Use of anti-pccb antibodies in the preparation of diagnostic products for sjogrens syndrome

Abstract

The application discloses application of anti-PCCB antibody in preparation of a diagnostic product for Sjogren's syndrome (SS), and 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 SS patients is significantly increased, the sensitivity of the anti-PCCB antibody as a biomarker for diagnosing SS is 32.89%, the specificity is 91.82%, and the anti-PCCB antibody has a significant diagnostic value. In anti-SSA antibody negative SS patients, the positive rate of the anti-PCCB antibody is 23.08%, and in anti-SSB antibody negative SS patients, the positive rate of the anti-PCCB antibody is 34.65%. In addition, the anti-PCCB antibody combined with the anti-SSA antibody / anti-SSB antibody also has good diagnostic value. The application further provides a PCCB polypeptide which can effectively recognize the anti-PCCB antibody in biological samples of SS patients, provides more choices for SS diagnosis, and fills the blank of the anti-SSA antibody and the anti-SSB antibody diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of disease diagnosis, and particularly relates to application of anti-PCCB antibody in preparation of a diagnostic product for Sjogren's syndrome. BACKGROUND

[0002] Sjogren's syndrome (SS) is a very common autoimmune disease in clinic, mainly affecting young and middle-aged women. The characteristics of SS are that lymphocytes gradually infiltrate and destroy lacrimal and salivary glands, causing dry eyes and dry mouth, but its harm and severity far exceed the typical manifestations of local dry mouth and dry eyes. The nature of SS is the persistent attack of the immune system on the body's exocrine glands and multiple organ tissues. The disease shows a tendency of systemic damage and a risk of malignant transformation, causing serious health challenges.

[0003] About 30% to 40% of primary SS patients will develop systemic damage. Due to the attack of the immune system on the body's exocrine glands, lymphocyte infiltration occurs in the epithelium of the exocrine glands, which can cause interstitial nephritis, autoimmune primary biliary cirrhosis and obstructive bronchiolitis, etc. Moreover, due to the persistent high response of B cells, immune complexes are deposited, which can cause extracorporeal symptoms such as glomerulonephritis associated with globulinemia, interstitial pneumonia and peripheral neuropathy, etc. In addition, significant fatigue can also seriously affect the quality of life of patients.

[0004] More seriously, due to the persistent chronic activation of B cells, the risk of B cell lymphoma in primary SS patients is 15 to 20 times that of the general population (lifetime risk of 5% to 10%). These lymphomas are mostly B cell non-Hodgkin's lymphoma, mainly low-grade marginal zone histology.

[0005] In addition to SS, dry eyes and dry mouth can also occur in other autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. At present, the diagnostic criteria formulated by the American College of Rheumatology (ACR) in 2012 emphasizes that the diagnosis of SS must be based on autoantibodies (anti-SSA antibody and anti-SSB antibody) or lip biopsy. Anti-SSA antibody has high sensitivity in the diagnosis of SS, and most (31%-77%) of primary SS patients can detect this antibody in their serum, so its negative result has a certain reference value for excluding the diagnosis. However, since anti-SSA antibody is also common in other autoimmune diseases, such as systemic lupus erythematosus (especially highly associated with neonatal lupus and cardiac conduction block), rheumatoid arthritis, dermatomyositis, etc., its specificity is relatively poor. Anti-SSB antibody has high specificity, but the positive rate of this antibody in SS patients is significantly lower than that of anti-SSA antibody (about 27%-55%), and a considerable part of SS patients is negative for this antibody, so its negative result cannot be used as a basis for excluding SS.

[0006] Due to the limitations of anti-SSA antibody and anti-SSB antibody diagnosis, patients negative for anti-SSA antibody and anti-SSB antibody often need to undergo lip biopsy for diagnosis. Lip biopsy is an invasive examination, which has risks such as local numbness. Patients have resistance to the examination, and the interpretation of biopsy results seriously depends on the professional knowledge level and experience of specialist doctors. The subjective differences of different doctors in interpreting biopsy results cannot be ignored. Obtaining and interpreting lip biopsy results has become a key link that seriously restricts the timely diagnosis of SS. Therefore, identifying a new biomarker of SS with sensitivity and specificity has important clinical significance. SUMMARY

[0007] The purpose of the present application is to solve the above technical problems existing in the current SS diagnosis, and provide the application of anti-PCCB antibody in the preparation of SS diagnostic products, to provide more choices for SS diagnosis and fill the gap of anti-SSA antibody and anti-SSB antibody diagnosis.

[0008] The technical scheme of the present application is described in detail as follows:

[0009] In a first aspect, the present application provides the application of anti-PCCB antibody in the preparation of SS diagnostic products, wherein the diagnostic product comprises a reagent for detecting anti-PCCB antibody.

[0010] Propionyl-CoA carboxylase (PCC) is a mitochondrial enzyme involved in the catabolism of odd-chain fatty acids, branched-chain amino acids isoleucine, threonine, methionine, valine and other metabolites. Propionyl-CoA carboxylase is usually composed of α and β subunits, which are encoded by PCCA and PCCB genes, respectively, and maintain metabolic homeostasis together. PCCB is expressed in tissues such as salivary glands and is composed of 539 amino acids, which is located in the mitochondrial matrix. Its amino-terminal contains a biotin-binding site, which is essential for enzyme activity. PCCB gene variation can cause type II propionic acidemia (an autosomal recessive congenital metabolic abnormality).

[0011] Compared with normal healthy people, systemic lupus erythematosus patients and rheumatoid arthritis patients, the content of anti-PCCB antibody in the biological sample of SS patients is significantly increased.

[0012] The SS patients include anti-SSA antibody negative and / or anti-SSB antibody negative SS patients.

[0013] The biological sample is serum and plasma.

[0014] The reagent for detecting anti-PCCB antibody comprises a PCCB polypeptide, and the amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] The amino acid sequence of SEQ ID NO: 1 is IFKGHENVEAAQAEY.

[0016] The amino acid sequence of SEQ ID NO: 2 is BSA-C-IFKGHENVEAAQAEY, that is, a carrier protein BSA is coupled to the N-terminus of SEQ ID NO: 1 through a cysteine residue.

[0017] The diagnostic product includes an ELISA kit.

[0018] In a second aspect, the present application provides use of an anti-PCCB antibody in joint preparation of a SS diagnostic product, which includes any of the following combinations:

[0019] (1) a reagent for detecting anti-PCCB antibody and a reagent for detecting anti-SSA antibody;

[0020] (2) a reagent for detecting anti-PCCB antibody and a reagent for detecting anti-SSB antibody;

[0021] (3) a reagent for detecting anti-PCCB antibody, a reagent for detecting anti-SSA antibody, and a reagent for detecting anti-SSB antibody.

[0022] The reagent for detecting anti-PCCB antibody includes a PCCB polypeptide, and the amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] In a third aspect, the present application further provides a PCCB polypeptide, and the amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] In the present application, the reagent for detecting anti-PCCB antibody can specifically recognize anti-PCCB antibody, for example, a PCCB antigen peptide. The reagent for detecting anti-SSA antibody and the reagent for detecting anti-SSB antibody are both commercialized products, which can be purchased.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application finds that the anti-PCCB antibody in SS patients is significantly higher than that in normal healthy people, systemic lupus erythematosus patients, and rheumatoid arthritis patients. The sensitivity of anti-PCCB antibody ELISA detection in diagnosing SS is 32.89%, and the specificity is 91.82%, which has significant diagnostic value.

[0027] In addition, the anti-PCCB antibody combined with the anti-SSA antibody / anti-SSB antibody also has excellent diagnostic value. For example, the sensitivity and specificity of the anti-PCCB antibody combined with the anti-SSA antibody for diagnosing SS are 86.58% and 76.82%, respectively, the sensitivity and specificity of the anti-PCCB antibody combined with the anti-SSB antibody for diagnosing SS are 55.70% and 90.45%, respectively, and the sensitivity and specificity of the anti-PCCB antibody combined with the anti-SSA antibody and the anti-SSB antibody for diagnosing SS are 86.58% and 76.82%, respectively.

[0028] The PCCB polypeptide provided by the application can effectively recognize the anti-PCCB antibody in the biological sample of an SS patient, the sequence functional region is only 15 amino acids, the sequence is short and easy to prepare, and the immunogenicity can be further increased after being coupled with BSA, and the detection accuracy is improved. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 For the statistical results of the content of the anti-PCCB antibody in the serum of different groups of people using the PCCB polypeptide as a detection antigen, the figure is p<0.05, p<0.01, p<0.001 (Kruskal-Wallis test method is used). 【AU value=(OD value of the serum to be tested / OD value of the positive serum) 100, the OD value is the detection value of the microplate reader at 450 nm-570 nm

[0030] Figure 2 Receiver operating characteristic curve (ROC) of the serum anti-PCCB antibody ELISA detection.

[0031] Figure 3 For the statistical results of the content of the anti-PCCB antibody in the serum of different groups of SS and healthy controls, the figure is p<0.05, p<0.01, p<0.001 (Kruskal-Wallis test method is used). 【AU value=(OD value of the serum to be tested / OD value of the positive serum) 100, the OD value is the detection value of the microplate reader at 450 nm-570 nm

[0032] Figure 4 The ROC analysis results of the serum anti-PCCB antibody ELISA detection, the serum anti-SSA antibody ELISA detection, the serum anti-SSB antibody ELISA detection, the serum anti-PCCB antibody and the serum anti-SSA antibody combined detection, the serum anti-PCCB antibody and the serum anti-SSB antibody combined detection, and the serum anti-PCCB antibody and the serum anti-SSA antibody and the serum anti-SSB antibody are respectively reflected.

[0033] Figure 5 For the use of PCCB polypeptide as detection antigen, the statistical results of detecting the content of anti-PCCB antibody in the plasma of different groups of people are shown in the figure p<0.05, p<0.01, p<0.001 (Kruskal-Wallis test method). 【AU value = (OD value of the tested plasma / OD value of the positive plasma) 100, OD value is the detection value of the microplate reader at 450 nm-570 nm DETAILED DESCRIPTION

[0034] In order to make the person skilled in the art better understand the scheme of the present application, the present application will be described clearly and completely in combination with the embodiments and the drawings. The described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the scope of protection of the present application. The instruments and reagents used in the embodiments are all from commercial channels, unless otherwise specified.

[0035] The carrier protein BSA is coupled to the N-terminal of the PCCB polypeptide sequence IFKGHENVEAAQAEY (SEQ ID NO: 1) through a cysteine residue. The amino acid sequence of the coupled PCCB polypeptide is BSA-C-IFKGHENVEAAQAEY (SEQ ID NO: 2). The polypeptide sequence of SEQ ID NO: 2 is synthesized by Shanghai Nuoyou Biological Technology Co., Ltd.

[0036] Through enzyme-linked immunosorbent assay (ELISA), the PCCB polypeptide sequence connected with BSA above SEQ ID NO: 2 is used as a detection antigen to detect anti-PCCB antibodies in the serum of SS patients, normal healthy people and patients with various rheumatic diseases.

[0037] In addition to the test serum samples, the main reagents used in ELISA detection include: coating antigen (PCCB polypeptide shown in SEQ ID NO: 2 above) combined with enzyme-labeled plates, detection antibody (i.e. enzyme-labeled secondary antibody, specifically recognizing human IgG and combined with horseradish peroxidase), color developing substrate (i.e. TMB reagent, which is colored after being catalyzed by horseradish peroxidase, and the experimental results are presented according to the color developing degree of the substrate), positive standard serum, and washing solution, blocking solution, termination solution, dilution solution, buffer solution, etc.

[0038] ELISA detection experimental steps:

[0039] (1) Plate coating: PCCB polypeptide was diluted to 10 μg / ml with 4°C carbonate as diluent, 100 μl / well, 4°C overnight;

[0040] (2) Washing plate: 0.05% Tween 20 PBS (PBS-T, washing solution) was used to wash the plate 3 times, 200 μl / well 2 min / time;

[0041] (3) Blocking: 3% BSA-PBST (blocking solution) was added, 200 μl / well, room temperature blocking for 2 hours;

[0042] (4) Washing plate: 0.05% PBS-T (washing solution) was used to wash the plate 3 times, 200 μl / well 2 min / time;

[0043] (5) Sample incubation: 1% BSA-PBST (diluent) was used to dilute the sample serum 1:100, 100 μl / well, 37°C incubation for 1 hour;

[0044] (6) Washing plate: 0.05% PBS-T (washing solution) was used to wash the plate 3 times, 200 μl / well 2 min / time;

[0045] (7) Secondary antibody incubation: horseradish peroxidase-labeled goat anti-human IgG (detection antibody) was added at a dilution of 1:10000, 100 μl, 37°C incubation for 30 minutes, washing plate 3 times, 200 μl / well 2 min / time;

[0046] (8) Color development: TMB color developing solution was added, 100 μl / well, light reaction for 10 min;

[0047] (9) Termination: 2 mol / L sulfuric acid (stop solution) was used to terminate the reaction, 100 μl / well, 450 nm, 570 nm respectively to read OD value.

[0048] (10) Result calculation:

[0049] 1) Selection of positive control standard serum: 10 cases of SS serum with OD value greater than 1.0 were selected, mixed in equal volume to obtain;

[0050] 2) AU value (Arbitrary units) was used, and the calculation formula was: AU = [OD peptide-OD non-specific background] test serum / [OD peptide-OD non-specific background] positive serum x 100.

[0051] Verification of the diagnostic value of anti-PCCB antibody in SS

[0052] 1. Anti-PCCB antibody as a biomarker for SS diagnosis alone

[0053] 149 SS patients, 64 systemic lupus erythematosus patients, 56 rheumatoid arthritis patients, and 100 healthy controls were selected, and their peripheral blood was obtained to separate serum. The content of anti-PCCB antibody in the serum samples of each group was detected by ELISA as described above, and the sensitivity and specificity of anti-PCCB antibody for SS diagnosis were analyzed.

[0054] The detection results are shown in Figure 1 and Figure 2 As shown in Figure 1 is the statistical result of the detection of the content of anti-PCCB antibody in the peripheral blood serum of different populations by ELISA using the PCCB polypeptide represented by SEQ ID NO: 2, and the results show that the anti-PCCB antibody in SS patients is significantly higher than that in normal people and related disease control groups, and has statistical significance. Figure 2 is the ROC curve of serum anti-PCCB antibody ELISA detection for SS patients, reflecting the mutual relationship between the sensitivity and specificity of ELISA diagnosis, and the AUC is 0.722. Figure 5 is the detection result of plasma samples, showing that the anti-PCCB antibody in SS patients is significantly higher than that in normal people and related disease control groups, and has statistical significance.

[0055] Further analysis of the sensitivity and specificity of anti-PCCB antibody as a marker for SS diagnosis by ELISA detection is shown in Table 1. The sensitivity of serum anti-PCCB antibody ELISA detection for SS diagnosis is 32.89%, and the specificity is 91.82%. Therefore, under the condition of ensuring high specificity, anti-PCCB antibody ELISA detection shows good diagnostic value.

[0056] Table 1. Diagnostic value of anti-PCCB antibody in SS patients

[0057]

[0058] 2. Diagnostic value of anti-PCCB antibody as a marker in serum-negative SS

[0059] Anti-SSA antibody and anti-SSB antibody are antibodies in the classification criteria for SS. We further analyzed the diagnostic value of anti-PCCB antibody as a marker in anti-SSA antibody-negative SS and / or anti-SSB antibody-negative SS.

[0060] 26 anti-SSA antibody-negative SS patients and 101 anti-SSB antibody-negative SS patients were selected, and the content of anti-PCCB antibody in the serum samples was detected by ELISA using the PCCB polypeptide represented by SEQ ID NO: 2 as the coating antigen, and the detection results are shown inFigure 3 As shown in Table 2, the diagnostic value was further analyzed.

[0061] Table 2. Diagnostic value of anti-PCCB antibodies in seronegative SS patients

[0062]

[0063] 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.

[0064] 3. Diagnostic value of anti-PCCB antibody combined with anti-SSA antibody / anti-SSB antibody as a biomarker in serum-negative SS

[0065] 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.

[0066] Table 3. Analysis of the combined diagnostic value of anti-PCCB antibody and anti-SSA antibody / anti-SSB antibody

[0067]

[0068] In serum samples:

[0069] The sensitivity and specificity of anti-PCCB antibody as a biomarker alone were 32.89% and 91.82%, respectively.

[0070] The sensitivity and specificity of anti-SSA antibodies as biomarkers alone were 82.55% and 83.64%, respectively.

[0071] The sensitivity and specificity of anti-SSB antibody as a biomarker alone were 32.21% and 98.64%, respectively.

[0072] 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.

[0073] 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.

[0074] The sensitivity and specificity of anti-PCCB antibody combined with anti-SSB antibody as a biomarker for diagnosing SS were 55.70% and 90.45%, respectively. Therefore, anti-PCCB antibody can increase the sensitivity of anti-SSB antibody in diagnosing SS by 23.49% while maintaining high specificity.

[0075] As shown in Figure 4 The area under the ROC curve (AUC) of anti-PCCB antibody and anti-SSB antibody was 0.722 and 0.731, respectively, and the AUC of anti-PCCB antibody combined with anti-SSB antibody for diagnosis can be increased to 0.823.

[0076] The sensitivity and specificity of anti-PCCB antibody combined with anti-SSA antibody and anti-SSB antibody were 86.58% and 76.82%, respectively.

[0077] As shown in Figure 4 The AUC of anti-PCCB antibody combined with anti-SSA antibody and anti-SSB antibody was 0.823.

[0078] The specific examples are applied in this paper to elaborate the inventive concept in detail, and the above examples are only used to help understand the core idea of the present application. It should be noted that any obvious modification, equivalent replacement or other improvement made by those skilled in the art without departing from the inventive concept should be included in the protection scope of the present application.

Claims

1. Use of an anti-PCCB antibody for the manufacture of a diagnostic product for Sjogren's syndrome, characterized in that, The diagnostic product comprises reagents for detecting anti-PCCB antibodies.

2. Use according to claim 1, characterized in that, The content of anti-PCCB antibodies in the biological sample of the Sjogren's syndrome patient is significantly higher than that in normal healthy people, systemic lupus erythematosus patients and rheumatoid arthritis patients.

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

4. Use according to claim 1, characterized in that, The reagents for detecting anti-PCCB antibodies comprise a PCCB polypeptide, and the amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. Use according to claim 4, characterized in that, The diagnostic product comprises an ELISA kit.

6. Use of an anti-PCCB antibody for the manufacture of a product for the conjoint diagnosis of Sjogren's syndrome, characterized in that, The diagnostic product comprises any one 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.

7. Use according to claim 6, characterized in that, The reagents for detecting anti-PCCB antibodies comprise a PCCB polypeptide, and the amino acid sequence of the PCCB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

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