Application of anti-PCCB antibody in preparation of sicca syndrome diagnosis product

Abstract

The invention discloses application of an anti-PCCB antibody in preparation of a sicca syndrome (SS) diagnostic product. The diagnostic product comprises a reagent for detecting the anti-PCCB antibody. It is found that the anti-PCCB antibody in serum and plasma of an SS patient is significantly increased, the sensitivity of the antibody as a biomarker for diagnosing SS is 32.89%, the specificity is 91.82%, and the antibody has significant diagnostic value. The positive rate of the anti-PCCB antibody is 23.08% in patients with negative SS of the anti-SSA antibody, and the positive rate of the anti-PCCB antibody is 34.65% in patients with negative SS of the anti-SSB antibody. In addition, the combination of the anti-PCCB antibody and the anti-SSA antibody / anti-SSB antibody also has a good diagnostic value. The invention also provides a PCCB polypeptide, which can effectively identify the anti-PCCB antibody in a biological specimen of a SS patient, provides more choices for SS diagnosis, and fills the blank of anti-SSA antibody and anti-SSB antibody diagnosis.

Description

Technical Field

[0001] This invention relates to the field of disease diagnostic technology, specifically to the application of anti-PCCB antibodies in the preparation of diagnostic products for Sjögren's syndrome. Background Technology

[0002] Sjögren's syndrome (SS) is a very common autoimmune disease that primarily affects young and middle-aged women. SS is characterized by the progressive infiltration and destruction of lacrimal and salivary glands by lymphocytes, leading to dry eyes and dry mouth, but its harm and severity far exceed the typical symptoms of localized dry mouth and dry eyes. Essentially, SS is a persistent attack by the immune system on its own exocrine glands and multiple organ tissues. The disease exhibits a tendency for systemic damage and a risk of malignant transformation, posing serious health challenges.

[0003] Approximately 30% to 40% of patients with primary SS develop systemic damage. Due to the immune system's attack on its own exocrine glands, lymphocyte infiltration occurs in the exocrine gland epithelium, which can lead to interstitial nephritis, autoimmune primary cholangitis, and obstructive bronchiolitis. Furthermore, persistent B-cell hyperresponsiveness can cause immune complex deposition, potentially triggering extraepithelial symptoms such as glomerulonephritis associated with glomerulonephritis, interstitial pneumonia, and peripheral neuropathy. In addition, significant fatigue severely impacts the patient's quality of life.

[0004] Even more concerning is that, due to the persistent chronic activation of B cells, patients with primary SS have a 15 to 20 times higher risk of developing B-cell lymphoma than the general population (a lifetime risk of 5% to 10%). Most of these lymphomas are B-cell non-Hodgkin lymphomas, primarily low-grade marginal zone histology.

[0005] Besides SS, dry eyes and dry mouth can also occur in other autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Currently, the diagnostic criteria established by the American College of Rheumatology (ACR) in 2012 emphasize that the diagnosis of SS must be based on autoantibodies (anti-SSA antibodies and anti-SSB antibodies) or lip gland biopsy. Anti-SSA antibodies have high sensitivity in the diagnosis of SS; this antibody can be detected in the serum of the vast majority (31%-77%) of patients with primary SS. Therefore, a negative result has some reference value for ruling out the diagnosis. However, because anti-SSA antibodies are also common in other autoimmune diseases, such as systemic lupus erythematosus (especially highly associated with neonatal lupus and heart conduction block), rheumatoid arthritis, and dermatomyositis, their specificity is relatively poor. Anti-SSB antibodies have high specificity, but the positive rate of this antibody in SS patients is significantly lower than that of anti-SSA antibodies (approximately 27%-55%). A considerable proportion of SS patients are negative for this antibody; therefore, a negative result cannot be used as a basis for ruling out SS.

[0006] Due to the limitations of anti-SSA and anti-SSB antibody diagnosis, patients who are negative for these antibodies often require a labial gland biopsy for definitive diagnosis. Labial gland biopsy is an invasive procedure with risks such as local numbness, leading to patient resistance. Furthermore, the interpretation of biopsy results heavily relies on the specialist's expertise and experience, and subjective differences in interpretation among physicians cannot be ignored. Obtaining and interpreting labial gland biopsy results has become a critical factor severely hindering the timely diagnosis of SJK (SS). Therefore, identifying sensitive and specific novel biomarkers for SJK is of significant clinical importance. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned technical problems in existing SS diagnostics by providing the application of anti-PCCB antibodies in the preparation of SS diagnostic products, offering more options for SS diagnosis, and filling the gap in diagnostics using anti-SSA and anti-SSB antibodies.

[0008] The technical solution of this invention is described in detail below: In a first aspect, the present invention provides the application of anti-PCCB antibodies in the preparation of SS diagnostic products, the diagnostic products comprising reagents for detecting anti-PCCB antibodies.

[0009] Propionyl-CoA carboxylase (PCC) is a mitochondrial enzyme involved in the catabolism of odd-chain fatty acids, branched-chain amino acids such as isoleucine, threonine, methionine, valine, and other metabolites. PCC typically consists of α and β subunits, encoded by the PCCA and PCCB genes, respectively, which work together to maintain metabolic homeostasis. PCCB, expressed in tissues such as salivary glands, is composed of 539 amino acids and is located in the mitochondrial matrix. Its N-terminus contains a biotin-binding site, which is crucial for enzyme activity. Variations in the PCCB gene can lead to type II propionic acidemia (an autosomal recessive inherited congenital metabolic disorder).

[0010] Compared to normal healthy individuals, patients with systemic lupus erythematosus (SLE), and patients with rheumatoid arthritis, the levels of anti-PCCB antibodies were significantly increased in the biological samples of SS patients.

[0011] The SS patients include SS patients who are negative for anti-SSA antibodies and / or negative for anti-SSB antibodies.

[0012] The biological samples are serum and plasma.

[0013] The reagent for detecting anti-PCCB antibodies includes a PCCB polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] The amino acid sequence of SEQ ID NO:1 is IFKGHENVEAAQAEY.

[0015] The amino acid sequence of SEQ ID NO:2 is BSA-C-IFKGHENVEAAQAEY, meaning that the carrier protein BSA is coupled to the N-terminus of SEQ ID NO:1 via a cysteine ​​residue.

[0016] The diagnostic products include ELISA kits.

[0017] Secondly, this invention provides the use of anti-PCCB antibodies in the combined preparation of SS diagnostic products, said diagnostic products comprising any of the following combinations: (1) Reagents for detecting anti-PCCB antibodies and reagents for detecting anti-SSA antibodies; (2) Reagents for detecting anti-PCCB antibodies and reagents for detecting anti-SSB antibodies; (3) Reagents for detecting anti-PCCB antibodies, reagents for detecting anti-SSA antibodies and reagents for detecting anti-SSB antibodies.

[0018] The reagent for detecting anti-PCCB antibodies includes a PCCB polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0019] Thirdly, the present invention also provides a PCCB polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0020] In this invention, the reagent for detecting anti-PCCB antibodies specifically recognizes anti-PCCB antibodies, such as PCCB antigen peptides. Both the reagents for detecting anti-SSA antibodies and anti-SSB antibodies are commercially available and readily available.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention found that anti-PCCB antibody levels were significantly higher in SS patients than in healthy individuals, patients with systemic lupus erythematosus, and patients with rheumatoid arthritis. The ELISA detection of anti-PCCB antibodies showed a sensitivity of 32.89% and a specificity of 91.82% in diagnosing SS, demonstrating significant diagnostic value.

[0022] Furthermore, the combination of anti-PCCB antibody and anti-SSA antibody / anti-SSB antibody also has excellent diagnostic value. For example, the sensitivity and specificity of anti-PCCB antibody combined with anti-SSA antibody in diagnosing SS are 86.58% and 76.82%, respectively; the sensitivity and specificity of anti-PCCB antibody combined with anti-SSB antibody in diagnosing SS are 55.70% and 90.45%, respectively; and the sensitivity and specificity of anti-PCCB antibody combined with anti-SSA antibody and anti-SSB antibody in diagnosing SS are 86.58% and 76.82%, respectively.

[0023] The PCCB polypeptide provided by this invention can effectively identify anti-PCCB antibodies in biological samples of SS patients. Its functional region consists of only 15 amino acids, making it short and easy to prepare. After conjugation with BSA, its immunogenicity can be further increased, thus improving the detection accuracy. Attached Figure Description

[0024] Figure 1 The figure shows the statistical results of detecting anti-PCCB antibody levels in serum of different groups using PCCB peptide as the detection antigen. p<0.05, p<0.01, p < 0.001 (Kruskal-Wallis test). [AU value = (OD value of serum sample / OD value of positive serum)] 100, OD value is the value detected by the microplate reader at 450nm - 570nm. Figure 2 Receiver operating characteristic (ROC) curve of serum anti-PCCB antibody ELISA detection.

[0025] Figure 3 The figure shows the statistical results of anti-PCCB antibody levels in the serum of different groups of SS and healthy controls. p<0.05, p<0.01, p < 0.001 (Kruskal-Wallis test). [AU value = (OD value of serum sample / OD value of positive serum)] 100, OD value is the value detected by the microplate reader at 450nm - 570nm. Figure 4 The results reflect the ROC analysis of serum anti-PCCB antibody ELISA detection, serum anti-SSA antibody ELISA detection, serum anti-SSB antibody ELISA detection, combined detection of serum anti-PCCB antibody and serum anti-SSA antibody, combined detection of serum anti-PCCB antibody and serum anti-SSB antibody, and serum anti-PCCB antibody, serum anti-SSA antibody and serum anti-SSB antibody, respectively.

[0026] Figure 5 The figure shows the statistical results of detecting anti-PCCB antibody levels in plasma from different groups using PCCB peptides as the detection antigen. p<0.05, p<0.01, p < 0.001 (Kruskal-Wallis test method). [AU value = (OD value of plasma to be tested / OD value of positive plasma)] 100, OD value is the value detected by the microplate reader at 450nm - 570nm. Detailed Implementation To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. The described embodiments are merely some, not all, of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial sources.

[0027] The carrier protein BSA is coupled to the N-terminus of the PCCB polypeptide sequence IFKGHENVEAAQAEY (SEQ ID NO:1) via a cysteine ​​residue, and the resulting PCCB polypeptide amino acid sequence is BSA-C-IFKGHENVEAAQAEY (SEQ ID NO:2). SEQ ID NO:2 was synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0028] Using enzyme-linked immunosorbent assay (ELISA), the above-mentioned PCCB polypeptide sequence SEQ ID NO:2 linked with BSA was used as the detection antigen to detect anti-PCCB antibodies in the serum of SS patients, normal healthy individuals, and patients with various rheumatic diseases.

[0029] In addition to the serum sample being tested, the main reagents used in ELISA testing include: the coating antigen (PCCB polypeptide shown in SEQ ID NO:2 above) bound to the enzyme-labeled plate, the detection antibody (i.e., enzyme-labeled secondary antibody, which specifically recognizes human IgG and binds to horseradish peroxidase), the chromogenic substrate (i.e., TMB reagent, which develops color after being catalyzed by horseradish peroxidase, and the experimental results are presented according to the degree of substrate color development), positive standard serum, as well as washing buffer, blocking buffer, stop buffer, diluent, buffer solution, etc.

[0030] ELISA detection experimental steps: (1) Plate coating: PCCB peptides were diluted to 10 μg / ml with carbonate at 4℃ and coated with 96-well plates at 100 μl / well. The plates were then coated overnight at 4℃. (2) Washing: Wash the plate 3 times with 0.05% Tween 20 PBS (PBS-T, washing buffer), 200 μl / well. 2 minutes / time; (3) Blocking: Add 3% BSA-PBST (blocking solution), 200 μl / well, and block at room temperature for 2 hours; (4) Washing: Wash the plate 3 times with 0.05% PBS-T (washing buffer), 200 μl / well. 2 minutes / time; (5) Sample incubation: Dilute the sample serum with 1% BSA-PBST (diluent) at a ratio of 1:100, 100 μl / well, and incubate at 37°C for 1 hour; (6) Washing: Wash the plate 3 times with 0.05% PBS-T (washing buffer), 200 μl / well 2 minutes / time; (7) Secondary antibody incubation: Add 100 μl of horseradish peroxidase-labeled 1:10000 diluted goat anti-human IgG (detection antibody), incubate at 37℃ for 30 minutes, wash the plate 3 times, and add 200 μl / well. 2 minutes / time; (8) Color development: Add TMB color development solution, 100 μl / well, and react in the dark for 10 min; (9) Termination: The reaction was terminated with 2 mol / L sulfuric acid (termination solution), 100 μl / well, and OD values ​​were read at 450 nm and 570 nm respectively.

[0031] (10) Calculation of results: 1) Selection of positive control standard serum: obtained by selecting 10 SS serum samples with OD values ​​greater than 1.0 and mixing them in equal volumes; 2) AU value (Arbitrary units) is expressed by the following formula: AU = [OD peptide - OD nonspecific background] test serum / [OD peptide - OD nonspecific background] positive serum × 100.

[0032] Validation of the diagnostic value of anti-PCCB antibodies in SS 1. Anti-PCCB antibody as a biomarker for SS diagnosis 149 patients with systemic lupus erythematosus (SS), 64 patients with systemic lupus erythematosus (SLE) and 56 patients with rheumatoid arthritis (RA) were selected as disease controls, and 100 healthy controls were included. Peripheral blood was obtained from all participants, and serum was separated. The levels of anti-PCCB antibodies in the serum samples of each group were detected by the above-mentioned ELISA method, and the sensitivity and specificity of the antibody for the diagnosis of SS were analyzed.

[0033] Test results as follows Figure 1 and Figure 2 As shown, Figure 1 The results show that the levels of anti-PCCB antibodies in peripheral blood serum of different populations were detected by ELISA using the PCCB peptide shown in SEQ ID NO:2. The results showed that the levels of anti-PCCB antibodies in SS patients were significantly higher than those in normal individuals and the control group of related diseases, and this was statistically significant. Figure 2 The ROC curve for serum anti-PCCB antibody ELISA detection in SS patients reflects the relationship between the sensitivity and specificity of ELISA diagnosis, with an AUC of 0.722. Figure 5 The results of plasma sample testing showed that anti-PCCB antibodies in SS patients were significantly higher than those in normal individuals and the relevant disease control group, and this was statistically significant.

[0034] Further analysis of the sensitivity and specificity of anti-PCCB antibody as a biomarker for the diagnosis of SS using ELISA was conducted. The results are shown in Table 1. The sensitivity of serum anti-PCCB antibody ELISA detection for diagnosing SS was 32.89%, and the specificity was 91.82%. Therefore, while ensuring high specificity, anti-PCCB antibody ELISA detection showed good diagnostic value.

[0035] Table 1. Diagnostic value of anti-PCCB antibodies in SS patients 2. Diagnostic value of anti-PCCB antibody as a marker in serum-negative SS Anti-SSA antibodies and anti-SSB antibodies are antibodies in the SS classification criteria. We further analyzed the diagnostic value of anti-PCCB antibodies as biomarkers in anti-SSA antibody-negative SS and / or anti-SSB antibody-negative SS.

[0036] Twenty-six patients with SS who were negative for anti-SSA antibodies and 101 patients with SS who were negative for anti-SSB antibodies were selected. The content of anti-PCCB antibodies in the serum samples was detected by the above-described ELISA using the PCCB polypeptide shown in SEQ ID NO:2 as the coating antigen. The detection results are as follows: Figure 3 As shown in Table 2, the diagnostic value was further analyzed.

[0037] Table 2. Diagnostic value of anti-PCCB antibodies in seronegative SS patients In serum samples from SS patients who were negative for anti-SSA antibodies, the positive rate of anti-PCCB antibodies was 23.08%, while in serum samples from SS patients who were negative for anti-SSB antibodies, the positive rate of anti-PCCB antibodies was 34.65%, indicating that anti-PCCB antibodies also have good diagnostic value in serum-negative SS.

[0038] 3. Diagnostic value of anti-PCCB antibody combined with anti-SSA antibody / anti-SSB antibody as a biomarker in serum-negative SS Given the good diagnostic value of anti-PCCB antibody in serum-negative SS patients, we conducted a combined diagnostic analysis of anti-PCCB antibody ELISA detection and anti-SSA antibody / anti-SSB antibody ELISA detection. Using the PCCB peptide shown in SEQ ID NO:2, serum samples from 149 SS patients were selected for ELISA detection, and the statistical results are shown in Table 3.

[0039] Table 3. Analysis of the combined diagnostic value of anti-PCCB antibody and anti-SSA antibody / anti-SSB antibody In serum samples: The sensitivity and specificity of anti-PCCB antibody as a biomarker alone were 32.89% and 91.82%, respectively. The sensitivity and specificity of anti-SSA antibodies as biomarkers alone were 82.55% and 83.64%, respectively. The sensitivity and specificity of anti-SSB antibody as a biomarker alone were 32.21% and 98.64%, respectively. The sensitivity and specificity of the combined use of anti-PCCB antibody and anti-SSA antibody as biomarkers for the diagnosis of SS were 86.58% and 76.82%, respectively.

[0040] like Figure 4 As shown, the area under the ROC curve (AUC) of anti-PCCB antibody and anti-SSA antibody were 0.722 and 0.859, respectively. The AUC of combined diagnosis of anti-PCCB antibody and anti-SSA antibody can be increased to 0.880.

[0041] The combined use of anti-PCCB antibody and anti-SSB antibody as biomarkers for diagnosing SS showed a sensitivity of 55.70% and a specificity of 90.45%, respectively. Therefore, anti-PCCB antibody can improve the sensitivity of anti-SSB antibody in diagnosing SS by 23.49% while maintaining high specificity.

[0042] like Figure 4As shown, the area under the ROC curve (AUC) of anti-PCCB antibody and anti-SSB antibody were 0.722 and 0.731, respectively. The AUC of the combined diagnosis of anti-PCCB antibody and anti-SSB antibody can be increased to 0.823.

[0043] The sensitivity and specificity of the combination of anti-PCCB antibody, anti-SSA antibody, and anti-SSB antibody were 86.58% and 76.82%, respectively.

[0044] like Figure 4 As shown, the AUC of the combination of anti-PCCB antibody, anti-SSA antibody, and anti-SSB antibody was 0.823.

[0045] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. The application of anti-PCCB antibody in the preparation of diagnostic products for Sjögren's syndrome, characterized in that, The diagnostic products include reagents for detecting anti-PCCB antibodies.

2. The application according to claim 1, characterized in that, Compared with normal healthy individuals, patients with systemic lupus erythematosus, and patients with rheumatoid arthritis, the levels of anti-PCCB antibodies were significantly increased in biological samples from patients with Sjögren's syndrome.

3. The application according to claim 2, characterized in that, The patients with Sjögren's syndrome include those who are negative for anti-SSA antibodies and / or negative for anti-SSB antibodies.

4. The application according to claim 2, characterized in that, The biological sample is serum or plasma.

5. The application according to claim 1, characterized in that, The reagent for detecting anti-PCCB antibodies includes a PCCB polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:

2.

6. The application according to claim 5, characterized in that, The diagnostic products include ELISA kits.

7. The use of anti-PCCB antibody in the preparation of combined diagnostic products for Sjögren's syndrome, characterized in that, The diagnostic product includes any combination of the following: (1) Reagents for detecting anti-PCCB antibodies and reagents for detecting anti-SSA antibodies; (2) Reagents for detecting anti-PCCB antibodies and reagents for detecting anti-SSB antibodies; (3) Reagents for detecting anti-PCCB antibodies, reagents for detecting anti-SSA antibodies and reagents for detecting anti-SSB antibodies.

8. The use according to claim 7, characterized in that, The reagent for detecting anti-PCCB antibodies includes a PCCB polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:

2.

9. A PCCB polypeptide, characterized in that, The amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.

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