Use of anti-bglf5 protein antibody level as nasopharyngeal carcinoma marker

Abstract

The application provides application of an anti-BGLF5 protein antibody level as a nasopharyngeal carcinoma marker. The application finds, through research and screening, that the anti-BGLF5 protein antibody level in a nasopharyngeal swab sample of a nasopharyngeal carcinoma patient is significantly higher than that of a healthy control group, indicating that the anti-BGLF5 protein antibody level can be used as a specific biomarker for nasopharyngeal carcinoma. Further receiver operating characteristic curve analysis results show that, when the anti-BGLF5 protein antibody is used as a biomarker to diagnose a nasopharyngeal carcinoma patient, the AUC is 0.88, the sensitivity is 84% when the cutoff value is set to 0.31, the specificity is 80%, the sensitivity and the specificity are relatively high, and the diagnostic accuracy is good. Therefore, the anti-BGLF5 protein antibody can be used as a specific biomarker for nasopharyngeal carcinoma, and a reagent for detecting the anti-BGLF5 protein antibody level in a biological sample can be used to prepare a nasopharyngeal carcinoma detection kit to assist clinical diagnosis of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular diagnostic technology, specifically relating to the application of anti-BGLF5 protein antibody levels as a biomarker for nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma is a malignant tumor originating from the epithelial tissue of the nasopharynx. It has a high incidence rate among head and neck tumors and exhibits a clear regional clustering pattern, being particularly common in southern my country and Southeast Asia. Epidemiological studies show that the incidence rate is significantly higher in men than in women, and it is more prevalent in middle-aged individuals. Its development is closely related to multiple factors, including persistent Epstein-Barr virus (EBV) infection, individual genetic background, and dietary and environmental exposures. Among these, EBV infection is considered to play a key role in tumorigenesis.

[0003] Because nasopharyngeal carcinoma lacks specific clinical manifestations in its early stages, most patients are already in the advanced stage at the time of diagnosis, affecting treatment outcomes and survival rates. Therefore, establishing a sensitive, specific, and population-appropriate early detection method is of great significance. Currently, the commonly used screening methods in clinical practice mainly rely on the detection of serum EBV-related antibodies, such as the combined detection of VCA-IgA and EBNA1-IgA. However, these methods have limited positive predictive ability and are prone to producing many false positive results, requiring further confirmation through nasopharyngoscopy or imaging examinations, which not only increases medical costs but also affects screening efficiency and population compliance. In recent years, although new serological markers and plasma EBV-DNA detection technologies have been continuously developed, improving screening performance to some extent, problems such as complex detection procedures, high costs, and difficulties in balancing sensitivity and specificity still exist, and they cannot fully meet the needs of large-scale early screening.

[0004] From a pathological perspective, nasopharyngeal carcinoma originates from local epithelial tissue, and EBV primarily lies dormant and replicates within the nasopharyngeal mucosal epithelial cells. Therefore, compared to peripheral blood samples, samples from the nasopharynx are more likely to directly reflect tumor-related viral infection status and local immune response characteristics. Based on this, using nasopharyngeal swabs to obtain local samples for testing has potential advantages. Nasopharyngeal swab sampling is a non-invasive procedure that directly collects secretions, exfoliated cells, and related biomolecules from the nasopharynx, offering advantages such as ease of operation, good subject tolerance, and suitability for widespread adoption at the grassroots level. Furthermore, this method has the potential for standardized and home-based sampling applications, which is beneficial for expanding screening coverage. However, current technologies for detecting nasopharyngeal swab samples primarily focus on the nucleic acid level and lack stable, reliable biomarker systems and standardized detection methods.

[0005] Therefore, developing efficient and specific diagnostic biomarkers for nasopharyngeal carcinoma based on EBV in nasopharyngeal swab samples is of great significance for improving the accuracy and feasibility of early screening for nasopharyngeal carcinoma. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide the application of anti-BGLF5 protein antibody levels as a biomarker for nasopharyngeal carcinoma.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] The first aspect of the present invention provides the use of a reagent for detecting the level of anti-BGLF5 protein antibody in a biological sample in the preparation of a nasopharyngeal carcinoma detection product.

[0009] In some embodiments, the reagents for detecting the level of anti-BGLF5 protein antibodies in biological samples include reagents used for detection by ELISA, protein chip assay, chemiluminescent immunoassay, radioimmunoassay, and fluorescence immunoassay.

[0010] In some embodiments, the reagent used to detect the level of anti-BGLF5 protein antibody in a biological sample is a reagent used for detection by ELISA.

[0011] In some embodiments, the reagent used to detect the level of anti-BGLF5 protein antibody in a biological sample includes the BGLF5 protein.

[0012] In some embodiments, the amino acid sequence of the BGLF5 protein is shown in SEQ ID NO: 2.

[0013] In some embodiments, the biological sample includes nasopharyngeal swabs, serum, plasma, saliva, and urine.

[0014] In some implementations, the biological sample is a nasopharyngeal swab.

[0015] In some implementations, the detection product is a test kit.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] This invention, through research and screening, discovered that the level of anti-BGLF5 protein antibody in nasopharyngeal swab samples from nasopharyngeal carcinoma patients was significantly higher than that in healthy controls, suggesting that it can serve as a specific biomarker for nasopharyngeal carcinoma. Further receiver operating characteristic (ROC) curve analysis showed that when using anti-BGLF5 protein antibody as a biomarker to diagnose nasopharyngeal carcinoma patients, the AUC was 0.88. With a cutoff value set at 0.31, the sensitivity was 84% ​​and the specificity was 80%, demonstrating high sensitivity and specificity, and good diagnostic accuracy.

[0018] Therefore, anti-BGLF5 protein antibodies can serve as specific biomarkers for nasopharyngeal carcinoma, and reagents for detecting the level of anti-BGLF5 protein antibodies in biological samples can be used to prepare diagnostic kits for nasopharyngeal carcinoma, thus aiding in the clinical diagnosis of nasopharyngeal carcinoma. Attached Figure Description

[0019] Figure 1 Preliminary screening results using EBV peptide detection.

[0020] Figure 2 The results are for the detection of candidate antibodies obtained from the initial screening.

[0021] Figure 3 The results of anti-BGLF5 protein antibody level detection were used to screen cohorts of nasopharyngeal carcinoma patients and healthy controls.

[0022] Figure 4 To validate the results of anti-BGLF5 protein antibody level detection in nasopharyngeal carcinoma patients and healthy controls in the cohort.

[0023] Figure 5 ROC curve analysis results for using anti-BGLF5 protein antibody as a biomarker to diagnose nasopharyngeal carcinoma. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0026] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0027] The anti-BGLF5 protein antibody described in this invention refers to an antibody that can specifically bind to the BGLF5 protein, and is also referred to below as a specific antibody against the BGLF5 protein.

[0028] The EB virus BGLF5 protein is a core multifunctional protein expressed in the early stage of EB virus lysis. It mainly functions as an alkaline exonuclease (AE) and has both DNase and RNase activities. Example 1

[0029] This embodiment utilizes B-cell epitope prediction combined with synthetic peptide technology to systematically screen for antigens encoded by open reading frames (ORFs) of the entire Epstein-Barr virus (EBV) genome. A total of 86 specific peptides derived from EBV proteins were designed and successfully synthesized, with biotinylated labels introduced at their ends. These biotinylated peptides can serve as molecular probes, specifically binding to EBV-specific antibodies in nasopharyngeal swab samples. To achieve standardized management, all peptides were uniformly numbered according to their synthesis sequence, using a naming convention of "protein name – sequence number," where the protein name represents the source protein of the peptide, and the sequence number indicates its position among all peptides. For example, "BYRF1-6" indicates a peptide derived from the BYRF1 protein, and this peptide is the 6th of the 86 synthesized peptides.

[0030] In the exploratory phase of biomarker screening, nasopharyngeal swab samples from 8 patients with pathologically confirmed nasopharyngeal carcinoma and 3 healthy controls were included. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IgA and IgG antibodies corresponding to 86 EB virus peptides.

[0031] The ELISA detection method includes the following steps: 1. Dilute the peptide to 1 mg / ml with PBS; 2. Add 50 ng of peptide per well to a 96-well microplate and incubate overnight at 4°C; 3. The next day, wash twice with PBST (PBS + 0.1% Tween); 4. Add 200 μL of blocking buffer (PBS containing 1% BSA) to each well, incubate at 37°C for 2 hours; 5. After blocking, discard the blocking solution, add 100 μL of diluted nasal secretions (diluted 10 times with PBS) to each well, and react at 37°C for 30 minutes; 6. After the reaction is complete, wash 5 times with PBST, and add diluted HRP-labeled goat anti-human IgA or IgG. React at 37°C for 30 minutes. 7. After washing 5 times with PBST, add 100 μL of TMB chromogenic solution to each well and incubate at 37°C for 15 minutes; 8. Add 50 μL of stop solution to each well, mix well, and then measure the absorbance at 450 and 630 nm using a microplate reader.

[0032] Preliminary screening results are as follows Figure 1 As shown, according to Figure 1 The results were further used to screen candidate markers. The screening criteria were as follows: for the same polypeptide, if the IgA and IgG antibodies showed statistically significant differences in the case group and the control group (P < 0.05), they were screened as potential candidate markers.

[0033] Subsequently, eight nasopharyngeal carcinoma patients and eight healthy controls were included in the expanded sample to conduct preliminary validation of the above candidate biomarkers, using the same detection method as above.

[0034] Based on the results of expression differential analysis and fold change calculation, the BGLF5-62 peptide (amino acid sequence information as shown in SEQ ID NO: 1) and its corresponding antibody were screened out. The IgA and IgG levels showed consistent differences between the two groups, suggesting its potential diagnostic value. Figure 2 ).

[0035] SEQ ID NO: 1: ETNEGLEYDEDSENDELLFL.

[0036] Based on this, the protein sequence of the key peptide BGLF5 obtained through screening was optimized, and the full-length antigen of the target protein (BGLF5 protein) was further synthesized (amino acid sequence information is shown in SEQ ID NO: 2), and biotinylate labeling was performed at the ends. Based on the streptavidin-biotin high-affinity system, the biotinylated antigen was directionally immobilized on the surface of a solid-phase carrier pre-coated with streptavidin, thereby constructing a stable and reproducible ELISA detection method. The ELISA detection method uses a double-antigen sandwich method, i.e., antigen-antibody-antigen, and can detect the level of total anti-BGLF5 protein antibodies (including different anti-BGLF5 protein Ig antibodies) in nasopharyngeal swab samples.

[0037] SEQ ID NO: 2: MADVDELEDPMEEMTSYTFARFLRSPETEAFVRNLDRPPQMPAMRYVYLYCLCKQIQEFSGETGFCDFVSSLVQENDSQDGPSLKSIYWGLQEATDEQRTVLCSYVESMTRGQSENL MWDILRNGIISSSKLLSTIKNGPTKVFEPAPISTNHYFGGPVAFGLRCEDTVKDIVCKLICGDASANRQFGFMISPTDGIFGVSLDLCVNVESQGDFILFTDRSCIYEIKCRFKYLFS KSEFDPIYPSYTALYKRPCKRSFIRFINSIARPTVEYVPDGRLPSEGDYLLTQDEAWNLKDVRKRKLGPGHDLVADSLAANRGVESMLYVMTDPSENAGRIGIKDRVPVNIFINPRHN YFYQVLLQYKIVGDYVRHSGGGKPGRDCSPRVNIVTAFFRKRSPLDPATCTLGSDLLLDASVEIPVAVLVTPVVLPDSVIRKTLSTAAGSWKAYADNTFDTAPWVPSGLFADDESTP.

[0038] The ELISA detection method includes the following steps: 1. Dilute BGLF5 protein to 1 mg / ml with PBS; 2. Add 50 ng of BGLF5 protein to each well of a 96-well microplate and incubate at 37°C for 2 hours; 3. After the reaction is complete, wash twice with PBST; 4. Add 200 μL of blocking solution to each well and incubate at 37°C for 2 hours; 5. After sealing is complete, discard the sealing solution; 6. Add 50 μL of the nasal secretion dilution buffer (diluted 10 times with PBS) and 50 μL of biotin-labeled BGLF5 protein dilution buffer (concentration of 1 mg / ml) to each well, and incubate at 37°C for 1 hour. 7. Washing: Wash the test plate 5 times with PBST and then blot dry; 8. Add SA-HRP: Add 1 / 5000 of SA-HRP to the microplate and incubate at 37°C for 30 minutes; 9. Washing: Wash the detection plate 5 times as above and pat it dry; 10. Color development: Add 100 μL of TMB to each well of the microplate and incubate at 37°C for 15 minutes. 11. Termination: Add the reaction termination solution to the reaction plate at a rate of 50 μL / well; 12. Reading: Detects absorbance at dual wavelengths of 450 nm and 630 nm.

[0039] Nasopharyngeal swab samples from 20 patients with nasopharyngeal carcinoma and 20 healthy controls were first tested. Results ( Figure 3 The results showed a significant difference in anti-BGLF5 protein antibody levels between the two groups, with significantly higher levels in nasopharyngeal swab samples from nasopharyngeal carcinoma patients compared to healthy controls. These findings suggest that anti-BGLF5 protein antibody could serve as a potential biomarker for nasopharyngeal carcinoma detection. Example 2

[0040] This embodiment further includes 92 nasopharyngeal carcinoma patients and 110 healthy controls as a validation cohort to detect and analyze the diagnostic efficacy of anti-BGLF5 protein antibody levels.

[0041] The method for detecting the level of anti-BGLF5 protein antibody is the same as in Example 1.

[0042] The results of the anti-BGLF5 protein antibody level detection were consistent with the examples; the level of anti-BGLF5 protein antibody in nasopharyngeal swab samples from nasopharyngeal carcinoma patients was significantly higher than that in healthy controls. Figure 4 ).

[0043] Receiver operating characteristic (ROC) curves were constructed based on ELISA test data from the validation cohort to evaluate the diagnostic efficacy of the anti-BGLF5 protein antibody. Results ( Figure 5 The results showed that the area under the curve (AUC) was 0.88, and the sensitivity was 84% ​​and the specificity was 80% when the cutoff value was set to 0.31. This indicates that the anti-BGLF5 protein antibody can be used as a relatively ideal diagnostic marker for nasopharyngeal carcinoma and has good diagnostic performance.

[0044] In conclusion, anti-BGLF5 protein antibodies have relatively ideal diagnostic performance in distinguishing nasopharyngeal carcinoma patients from healthy individuals and can be used as a specific detection biomarker for nasopharyngeal carcinoma.

[0045] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0046] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of reagents for detecting anti-BGLF5 protein antibody levels in biological samples in the preparation of nasopharyngeal carcinoma detection products.

2. The application as described in claim 1, characterized in that, The reagents used to detect the level of anti-BGLF5 protein antibodies in biological samples include reagents used in ELISA, protein chip assay, chemiluminescent immunoassay, radioimmunoassay, and fluorescence immunoassay.

3. The application as described in claim 2, characterized in that, The reagent used to detect the level of anti-BGLF5 protein antibody in biological samples is the same reagent used for ELISA detection.

4. The application as described in claim 2 or 3, characterized in that, The reagent used to detect the level of anti-BGLF5 protein antibody in biological samples includes the BGLF5 protein.

5. The application as described in claim 4, characterized in that, The amino acid sequence of the BGLF5 protein is shown in SEQ ID NO:

2.

6. The application as described in claim 1, characterized in that, The biological samples include nasopharyngeal swabs, serum, plasma, saliva, and urine.

7. The application as described in claim 6, characterized in that, The biological sample was a nasopharyngeal swab.

8. The application as described in any one of claims 1 to 7, characterized in that, The testing product is a test kit.

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