Application of anti-BBRF2 protein antibody levels as a biomarker for nasopharyngeal carcinoma

Abstract

This invention provides the application of anti-BBRF2 protein antibody levels as a biomarker for nasopharyngeal carcinoma. Through research and screening, this invention found that the level of anti-BBRF2 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 the AUC was 0.83 when using anti-BBRF2 protein antibody as a biomarker to diagnose nasopharyngeal carcinoma patients; when the cutoff value was set to 0.24, the corresponding sensitivity was 82%, and the specificity was 83%, indicating high diagnostic accuracy. Therefore, anti-BBRF2 protein antibody can serve as a specific biomarker for nasopharyngeal carcinoma, possessing high sensitivity and specificity, and can be used to assist in the diagnosis of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to the application of anti-BBRF2 protein antibody levels as a biomarker for nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma is a type of malignant tumor with a distinct geographical distribution, exhibiting a higher incidence in southern China. Its occurrence is closely related to persistent Epstein-Barr virus (EBV) infection. EBV, a B-cell and epithelial cell-tropic herpesvirus, is considered to have an important molecular basis for the development of nasopharyngeal carcinoma due to its latent infection and reactivation process in nasopharyngeal epithelial cells.

[0003] Currently, the most widely used clinical methods for early screening and auxiliary diagnosis of nasopharyngeal carcinoma based on EBV-related biomarkers mainly include serum EBV antibody detection and plasma EBV DNA quantification. Serological testing methods have advantages such as simplicity and low cost, but their results are easily affected by individual immune differences and other infectious factors, and their specificity is somewhat limited. Furthermore, the EBV genome encodes various structural and non-structural proteins, which express differently at different stages of infection, resulting in diverse host immune responses. Existing detection methods mostly focus on a few classic antigens, which may not fully reflect the complexity of EBV infection.

[0004] Therefore, there is a need in this field for more sensitive and specific indicators to aid in the diagnosis of nasopharyngeal carcinoma. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide the application of anti-BBRF2 protein antibody levels as a biomarker for nasopharyngeal carcinoma.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention provides the use of a reagent for detecting the level of anti-BBRF2 protein antibody in a biological sample in the preparation of a nasopharyngeal carcinoma detection product.

[0008] In some embodiments, the reagents for detecting the level of anti-BBRF2 protein antibodies in biological samples include reagents used for detection by ELISA, protein chip assay, chemiluminescent immunoassay, radioimmunoassay, and fluorescence immunoassay.

[0009] In some embodiments, the reagent used to detect the level of anti-BBRF2 protein antibody in a biological sample is a reagent used for detection by ELISA.

[0010] In some embodiments, the reagent used to detect the level of anti-BBRF2 protein antibodies in a biological sample includes the BBRF2 protein.

[0011] In some embodiments, the amino acid sequence of the BBRF2 protein is shown in SEQ ID NO: 2.

[0012] In some embodiments, the biological sample includes nasopharyngeal swabs, serum, plasma, saliva, and urine.

[0013] In some implementations, the biological sample is a nasopharyngeal swab.

[0014] In some implementations, the detection product is a test kit.

[0015] Compared with the prior art, the present invention has the following beneficial effects.

[0016] This invention, through research and screening, discovered that the level of anti-BBRF2 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 the AUC was 0.83 when using anti-BBRF2 protein antibody as a biomarker to diagnose nasopharyngeal carcinoma patients; when the cutoff value was set to 0.24, the corresponding sensitivity was 82%, the specificity was 83%, and the diagnostic accuracy was high.

[0017] Therefore, anti-BBRF2 protein antibodies can serve as a specific biomarker for nasopharyngeal carcinoma, exhibiting high sensitivity and specificity, and can be used to assist in the diagnosis of nasopharyngeal carcinoma. Reagents for detecting anti-BBRF2 protein antibody levels in biological samples can be used to prepare diagnostic kits for nasopharyngeal carcinoma. Attached Figure Description

[0018] Figure 1 These are the preliminary screening results for EBV peptides.

[0019] Figure 2 The results are for the detection of candidate biomarkers obtained from the initial screening.

[0020] Figure 3 The results show the levels of anti-BBRF2 protein antibodies in nasopharyngeal carcinoma patients and healthy controls in the initial screening cohort.

[0021] Figure 4 To validate the results of anti-BBRF2 protein antibody level detection in nasopharyngeal carcinoma patients and healthy controls in the cohort.

[0022] Figure 5 The results are from the ROC curve analysis. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The anti-BBRF2 protein antibody described in this invention refers to an antibody that can specifically bind to the BBRF2 protein, and is also referred to below as a specific antibody against the BBRF2 protein.

[0027] The EB virus BBRF2 protein is a mesenchymal protein expressed during the lysis phase of EB virus, and its core functions are concentrated in viral assembly, enhanced infectivity, and immune evasion. Example 1

[0028] This embodiment uses B-cell epitope prediction as a theoretical basis and combines peptide synthesis technology to systematically mine and screen for antigens encoded by open reading frames (ORFs) of the Epstein-Barr virus (EBV) genome. Through analysis and design of candidate antigen sequences, 86 specific peptides derived from different EBV proteins were obtained, and each peptide was modified with biotin at its end. These biotinylated peptides can be used as detection probes to specifically bind to EBV-specific antibodies present in nasopharyngeal swab samples. To facilitate subsequent experimental analysis and data management, all peptides were numbered in a predetermined order and named using a "source protein name-number" format, where the protein name indicates the peptide's origin, and the number represents its sequence number among all candidate peptides. For example, "BYRF1-6" indicates that the peptide originates from the BYRF1 protein and is the 6th of the 86 synthesized peptides.

[0029] In the initial screening stage, 8 patients with pathologically confirmed nasopharyngeal carcinoma and 3 healthy controls were selected. Nasopharyngeal swab samples were collected from them, and the levels of IgA and IgG antibodies corresponding to 86 peptides were detected by enzyme-linked immunosorbent assay (ELISA).

[0030] 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.

[0031] 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: the IgA and IgG antibodies corresponding to the same polypeptide showed statistically significant differences between the case group and the control group (P<0.05). Those that met the criteria were selected as candidate markers and entered the next step of analysis.

[0032] Based on the initial screening, the sample size was further expanded to include 8 nasopharyngeal carcinoma patients and 8 healthy controls. The levels of the candidate indicators obtained from the initial screening were initially verified using the same detection method as above.

[0033] Based on the results of differential expression analysis and the fold change index, candidate peptides were comprehensively evaluated, and peptide number 55 (BBRF2-55, whose amino acid sequence is shown in SEQ ID NO:1) and its corresponding antibody, derived from the BBRF2 protein, were finally screened. Results ( Figure 2 The results showed that the levels of IgA and IgG corresponding to the polypeptide exhibited a consistent trend of difference between the two groups, suggesting that it has potential diagnostic application value.

[0034] SEQ ID NO: 1: TSGKMGCLARSPKDYCADLN.

[0035] Based on the above screening results, the key candidate antigen BBRF2 was further investigated. By optimizing its protein sequence, the corresponding full-length antigen protein (BBRF2 protein, amino acid sequence as shown in SEQ ID NO: 2) was successfully prepared, and a biotinylate label was introduced at its end. Utilizing the high affinity between streptavidin and biotin, this biotinylated antigen was directionally immobilized on the surface of a solid-phase carrier pre-coated with streptavidin, thereby establishing an ELISA detection method. This ELISA detection method employs a double-antigen sandwich method, i.e., antigen-antibody-antigen, and can detect the level of total anti-BBRF2 protein antibodies (including different anti-BBRF2 protein Ig antibodies) in nasopharyngeal swab samples.

[0036] SEQ ID NO: 2: MASGKHHQPGGTRSLTMQKVSLRVTPRLVLEVNRHNAICVATNVPEFYNARGDLNVRDLRAHVKARMISSQFCGYILVSLLDSEDQVDHLNIFPHVFSERMILYKPNNVNLMEMCALLSMIENAKSPSIGLCREVLGRL TLLHSKCNNLDSLFLYNGARTLLSTLVKYHDLEEGAATPGPWNEGLSLFKLHKELKRAPSEARDLMQSLFLTSGKMGCLARSPKDYCADLNKEEDANSGFTFNLFYQDSLLTKHFQCQTVLQTLRRKCLGSDTVSKIIP.

[0037] The ELISA detection method includes the following steps: 1. Dilute BBRF2 protein with PBS to 1 mg / ml; 2. Add 50 ng of BBRF2 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 BBRF2 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.

[0038] Nasopharyngeal swab samples from 28 patients with nasopharyngeal carcinoma and 30 healthy controls were selected for analysis. Results ( Figure 3 The results showed that there was a significant difference in the level of specific antibodies against BBRF2 protein between the two groups, which preliminarily verified the application value of this indicator in the detection of nasopharyngeal carcinoma and suggested that it can be used as a specific detection biomarker for nasopharyngeal carcinoma. Example 2

[0039] This embodiment further validates the study with a larger sample size. Ninety nasopharyngeal carcinoma patients and 90 healthy controls were reinstated as the validation cohort, and anti-BBRF2 protein antibodies in nasopharyngeal swab samples from both groups were detected and analyzed. The detection method for anti-BBRF2 protein antibodies was the same as in Example 1.

[0040] Test results ( Figure 4 Consistent with Example 1, the level of anti-BBRF2 protein antibody in nasopharyngeal swab samples from nasopharyngeal carcinoma patients was significantly higher than that in healthy controls. Based on this, receiver operating characteristic (ROC) curves were constructed using ELISA data from the validation cohort to further evaluate the diagnostic efficacy of anti-BBRF2 protein antibodies against nasopharyngeal carcinoma. Results ( Figure 5 The results show that the area under the curve (AUC) is 0.83; when the cutoff value is set to 0.24, the corresponding sensitivity is 82% and the specificity is 83%, indicating high detection accuracy.

[0041] In conclusion, anti-BBRF2 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.

[0042] 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.

[0043] 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-BBRF2 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-BBRF2 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-BBRF2 protein antibody in biological samples is the same reagent used for ELISA detection.

4. The application as described in claim 2, characterized in that, The reagent used to detect the level of anti-BBRF2 protein antibodies in biological samples includes the BBRF2 protein.

5. The application as described in claim 4, characterized in that, The amino acid sequence of the BBRF2 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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