcinoma were not simultaneously infected with Epstein-Barr virus.

[0190] Using the antibody combination detection model of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21 and BKRF1-IgA, BMRF1-IgA, BALF2-IgA), serum was tested by enzyme-linked immunosorbent assay (ELISA). The obtained test data were analyzed for the ability of the antibody combination to detect different histopathological subtypes of nasopharyngeal carcinoma, regardless of whether the nasopharyngeal carcinoma patients were infected with Epstein-Barr virus. The reference values ​​in Table 8 were used for evaluation.

[0191] Rules for judging yin and yang:

[0192] Any OD450 value detected by one or more indicators was greater than or equal to its corresponding CUT-OFF value and was recorded as positive;

[0193] All the indexes tested with OD450 values ​​less than their corresponding CUT-OFF values ​​were recorded as negative;

[0194] The detection model of the present invention has a sensitivity of 100% for non-keratinizing nasopharyngeal carcinoma, 97.50% for keratinizing squamous cell carcinoma of the nasopharynx, and 96.67% for nasopharyngeal adenocarcinoma. The sensitivity is 100% for nasopharyngeal carcinoma patients who are also infected with Epstein-Barr virus (EBV), and 97.18% for nasopharyngeal carcinoma patients who are not infected with EBV. The detection model of the present invention has good sensitivity for all nasopharyngeal carcinoma subtypes, regardless of whether the nasopharyngeal carcinoma patients are infected with EBV.

[0195] 1.7 Detection of tumor autoantibodies and EB virus antibodies using magnetic microparticle chemiluminescence The combined detection model of the present invention has ideal detection capabilities for early-stage nasopharyngeal carcinoma and different subtypes of nasopharyngeal carcinoma

[0196] The inventors of the present invention classified and statistically analyzed the test results for different NPC stages and pathological subtypes. The different NPC stages included 72 patients with early-stage (stage 0, I, and II) NPC and 58 patients with advanced (stage III and IV) NPC, for a total of 130 NPC patients. Of these, 59 NPC patients were co-infected with Epstein-Barr virus (EBV), while 71 were not. The different histopathological subtypes included 60 patients with non-keratinizing NPC, 40 with keratinizing squamous cell carcinoma, and 30 with NPC adenocarcinoma. Using magnetic microparticle chemiluminescence, the antibody combination detection model of the present invention (anti-TPI1, anti-PRKACA, anti-TRIM21, and BKRF1-IgA, BMRF1-IgA, and BALF2-IgA) was analyzed for its ability to detect NPC at different stages and histopathological subtypes.

[0197] Evaluate according to the reference values ​​in Table 10 below.

[0198] Table 10

[0199]

[0200] Rules for judging yin and yang:

[0201] Any RLU value of one or more indicators detected is greater than or equal to its corresponding CUT-OFF value and is recorded as positive;

[0202] The RLU values ​​of all indicators tested were less than their corresponding CUT-OFF values ​​and were recorded as negative;

[0203] The detection model of the present invention has a sensitivity of 97.22% for patients with early-stage NPC (stages 0, I, and II) and 98.28% for patients with late-stage NPC (stages III and IV). The sensitivity for detecting NPC patients with concurrent EBV infection is 98.59%, while the sensitivity for detecting NPC patients without concurrent EBV infection is 100%. The detection model of the present invention has high sensitivity for all NPC stages, regardless of whether the patient is infected with EBV.

[0204] The detection model of the present invention has a sensitivity of 98.33% for non-keratinizing nasopharyngeal carcinoma, 97.50% for keratinizing squamous cell carcinoma of the nasopharynx, and 96.67% for nasopharyngeal adenocarcinoma. The sensitivity is 98.59% for nasopharyngeal carcinoma patients who are also infected with Epstein-Barr virus (EBV), and 100% for nasopharyngeal carcinoma patients who are not infected with EBV. The detection model of the present invention has good sensitivity for various nasopharyngeal carcinoma subtypes, regardless of whether the nasopharyngeal carcinoma patients are infected with EBV.

[0205] Example 2 Validation Cohort Validation of Antibody Combination Sensitivity and Specificity

[0206] 2.1 Validation cohort sample information

[0207] This example includes 150 healthy individuals (age range: 18-88, mean age 56) and 150 nasopharyngeal carcinoma patients undergoing physical examinations. The healthy individuals were recruited from at least three different physical examination centers. All nasopharyngeal carcinoma patient serum was collected at the time of diagnosis and before any chemotherapy, radiotherapy, or surgery, and stored at -80°C. Information on the nasopharyngeal carcinoma patients in this example is shown in Table 11.

[0208] Table 11 Sample information of the validation cohort subjects

[0209]

[0210] 2.2 Number of subjects meeting the cut-off values ​​measured by enzyme-linked immunosorbent assay and chemiluminescence assay

[0211] In this example, two methods were used in the validation cohort to measure the number of subjects with a cut-off value for each autoantibody marker, as shown in Tables 12-13.

[0212] Table 12 Distribution of individual antibodies in the ELISA assay in the validation cohort (number of subjects)

[0213] (For each autoantibody, number of subjects with ≥ cut-off value)

[0214]

[0215] Table 13 Distribution of individual antibodies in the chemiluminescence assay in the validation cohort (number of subjects)

[0216] (For each autoantibody, number of subjects with ≥ cut-off value)

[0217]

[0218] 2.3 Enzyme-linked immunosorbent assay (ELISA) to analyze the sensitivity and specificity of antibody combinations

[0219] Based on the enzyme-linked immunosorbent assay and CUT-OFF values ​​determined by the training cohort, 150 samples from nasopharyngeal carcinoma patients and 150 samples from healthy people undergoing physical examinations were tested, and the sensitivity and specificity of the antibody combination were analyzed.

[0220] The sensitivity and specificity of the antibody combination of this example are shown in Table 14.

[0221] Table 14 Sensitivity and specificity of the enzyme-linked immunosorbent assay validation cohort

[0222]

[0223] Note: The detection sensitivity of early nasopharyngeal carcinoma, advanced nasopharyngeal carcinoma, non-keratinizing nasopharyngeal carcinoma, keratinizing squamous cell carcinoma of the nasopharynx, and nasopharyngeal adenocarcinoma is based on the test results of the antibody combination (anti-TPI1, anti-PRKACA, anti-TRIM21 and BKRF1-IgA, BMRF1-IgA, BALF2-IgA).

[0224] From the above results, it can be seen that the sensitivity and specificity of the detection results of the validation cohort subjects using enzyme-linked immunosorbent assay are consistent with those of the training cohort detection results.

[0225] This example uses an enzyme-linked immunosorbent assay (ELISA) with a sensitivity of 98.80% for early-stage (stage 0, I, and II) NPC patients and 98.51% for late-stage (stage III and IV) NPC patients. The sensitivity for non-keratinizing NPC is 100%, for keratinizing squamous cell carcinoma of the NPC is 97.78%, and for nasopharyngeal adenocarcinoma is 97.14%. The sensitivity for NPC patients co-infected with Epstein-Barr virus (EBV) is 100%, and for NPC patients co-infected with EBV is 97.73%. The detection model of this invention has excellent sensitivity for various NPC stages and histopathological subtypes, regardless of whether the NPC patient is infected with EBV.

[0226] 2.4 Magnetic Particle Chemiluminescence Detection and Analysis of Antibody Combination Sensitivity and Specificity

[0227] Based on the magnetic microparticle chemiluminescence assay and cut-off values ​​determined for the training cohort, 150 samples from nasopharyngeal carcinoma patients and 150 samples from healthy individuals undergoing physical examinations were tested to analyze the sensitivity and specificity of the antibody combination. The sensitivity and specificity of the antibody combination in this example are shown in Table 15.

[0228] Table 15 Sensitivity and specificity of the magnetic microparticle chemiluminescence assay in the validation cohort

[0229]

[0230] Note: The detection sensitivity of early nasopharyngeal carcinoma, advanced nasopharyngeal carcinoma, non-keratinizing nasopharyngeal carcinoma, keratinizing squamous cell carcinoma of the nasopharynx, and nasopharyngeal adenocarcinoma is based on the test results of the antibody combination (anti-TPI1, anti-PRKACA, anti-TRIM21 and BKRF1-IgA, BMRF1-IgA, BALF2-IgA).

[0231] From the above results, it can be seen that the sensitivity and specificity of the detection results of the validation cohort subjects using the magnetic microparticle chemiluminescence method are consistent with those of the training cohort detection results.

[0232] This example uses magnetic microparticle chemiluminescence detection technology, achieving a sensitivity of 100% for early-stage (stage 0, I, and II) NPC patients and 95.56% for late-stage (stage III and IV) NPC patients. The sensitivity for non-keratinizing NPC was 100%, for keratinizing squamous cell carcinoma of the NPC was 97.78%, and for nasopharyngeal adenocarcinoma was 97.14%. The sensitivity for NPC patients co-infected with Epstein-Barr virus (EBV) was 98.39%, while the sensitivity for NPC patients co-infected with EBV was 97.73%. The detection model of this invention demonstrates excellent sensitivity across various NPC stages and histopathological subtypes, regardless of whether the patient is infected with EBV.

[0233] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of a nasopharyngeal carcinoma antibody marker or a detection reagent thereof in the preparation of a diagnostic reagent or a diagnostic kit for determining the risk of nasopharyngeal carcinoma, characterized in that: The nasopharyngeal carcinoma antibody markers include a combination of the following antibodies: (B) EBV antibodies: (B1) BKRF1-IgA; (B2) BMRF1-IgA; and (B3) BALF2-IgA; and (A) An autoantibody, wherein the autoantibody comprises 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, characterized in that The autoantibody further includes any 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 marker is an antibody combination 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, wherein 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 4, 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, wherein 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 nasopharyngeal carcinoma antibody markers; wherein 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) An autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21.

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

9. The collection according to claim 8, wherein The autoantibody further includes any autoantibody selected from the following group, or a combination thereof: (A4) anti-SOX2; (A5) anti-BMI; (A6) anti-PDE4DIP; (A7) anti-GATA3.

10. A nasopharyngeal carcinoma risk assessment device, characterized in that: The device comprises: (a) An input module configured to input nasopharyngeal carcinoma antibody marker data in a sample to be tested; the nasopharyngeal carcinoma antibody marker data is the level of the nasopharyngeal carcinoma antibody marker 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) an autoantibody, wherein the autoantibody comprises any one autoantibody selected from the following group, or a combination thereof: (A1) anti-TPI1; (A2) anti-PRKACA; (A3) anti-TRIM21; (b) a risk assessment module, wherein the risk assessment module is configured to evaluate the input nasopharyngeal carcinoma antibody marker data to obtain a risk assessment result; the evaluation includes: Comparing the nasopharyngeal carcinoma antibody marker level with a control reference value or a cut-off value; when the nasopharyngeal carcinoma antibody marker level is ≥ the control reference value or the cut-off value, it indicates that the subject is at high risk of nasopharyngeal carcinoma; otherwise, it indicates that the subject is not at high risk of nasopharyngeal carcinoma; (c) an output module, wherein the output module is configured to output the evaluation result.

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