Use of COBLL1 and CD79B biomarkers in diagnosis of lupus nephritis

Abstract

The invention relates to the technical field of biomolecular detection, in particular to application of COBLL1 and CD79B biomarkers in diagnosis of lupus nephritis. The invention discloses application of a reagent for detecting the level of one or more biomarkers in preparation of a lupus nephritis diagnosis product, the biomarkers comprise COBLL1 and CD79B, lupus nephritis patients and normal people can be effectively distinguished, a new scheme and approach are provided for diagnosis and treatment of lupus nephritis, and the reagent has remarkable clinical value.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202311205362.X and title “Lupus Nephritis Biomarkers and Their Applications” filed with the Chinese Patent Office on September 18, 2023, the entire contents of which are incorporated into this application by application. Technical Field

[0003] The present application relates to the field of biomolecular detection technology, and in particular to the use of COBLL1 and CD79B biomarkers in diagnosing lupus nephritis. Background Art

[0004] Systemic lupus erythematosus (SLE) is a common chronic systemic autoimmune disease, with the main clinical characteristics of the production of multiple autoantibodies, multi-organ and multi-system involvement, and high disability rate. With the vigorous development of modern immunology, systemic lupus erythematosus, as the prototype of systemic autoimmune diseases, has become a hot spot and focus of research in the field of rheumatology and immunology. Studies have shown that 30-80% of SLE patients will develop lupus nephritis (Lupus nephritis, LN), which often leads to chronic kidney disease (Chronic Renal Disease, CKD) and end-stage renal disease (End Stage Renal Disease, ESRD). In clinical practice, hormones and immunosuppressants are usually used for treatment, but some LN patients are insensitive to drugs and transform into chronic nephritis, and can only survive for 5-10 years. In addition, hormone therapy can also produce significant side effects. Some patients progress extremely rapidly and survive for only 2-3 years.

[0005] At present, renal histological biopsy is still the gold standard for diagnosing and distinguishing different stages of LN; however, renal biopsy has many disadvantages and problems, such as less tissue is obtained. If the renal lesions are focal or unevenly distributed, it may lead to misdiagnosis due to deviations; and renal histological biopsy may leave trauma after surgery, which is easy to aggravate hematuria, and even cause massive bleeding and death in severe cases. Renal puncture can clarify the pathological type of kidney disease and is helpful for clinical research, but it has little significance for clinical treatment guidance for patients. There are many contraindications, and many patients are not suitable for renal puncture. In 2008, Lane et al. from the Cleveland Clinic in Ohio, USA, reported the results of a systematic review of English literature on renal tumor puncture biopsy published before 2001: In this systematic review, insufficient tissue obtained by puncture for pathological diagnosis was defined as puncture failure, and tissue obtained by puncture and the pathological diagnosis of surgical resection specimens were inconsistent, which was defined as inaccurate puncture. The researchers collected 27 relevant articles, including 2474 cases. The results showed that the average false negative rate of biopsy was 4.4% (0-25%), the average false positive rate was 1.2% (0-8.2%), the average puncture failure rate was 8.9% (0-22%), the average pathological diagnosis rate was 5.5% (0-36%), the average diagnostic accuracy was 88.9% (40%-100%), and the incidence of complications of renal tumor biopsy was <5%, including bleeding, infection, arteriovenous fistula, pneumothorax, etc. CT scans after renal puncture revealed that 85%-90% of patients had perirenal bleeding, but only 1%-2% of patients required blood transfusions.

[0006] Currently, there is no clinically established non-invasive method to diagnose and guide the treatment of LN. Traditional SLE biomarkers such as anti-DNA antibodies and complement deficiency are widely accepted as diagnostic tools for assessing SLE activity. So far, double-stranded anti-DNA antibodies (anti-dsDNA) and serum complement are the only non-invasive conventional biomarkers for monitoring renal activity in LN patients. However, these markers are only a reflection of the immune activity of the disease, not a marker of renal damage or poor prognosis.

[0007] Therefore, it is of great significance to the diagnosis and treatment of lupus nephritis to continue to explore the potential therapeutic or diagnostic targets of lupus nephritis, explore the mechanism of target action, and promote the development of new drugs and clinical transformation. Summary of the invention

[0008] The purpose of this application is to provide a new biomarker for lupus nephritis and its application.

[0009] In order to achieve the above objectives, this application adopts the following technical solutions:

[0010] The first aspect of the present application discloses the use of COBLL1 and CD79B as biomarkers for lupus nephritis; or the use of reagents for detecting the levels of one or more of the following biomarkers in the preparation of lupus nephritis diagnostic products, wherein the biomarkers include COBLL1 and CD79B.

[0011] It should be noted that the key to this application is that the study found that COBLL1 and CD79B have expression differences between lupus nephritis patients and healthy people, and verified that this expression difference only exists in lupus nephritis patients, and there is no differential expression in systemic lupus erythematosus patients without lupus nephritis. COBLL1 and CD79B can both be used as independent indications for the diagnosis of lupus nephritis. Therefore, this application is the first to creatively propose to use COBLL1 and CD79B as biomarkers for lupus nephritis; on this basis, the reagents for detecting COBLL1 and CD79B can be used as lupus nephritis diagnostic reagents, that is, the detection reagents or components for obtaining the level of COBLL1 or CD79B in the sample are used to prepare lupus nephritis diagnostic products.

[0012] In one implementation of the present application, the biomarkers used to diagnose lupus nephritis include one or more of COBLL1 and CD79B.

[0013] Furthermore, COBLL1 and CD79B can both serve as independent indications for the diagnosis of lupus nephritis.

[0014] It should also be noted that the present application uses COBLL1 and CD79B as biomarkers for lupus nephritis, which can be nucleic acids or proteins. For example, lupus nephritis can be diagnosed based on the nucleic acid expression level of the biomarker, or the protein expression level of the biomarker. Among them, the nucleic acid expression level can be determined by detecting DNA, RNA or mRNA; the protein expression level can be characterized by detecting the level of binding between the protein and its specific polypeptide. In the present application, the analysis object can be a collected sample, for example, lupus nephritis can be diagnosed by detecting the nucleic acid expression level or protein expression level of the biomarker in the collected sample; or, the nucleic acid expression level or protein expression level of the biomarker can be directly analyzed based on the existing data to diagnose lupus nephritis.

[0015] Furthermore, the nucleic acid expression level or protein expression level includes but is not limited to the abundance or concentration of the nucleic acid or protein, and in some aspects may also include the abundance or concentration of mutations of the nucleic acid or protein.

[0016] Furthermore, the nucleic acid expression level of the present application is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biomass spectrometry technology or chromatography technology.

[0017] Furthermore, methods for obtaining nucleic acid expression levels include, but are not limited to, any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0018] Furthermore, the protein expression level of the present application is obtained by sequencing technology, immunoassay technology, electrophoresis technology, biomass spectrometry technology or chromatography technology.

[0019] Furthermore, methods for obtaining protein expression levels include, but are not limited to, any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling or liquid chromatography.

[0020] The second aspect of the present application discloses a product for diagnosing lupus nephritis, which contains reagents for detecting biomarker nucleic acids and / or reagents for detecting biomarker proteins, wherein the aforementioned biomarkers are selected from one or more of COBLL1 and CD79B.

[0021] In some aspects, the article of manufacture includes, but is not limited to, a kit form, a system device form, a computer readable medium, or a computer system form.

[0022] It should be noted that the key to this application is the research discovery that COBLL1 or CD79B can be used as a biomarker for lupus nephritis; therefore, reagents for detecting one or more biomarker nucleic acids and / or proteins selected from COBLL1 and CD79B can be used as components of lupus nephritis diagnostic products.

[0023] In one implementation of the present application, the reagents for detecting biomarker nucleic acids include specific primers and / or probes designed for COBLL1 or CD79B nucleic acids; the reagents for detecting biomarker proteins include polypeptides that specifically bind to or recognize COBLL1 or CD79B proteins.

[0024] It should be noted that the specific primers of the present application refer to primers designed for the biomarker nucleic acids of the present application, such as primers for gene sequencing methods, primers for polymerase chain reaction methods, primers for isothermal amplification reaction methods, or other primers capable of detecting the biomarker nucleic acids of the present application; specific probes refer to probes designed for the biomarker nucleic acids of the present application, such as real-time fluorescence quantitative PCR probes, gene chip detection probes, specific probes for probe hybridization methods, or other probes capable of detecting the biomarker nucleic acids of the present application.

[0025] It should also be noted that the key to the product of the present application is the specific primers and / or probes designed for the biomarker nucleic acid of the present application, or the polypeptides that specifically bind to or recognize the biomarker protein of the present application; for ease of use, the reagents for detecting the biomarker nucleic acid of the present application may also include conventional reagents or components corresponding to the specific detection method used, such as gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, RNA blotting, nucleic acid mass spectrometry or liquid chromatography conventional reagents or components; similarly, the reagents for detecting the biomarker protein of the present application may also include amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrum or liquid chromatography conventional reagents or components. It can be understood that these conventional reagents or components corresponding to the specific detection methods used can be purchased commercially or integrated into the product of the present application.

[0026] In one implementation of the present application, the biomarker nucleic acid of the present application includes the biomarker gene, a DNA fragment extracted from the biomarker gene, and the RNA, mRNA, miRNA or LcRNA corresponding to the biomarker gene or its DNA fragment; the polypeptide includes at least one of an antibody, an antigen-binding fragment, a single-chain antibody and a ligand that specifically binds to the biomarker protein of the present application.

[0027] It should be noted that the key to this application is to detect the level of the biomarker of this application. The detection object can be the biomarker nucleic acid of this application, including various DNA or RNA, or the biomarker protein of this application. When the detection object is a protein, it can be detected by various polypeptides that specifically bind to it, such as but not limited to antibodies, antigen binding fragments, single-chain antibodies, ligands that specifically bind to the biomarker protein of this application, etc.

[0028] In one implementation of the present application, the product of the present application also includes reagents or components for detecting other markers.

[0029] Furthermore, the reagents for detecting the biomarkers of the present application can be used in combination with reagents or components for detecting other markers. More specifically, other markers include but are not limited to one or more of C3, C4, dsDNA, EGLN1, FAM129C, HLA-DOB, SLPI, GPR18, VPREB3, E2F5, FBXL13, and C3orf62.

[0030] In some aspects, the product of the present application also includes a sample processing reagent. The sample processing reagent depends on the specific sample, for example, more specifically, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0031] Furthermore, the product of the present application also includes at least one of a standard substance, a calibrator, a reference substance, and a buffer solution.

[0032] Furthermore, the product of the present application may be supplemented with tubes for mixing the components, microplates, and instruction materials recording the method of use, etc., as needed.

[0033] In some aspects, the sample of the present application includes tissue, cell, urine, saliva, pleural effusion, ascites, feces, synovial fluid or various body fluids. Wherein, body fluid refers to intracellular fluid and extracellular fluid, including tissue fluid, plasma, blood (such as whole blood, serum, or plasma), lymph, cerebrospinal fluid, semen, aqueous humor, etc. In some embodiments, the sample includes peripheral blood mononuclear cells.

[0034] The third aspect of the present application discloses a method for diagnosing lupus nephritis, comprising the following steps:

[0035] (i) obtaining the level of a biomarker in the sample to be tested, wherein the biomarker is selected from one or more of the following: COBLL1 and CD79B;

[0036] (ii) comparing the biomarker level in the test sample with the corresponding biomarker level in the control sample; if there is a significant difference, the subject from which the test sample is derived has an indication of lupus nephritis; or, comparing the biomarker level in the test sample with a set threshold; if the biomarker level in the test sample is lower than the threshold, the subject from which the test sample is derived has an indication of lupus nephritis.

[0037] In the present application, for example, the control sample is a sample from a healthy population. At this time, the diagnostic criteria for lupus nephritis are that the biomarker level of the present application in the sample to be tested is significantly lower than the corresponding biomarker level in the control sample. Alternatively, a threshold is set in advance according to the biomarker level of the control sample, and the biomarker level of the present application in the sample to be tested is compared with the set threshold. Therefore, in the present application, the threshold is a threshold set according to the biomarker level of the control sample, that is, a threshold is set according to the biomarker level in the sample from a healthy population.

[0038] Furthermore, the biomarker levels of the present application include nucleic acid expression levels or protein expression levels.

[0039] Furthermore, the biomarkers COBLL1 and CD79B of the present application can both independently serve as indications for the diagnosis of lupus nephritis.

[0040] Furthermore, the biomarkers of the present application can not only be used as markers to distinguish lupus nephritis patients (LN) from healthy people, but also as markers to distinguish systemic lupus erythematosus patients (NoLN) from LN patients.

[0041] In some aspects, the above diagnosis includes but is not limited to screening, auxiliary diagnosis, diagnosis, prediction / judgment of disease severity, assessment of disease activity, and monitoring of treatment for lupus nephritis.

[0042] The fourth aspect of the present application discloses a method for evaluating or screening lupus nephritis markers, comprising performing a correlation or consistency analysis between the candidate biomarkers and the biomarker levels of the present application, and verifying the diagnostic value of the potential markers through the conclusions of the correlation or consistency.

[0043] It can be understood that the present application study found that COBLL1 or CD79B can be used as a lupus nephritis marker; therefore, COBLL1 or CD79B can be used as a positive control for screening other lupus nephritis markers; for example, based on the correlation or consistency between the candidate biomarker and the COBLL1 or CD79B level, lupus nephritis markers can be screened.

[0044] It should be noted that the biomarkers COBLL1 and CD79B of the present application can both be independently used as indications for screening other lupus nephritis markers.

[0045] The fifth aspect of the present application discloses a method for evaluating or screening a therapeutic agent for lupus nephritis, comprising evaluating the level of a biomarker in a sample treated with the therapeutic agent, wherein the biomarker is selected from one or more of the following: COBLL1 and CD79B.

[0046] It should be noted that the present application study found that there are expression differences in COBLL1 and CD79B between lupus nephritis patients and healthy people, so the aforementioned biomarkers are used as lupus nephritis markers; on this basis, it can be understood that if the treatment of lupus nephritis patients is successful, their COBLL1 and CD79B expression levels will also be different from before treatment. Therefore, the present application creatively proposes to use COBLL1 and CD79B levels to evaluate or screen lupus nephritis therapeutic agents. For example, if the COBLL1 and CD79B levels of the sample are significantly lower than before the treatment with the therapeutic agent after treatment with the therapeutic agent, it is judged that the therapeutic agent has a good therapeutic effect on lupus nephritis; otherwise, it indicates that before and after the treatment with the therapeutic agent, the lupus nephritis patient has not improved or the effect is not obvious; thereby achieving the screening of lupus nephritis therapeutic agents or the evaluation of the efficacy of therapeutic agents.

[0047] It should also be noted that the biomarkers COBLL1 and CD79B of the present application can both independently be used as indications for evaluating or screening lupus nephritis therapeutic agents.

[0048] In the present application, the level of a biomarker refers to the level of nucleic acid expression or protein expression. For example, the level of nucleic acid expression is the level of expression of DNA, RNA or mRNA, and the level of protein expression is the level of binding between the protein of the biomarker and its specific polypeptide.

[0049] Furthermore, it also includes one or more nucleic acids or proteins of C3, C4, dsDNA, EGLN1, FAM129C, HLA-DOB, SLPI, GPR18, VPREB3, E2F5, FBXL13, and C3orf62.

[0050] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0051] The present application provides a new biomarker for lupus nephritis. By analyzing the levels of one or more of COBLL1 and CD79B, it can effectively distinguish lupus nephritis patients from normal people, and provides a new solution and approach for the diagnosis and treatment of lupus nephritis, which makes up for the lack of existing clinical non-invasive detection methods for lupus nephritis and has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 contains Figure 1A and Figure 1B , which is the GEO discovery set data analysis result in the embodiment of this application. Figure 1A : Compared with normal controls, the expression of COBLL1 gene was decreased in peripheral blood mononuclear cells (PBMCs) of patients with lupus nephritis (LN); Figure 1B : Compared with normal people, the expression of CD79B gene in PBMC of LN patients is reduced.

[0053] Figure 2 contains Figure 2A and Figure 2B , is the ROC curve analysis result of the discovery set in the examples of this application. Figure 2A : The data from the discovery set showed that the COBLL1 gene had a good ability to distinguish LN patients from normal people; Figure 2B : The discovery set data showed that the CD79B gene had a good ability to distinguish LN patients from normal people.

[0054] Figure 3 contains Figure 3A and Figure 3B , which is the analysis result of the GEO validation set in the embodiment of this application. Figure 3A : Compared with normal controls, the expression of COBLL1 gene in peripheral blood mononuclear cells (PBMC) of LN patients was significantly decreased; Figure 3B: Compared with normal people, the expression of CD79B gene in PBMC of LN patients was significantly reduced.

[0055] Figure 4 contains Figure 4A and Figure 4B , is the ROC curve analysis result of the validation set in the embodiment of this application. Figure 4A : The validation set data showed that the COBLL1 gene had good discrimination ability between LN patients and normal people; Figure 4B : The validation set data showed that the CD79B gene had good discrimination ability between LN patients and normal people.

[0056] Figure 5 contains Figure 5A to Figure 5B , which is the GEO discovery set analysis result in the embodiment of this application. Figure 5A : The data from the discovery set showed that the expression of COBLL1 gene in PBMCs of patients with systemic lupus erythematosus without lupus nephritis (NoLN) and normal subjects did not reach a 1.5-fold difference, and was not a differentially expressed gene; Figure 5B : The data from the discovery set showed that the expression of the CD79B gene in PBMCs of NoLN patients and normal controls did not reach a 1.5-fold difference, and was not a differentially expressed gene.

[0057] Figure 6 contains Figure 6A to Figure 6B , which is the analysis result of the GEO validation set in the embodiment of this application. Fig. 6A : The validation set data showed that the expression of COBLL1 gene in PBMC of NoLN patients and normal subjects did not reach 1.5 times difference, and it was not a differentially expressed gene; Figure 6B : The validation set data showed that the expression of CD79B gene in PBMCs of NoLN patients and normal subjects did not reach 1.5 times difference, and it was not a differentially expressed gene.

[0058] Figure 7 contains Fig. 7A and Figure 7B , which is the GEO discovery set analysis result in the embodiment of this application. Fig. 7A : The data from the discovery set showed that the COBLL1 gene was differentially expressed between NoLN patients and LN patients; Figure 7B : The discovery set data showed that the CD79B gene was differentially expressed in NoLN patients and LN patients.

[0059] Figure 8 contains Fig. 8A and Figure 8B , which is the analysis result of the GEO validation set in the embodiment of this application. Fig. 8A : The validation set data showed that the COBLL1 gene was differentially expressed in NoLN patients and LN patients; Figure 8B : The validation set data showed that the CD79B gene was differentially expressed in NoLN patients and LN patients.

[0060] Figure 9 contains FIG. 9A to FIG. 9D , is the ROC curve analysis result of the discovery set in the examples of this application. Fig. 9A : COBLL1+CD79B combined detection can well distinguish LN patients from normal people; Fig. 9B :CD79B+EGLN1 combined detection has good discrimination between LN patients and normal people; Fig. 9C : COBLL1+FAM129C combined detection has good discrimination between LN patients and normal people; Fig.9D : The combined detection of COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 has good discrimination between LN patients and normal people.

[0061] Figure 10 contains FIG. 10A to FIG. 10D , is the ROC curve analysis result of the validation set in the embodiment of this application. Fig. 9A : COBLL1+CD79B combined detection can well distinguish LN patients from normal people; Fig. 9B :CD79B+EGLN1 combined detection has good discrimination between LN patients and normal people; Fig. 9C : COBLL1+FAM129C combined detection has good discrimination between LN patients and normal people; Fig.9D : The combined detection of COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 has good discrimination between LN patients and normal people.

[0062] Figure 11 contains FIG. 11A to FIG. 11D , are the clinical sample test results in the examples of this application. Fig.11A : Compared with normal people, the expression of COBLL1 gene in LN patients is significantly reduced. Fig. 11B : Compared with normal people, the expression of CD79B gene in LN patients is significantly reduced. Fig. 11C : Compared with normal subjects, there was no significant difference in the expression of COBLL1 gene in patients with systemic lupus erythematosus and without lupus nephritis; Fig.11D : Compared with normal people, there is no significant difference in the expression of CD79B gene in patients with systemic lupus erythematosus without lupus nephritis. DETAILED DESCRIPTION

[0063] At present, there is no non-invasive detection method for lupus nephritis in clinical practice; therefore, this application creatively downloads 1038 samples from the GeneExpression Omnibus (GEO) database, hoping to screen genes or their proteins that are differentially expressed in peripheral blood mononuclear cells of lupus nephritis patients and normal people through a series of bioinformatics methods. In the end, it was surprisingly found that COBLL1 and CD79B can be used as indications for lupus nephritis. Therefore, it is proposed to use COBLL1 and CD79B genes or proteins as biomarkers for the diagnosis of lupus nephritis, which makes up for the shortcomings of existing lupus nephritis-related indicators and has significant clinical value. This application is completed on this basis.

[0064] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0065] The terms "comprise", "include", "have", "contain" or "involve" used in this application are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0066] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" is used, this includes a plural of that noun.

[0067] The terms "approximately" and "substantially" used in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10%, preferably ±5%, of the indicated value.

[0068] The terms "probe" and "primer" used in this application refer to oligonucleotides complementary to the target position of the target nucleic acid, preferably single-stranded deoxyribonucleotides, including naturally occurring dNMPs (dAMP, dGMP, dCMP and dTMP), deformed nucleotides or non-natural nucleotides, and may also contain ribonucleotides. Of course, the "probe" and "primer" can be modified or sequences unrelated to the target nucleic acid can be added as needed; for example, in real-time fluorescence quantitative PCR amplification, the 5' end of the probe can be modified with a fluorescent group and the 3' end can be modified with a fluorescent quenching group; for example, in the sequencing process, a label sequence can be added to the 5' end of the primer or a corresponding adapter sequence can be added according to the sequencing platform.

[0069] In the present application, the meaning of "complementary" refers to the sufficient complementarity that a primer or a probe selectively hybridizes with a target nucleic acid sequence under hybridization conditions, and has the meaning of including both substantially complementary and perfectly complementary, preferably perfectly complementary. The term "substantially complementary sequences" used in the present specification includes not only completely identical sequences, but also sequences that can function as primers on a specific target sequence and are partially inconsistent with the sequence to be compared.

[0070] The sequences of gene probes and primers do not need to have sequences that are completely complementary to a portion of the sequence of the template, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. Therefore, the gene probes and primers in the present invention do not need to have sequences that are completely complementary to the above-mentioned nucleotide sequences as templates, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. The design of primers and gene probes is a skill mastered by those skilled in the art, for example, a primer design program (e.g., PRIMER 3 program) can be utilized.

[0071] The term "peptide" used in this application is a compound formed by α-amino acids linked together by peptide bonds. A peptide composed of three or more amino acid molecules is called a polypeptide; a protein is a substance with a certain spatial structure formed by the winding and folding of a polypeptide chain. "Antibody" refers to a protein that specifically recognizes and binds to an antigen protein. "Antigen binding fragment" refers to a polypeptide chain that can specifically bind to an antigen protein. In this application, the antigen protein refers to COBLL1 or CD79B protein.

[0072] The terms "COBLL1" and "CD79B" used in the present application refer to nucleic acid or protein thereof.

[0073] This application found through differential gene expression analysis that COBLL1 and CD79B were expressed less in peripheral blood mononuclear cells of patients with lupus nephritis, revealing that the above gene expressions can distinguish LN patients from normal people, thereby making a judgment on LN patients and assisting doctors in making decisions on kidney biopsy. The known functions of these genes are shown in Table 1.

[0074] Therefore, in various aspects of the present application, COBLL1 and CD79B are provided as markers for the diagnosis of lupus nephritis (LN), and the use of a detection reagent for detecting COBLL1 or CD79B in a sample in the preparation of a product for diagnosing lupus nephritis is provided.

[0075] The terms "lupus nephritis" and "LN" used in this application are interchangeable and widely occur in patients with systemic lupus erythematosus. 30-80% of SLE patients will develop lupus nephritis (Lupus nephritis, LN), often leading to chronic kidney disease (Chronic Renal Disease, CKD) and end-stage renal disease (End Stage Renal Disease, ESRD). Poor prognosis and high mortality. The onset is related to factors such as heredity, sex hormones, immunity, and environment. Specifically, it can refer to the criteria for diagnosing LN set by the American College of Rheumatology (ACR) in 1982 and / or the criteria revised by the Systemic Lupus Collaborative Clinic (SLICC) (reviewed in Petri et al. (Arthritis and Rheumatism, 2012, Vol.64, pp. 2677-2686)) characterized by autoimmune diseases.

[0076] The term "diagnosis" used in this application refers to the process of identifying a medical disorder or disease (such as LN) through its signs, symptoms, and especially the results of various diagnostic processes (including detecting the level of CD79B in a biological sample (such as serum) obtained from an individual). Moreover, the term "diagnosis" as used herein includes screening for diseases, auxiliary diagnosis, diagnosis (determining whether a disease exists), predicting or determining the severity of a disease, assessing disease activity, monitoring treatment, such as monitoring disease progression or recurrence during treatment, evaluating the therapeutic efficacy of the disease, and the selection of a given treatment regimen, etc. Without limitation, in some specific embodiments, the present application has fully demonstrated that COBLL1 or CD79B levels can be used to determine the presence or absence of LN in a subject, and can then be used for screening, auxiliary diagnosis, and diagnosis.

[0077] The term "treatment" as used in this application includes any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing partial or complete improvement of the disease. The therapeutic agent can be administered before, during, or after the onset of the disease or injury.

[0078] As used herein, the terms "individual," "subject," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, from whom a biological sample is collected.

[0079] The terms "sample", "sample", "test sample", "subject sample" and the like used in this application include various sample types obtained from a patient, individual or subject and can be used for diagnosis or monitoring determination. Patient samples can be obtained from healthy subjects, sick patients or patients with LN related symptoms. In addition, the sample obtained from the patient can be divided into parts, and only a part can be used for diagnosis. In addition, the sample or a part thereof can be stored under the condition of keeping the sample for subsequent analysis. This definition specifically includes other liquid samples of blood and biological origin (including but not limited to tissue, cells (such as PMBC cells, T cells, etc.), body fluids, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid, etc.). In a specific embodiment, the sample includes a blood sample. In another embodiment, a serum sample is used. This definition also includes samples that are manipulated in any way after the sample is obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells, cell supernatants, tissue samples, organs, etc. in culture. Samples may also include fresh frozen and / or formalin fixed, paraffin embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or research by immunohistochemistry. Samples may be tested immediately after collection, stored at RT, 4 degrees Celsius, -20 degrees Celsius, or -80 degrees Celsius, and tested after storage for 24 hours, 1 week, 1 month, 1 year, 10 years, or up to 30 years.

[0080] The term "ROC" or "ROC curve" used in this application refers to a receiver operating characteristic curve, also known as a sensitivity curve, which can be a graphical representation of the performance of a binary classifier system. For any given method, a ROC curve can be generated by plotting sensitivity for specificity under a variety of threshold settings. In addition, as long as at least one of the three parameters (e.g., sensitivity, specificity, and threshold settings) is provided, and the ROC curve can determine the value or expected value of any unknown parameter, the unknown parameter can be determined using a curve for ROC curve fitting.

[0081] The term "AUC" or "ROC-AUC" used in this application generally refers to the area under the receiver operating characteristic curve, i.e., Area Under Curve. This metric takes into account the sensitivity and specificity of the method and can provide a measure of the diagnostic utility of the method. Typically, ROC-AUC is in the range of 0.5 to 1.0, where values ​​closer to 0.5 indicate that the method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and values ​​closer to 1.0 indicate that the method has greater diagnostic utility (e.g., higher sensitivity and / or specificity). See, e.g., Pepe et al., "Limitations of the Odds Ratio in Gauging the Performance of a Diagnostic, Prognostic, or Screening Marker", Am. J. Epidemiol 2004, 159 (9): 882-890, which is incorporated herein by reference in its entirety. Other methods of characterizing diagnostic utility using likelihood functions, odds ratios, information theory, predictive values, calibration (including goodness of fit), and reclassification measures are summarized in Cook, "Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction), Circulation 2007, 115:928-935, which is incorporated herein by reference in its entirety.

[0082] The detection agents or components for obtaining the levels of biomarkers in a sample can be understood to include detection agents that directly obtain the levels of biomarkers in a sample, or components that indirectly obtain the levels of biomarkers in a sample (such as a computer program that directly obtains an indicator of the levels of biomarkers in a sample that has already been detected).

[0083] It can be understood that there are many ways to detect nucleic acids in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography. It can be understood that nucleic acids include DNA or RNA levels.

[0084] For example, in some embodiments, gene sequencing can be transcriptome sequencing or genome sequencing. Genome sequencing can adopt first-generation Sanger sequencing, second-generation sequencing ("NGS") or third-generation sequencing reagents, etc. Second-generation sequencing ("NGS") is different from "Sanger sequencing" (first-generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary change to traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. The sequencing platform of NGS that can be used for the present application is commercially available, including but not limited to Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Applied Biosystems SOLID system, etc. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a certain species' specific cells or tissues in a certain state through the second-generation sequencing platform, which can be used to study gene expression, gene function, structure, alternative splicing and new transcript prediction, etc. In some other embodiments of the present application, the nucleic acid sequencing method can be single-molecule real-time sequencing. Single-molecule DNA sequencing technology is a new generation of sequencing technology developed in the past 10 years, also known as the third-generation sequencing technology, including single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing and other technologies.

[0085] For example, in some embodiments, the polymerase chain reaction method includes primers for biomarker amplification, wherein the primer refers to a primer that can specifically amplify the biomarker gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least part of the template sequence and serve as the starting point for synthesizing the primer extension product. In some embodiments, the probe hybridization method includes a probe for a biomarker, wherein the probe refers to a probe that can specifically identify the CD79B gene or the transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other part of another molecule, and generally refers to a nucleic acid probe that binds to another nucleic acid (i.e., a biomarker-specific sequence as a target nucleotide) through complementary base pairing. In some embodiments, a gene chip refers to a composite structure formed by an array of the aforementioned probes fixed on a substrate material (specifically including but not limited to polymers such as nylon membranes, nitrocellulose membranes, and glass, etc.). In some embodiments, when RNA needs to be detected, cDNA needs to be obtained by reverse transcription, and then the detection is implemented by the primers for biomarker amplification.

[0086] It is understood that there are many protein detection methods in the art, including but not limited to sequencing technology, immunological technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0087] For example, in some embodiments, a method for quantitatively detecting biomarkers at the protein expression level includes a specific antibody. Among them, a specific antibody refers to an antibody that can specifically recognize a biomarker-encoded protein, specifically including but not limited to a monoclonal antibody (e.g., a full-length or complete monoclonal antibody), a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., a bispecific antibody with expected biological activity), a nanobody, or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity-matured antibody. According to the experimental data of the present application, COBLL1 and CD79B can be used as independent indications for the diagnosis of lupus nephritis, that is, effective diagnosis of lupus nephritis can be achieved based solely on either COBLL1 or CD79B biomarkers.

[0088] It is understandable that in order to further enhance the diagnostic effect of lupus nephritis, the biomarkers detected by the reagents can also be other biological materials that can be used as markers, such as nucleic acid fragments, proteins, metabolites, etc. with diagnostic effects known to those skilled in the art. In this way, a combination of multiple biomarkers can be used, and they can cooperate with each other to achieve a better diagnostic effect on lupus nephritis patients, thereby achieving a more effective diagnostic evaluation. Therefore, in addition to being combined with each other as markers for the joint detection of lupus nephritis, COBLL1 and CD79B can also be combined with other existing known diagnostic markers for detection, thereby improving diagnostic efficiency.

[0089] For example, in some specific embodiments, the diagnosis also includes diagnostic reagents for detecting other genes or proteins, such as those mentioned in this application. Combined detection can be a combination of any one or more of them. In a preferred embodiment of this application, when COBLL1, CD79B, VPREB3, E2F5, FBXL13, and C3orf62 are used for the diagnosis and evaluation of lupus nephritis at the same time, it has a higher AUC value of 0.864, which significantly improves the effect of distinguishing lupus nephritis patients from normal people.

[0090] In the present application, the control samples can be selected according to actual needs. For example, in disease diagnosis, the control samples are samples from the normal population, while when used for prognosis assessment, they can be control samples of different prognostic conditions.

[0091] In some specific embodiments, a set value of a biomarker level can be given. For example, the set value can be determined based on the expression level of CD79B in normal samples of normal people and / or non-lupus nephritis patients, for example, the average value of the expression level of CD79B of normal samples of a suitable number of samples is selected, or a reasonable multiple is set based on the average value, such as 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the expression level of CD79B of the subject is lower than the set value, it is judged as lupus nephritis. It is understandable that the set value determined based on the average value, or the multiple of the average value needs to have good classification significance, and the known samples can be tested by using commonly used statistical test methods through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard.

[0092] The level of a biomarker refers to a value derived from direct measurement or further indirect measurement of the biomarker for a subject, which is usually derived at least in part from the abundance or concentration of the biomarker in the subject's sample. Indirectly derived values ​​include those derived by applying a function to the measured value of the biomarker. Directly measured values ​​include, but are not limited to, values ​​of biomarkers determined by at least one of sequencing, hybridization, mass spectrometry, immunoassay, immunofluorescence, flow cytometry, etc.

[0093] According to the core diagnostic use of the present application, the process for detecting the level of the biomarker of the present application can be configured into a corresponding product form for the diagnosis or prediction of lupus nephritis. The product includes reagents or components for detecting the biomarker of the present application. It is understood that such product forms are diverse, including but not limited to kit forms, system devices, computer readable media or computer system forms.

[0094] In some embodiments of the present application, a kit for detecting or analyzing the biomarker level of the present application to predict LN disease is also disclosed. The kit may include reagents for detecting the level of one or more markers and instructions for predicting LN disease based on the detected levels.

[0095] The kit of the present application may include a set of reagents for generating a data set by at least one determination. The set of reagents can detect any biomarker level of the quantitative present application. The set of reagents can also further detect other one or more biomarker levels. In some aspects, the detection reagent is used for the level detection of nucleic acids or proteins.

[0096] When the detection reagent is used for nucleic acid expression level detection, it can be understood that there are many nucleic acid detection methods in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0097] For example, in some embodiments, the reagent may be a sequencing reagent, such as a first-generation Sanger sequencing reagent, a second-generation sequencing (NGS) reagent, a third-generation sequencing reagent, etc. In other embodiments, the reagent may be a PCR primer reagent, wherein the primer refers to a primer that can specifically amplify a biomarker gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting site for synthesizing primer extension products. In other embodiments, the reagent may be a probe reagent, wherein the probe refers to a probe that can specifically identify a biomarker gene or a transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other portion of another molecule, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a biomarker-specific sequence as a target nucleotide) through complementary base pairing.

[0098] When the detection reagent is used for protein expression level detection, it can be understood that there are many protein detection methods in the art, including but not limited to sequencing technology, immune technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0099] For example, in some embodiments, the reagent can be a specific antibody, wherein the specific antibody refers to an antibody that can specifically recognize the protein encoded by the biomarker, specifically including but not limited to monoclonal antibodies (such as full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies with expected biological activity) or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity matured antibody.

[0100] In some embodiments, the kit may also include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which contains one of the individual elements to be used in the method. The kit of the present application may include the above-mentioned containers and one or more other containers containing materials required from the standpoint of a commercial end user, including buffers, diluents, filters, and package inserts with instructions for use.

[0101] In some embodiments, the kit further includes a sample processing reagent, which may include at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0102] In some embodiments, the kit further includes at least one of a standard, a calibrator, a reference substance, and a buffer. The reference substance is a control substance for testing the effectiveness of the experiment and serves as a control for judging the results. The buffer can be any solution known in the art that can provide appropriate buffer conditions during the detection process.

[0103] In some embodiments, the kit may include instructions for use of a set of reagents. For example, the kit may include instructions for performing at least one assay, such as an immunoassay, a protein binding assay, an antibody-based assay, an antigen binding protein-based assay, a protein-based array, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein array, blots, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, enzyme activity, and an immunoassay selected from RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassay, and immunoprecipitation.

[0104] In addition to the above-mentioned components, the kit will further include instructions for implementing the subject method. These instructions can be present in the subject kit in various forms, one or more of which can be present in the kit. One form in which these instructions can exist is that they are printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, the paper is in the packaging of the kit or in the form of a package insert, etc.

[0105] In some embodiments of the present application, a system for detecting or analyzing biomarker levels to predict the severity of LN disease is also disclosed. The system may include a set of reagents for detecting the level of one or more markers; a device configured to receive a mixture of one or more reagents and a test sample obtained from a subject to measure the biomarker level of the present application; and a computer system that is coupled to the device in communication to obtain the measured level and determine a score that predicts the likelihood of LN disease severity in LN patients.

[0106] In some embodiments, the device is configured to detect the biomarker level in the mixture of reagent and test sample.For example, the device can determine the biomarker level of the present application by immunoassay or the assay for nucleic acid detection.The mixture of reagent and test sample can be provided to the device by various containers, and the examples of these containers include the hole, bottle or tube of the orifice plate (such as 96 orifice plate).Because of this, the device can have an opening (such as a slot, a cavity, an opening, a sliding tray), which can receive the container for accommodating the reagent test sample mixture and read to generate the quantitative expression value of a soluble medium.The example of the device includes an ELISA reader (such as a luminescent ELISA reader, an absorbent ELISA reader, a fluorescent ELISA reader), a spectrometer and a spectrophotometer.

[0107] In some embodiments, the computer system communicates with the device to receive the quantitative expression values ​​of the soluble mediators, and the computer system analyzes the quantitative expression values ​​and determines the likelihood of a LN disease activity event in the subject by applying a predictive model.

[0108] In some embodiments of the present application, the method or use of the present application can be performed on a computer, including detecting or analyzing the level of the biomarkers of the present application by a computer to predict LN disease.

[0109] For example, the construction and execution of a predictive model for generating a score (e.g., an LFPI score) can be implemented in hardware or software or a combination of both. In one embodiment, a readable storage medium is provided, such as a medium including a data storage material encoded with machine-readable data, which, when used with a machine programmed with instructions of the data, can display any data set and execution and results of the predictive model of the present application. Such data can be used for a variety of purposes, such as LN patient monitoring, diagnosis, treatment considerations, etc. The implementation scheme of the above method can be implemented in a computer program executed on a programmable computer, the computer including a processor, a data storage system, a graphics adapter, a network adapter, at least one input device and at least one output device, etc. The display is coupled to the graphics adapter. The program code is applied to the input data to perform the above functions and generate output information. The output information is applied to one or more output devices in a known manner. The computer can be a personal computer, a microcomputer, or a workstation of conventional design.

[0110] Each program can be implemented in a high-level program or object-oriented programming language to communicate with a computer system. However, if desired, the program can be implemented in assembly or machine language. In any case, the language can be a compiled language or an interpreted language. Each such computer program is preferably stored in a storage medium or device (such as a ROM or disk) readable by a general or special programmable computer, for configuring and operating the computer to execute the program of the present application when the computer reads the storage medium or device.

[0111] The system may also be considered to be implemented in the form of a computer-readable storage medium configured with a computer program, wherein the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions of the present application.

[0112] In some embodiments, feature patterns and their databases can be provided in various media to facilitate their use. The term "medium" refers to a manufactured product containing feature pattern information of the present application. The database of the present application can be recorded on a computer-readable medium, such as any medium that a computer can directly read and access. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD ROMs; electronic storage media such as RAM and ROM; and hybrids of these categories, such as magnetic / optical storage media. Those skilled in the art can easily understand how to use any currently known computer-readable medium to produce a record containing current database information. The term "record" refers to the process of storing information on a computer-readable medium using any such method known in the art. Based on the method used to access the stored information, any convenient data storage structure can be selected. A variety of data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0113] It can be understood that, based on the belief that the biomarkers of the present application can be used as diagnostic markers for lupus nephritis, technical personnel in this field can evaluate or screen other potential diagnostic markers for lupus nephritis based on this, and usually evaluate the potential biomarkers with the biomarkers of the present application through correlation, and on the basis of reaching the conclusion of positive correlation, it helps to illustrate the value of the potential markers.

[0114] The present application also relates to a method for detecting a marker in a subject suffering from or suspected of suffering from lupus nephritis in vivo or in vitro, the method comprising determining or detecting the level of the biomarker of the present application in a biological sample from the subject, and may include uses other than disease diagnosis.

[0115] In addition, the present application further relates to a method for in vivo or in vitro screening of lupus nephritis therapeutic agents, the method comprising the step of evaluating the biomarker level of the present application on a sample treated with the therapeutic agent, and evaluating the therapeutic effect of the therapeutic agent based on the biomarker level. For example, when the expression level of CD79B decreases, the therapeutic effect of the therapeutic agent can be determined.

[0116] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0117] This application downloads 2 expression matrix files of peripheral blood mononuclear cell samples of lupus nephritis patients from the Gene Expression Omnibus (GEO) database, namely GSE99967 and GSE65391. Among them, the GSE99967 data set contains 29 SLE samples with LN and 13 SLE samples without LN, and the GSE65391 data set contains 924 SLE patients with or without LN and 72 control samples. After merging and normalizing the GSE99967 and GSE65391 data, all samples are allocated as discovery set and validation set according to 7:3.

[0118] Example 1. Expression analysis of COBLL1 and CD79B in peripheral blood mononuclear cells (PBMC) of patients with lupus nephritis

[0119] The screening conditions for differentially expressed genes in this example are: difference fold>1.5, p<0.05. It was found that COBLL1 and CD79B were expressed lower in the disease group of the discovery set than in the normal group samples, that is, 541 LN samples and 49 control samples. The results are shown in Figure 1. The gene COBLL1 ( Figure 1A ) and CD79B( Figure 1B ) were expressed in peripheral blood mononuclear cells (PBMCs) of patients with lupus nephritis, indicating that both genes can be used as diagnostic markers for potential lupus nephritis.

[0120] Example 2: ROC-based evaluation analysis

[0121] In order to test the diagnostic efficacy of the candidate markers, the present example plotted the receiver operating characteristic (ROC) curves of COBLL1 and CD79B in the discovery data set, and the results are shown in FIG2 .

[0122] The results in Figure 2 show that in the discovery set, the area under the ROC curve (AUC) value of COBLL1 is 0.807 ( Figure 2A ), the AUC value of CD79B was 0.824 ( Figure 2B ), indicating that COBLL1 and CD79B have good performance in distinguishing patients with lupus nephritis from normal people.

[0123] Example 3: Verification of the verification set

[0124] In this example, 154 LN samples and 22 control sample data cohorts were used for statistical analysis, and the screening conditions for differentially expressed genes were: difference fold>1.5, p<0.05, and the results are shown in Figure 3. ROC curves of COBLL1 and CD79B in the validation set were drawn, and the results are shown in Figure 4.

[0125] The results in Figure 3 show that the gene COBLL1( Figure 3A ) and CD79B( Figure 3B ) was significantly downregulated in PBMC of patients with lupus nephritis; at the same time, as shown in Figure 4, ROC analysis in this cohort revealed that the AUC of COBLL1 could reach 0.797 ( Figure 4A ), the AUC value of CD79B was 0.813 ( Figure 4B ); fully demonstrated the specificity and sensitivity of COBLL1 and CD79B in the diagnosis of lupus nephritis.

[0126] Example 4: Analysis of expression in patients with systemic lupus erythematosus without lupus nephritis (NoLN)

[0127] 4.1 Differences in gene expression between NoLN patients and normal subjects

[0128] In this case, 189 NoLN samples and 49 control samples were used for expression difference statistical analysis, and the screening condition was: difference fold > 1.5. The results are shown in Figure 5. In the discovery set, COBLL1 ( Figure 5A ) and CD79B( Figure 5B ) had an expression difference of less than 1.5 times in PBMCs of NoLN patients and normal controls, indicating that COBLL1 and CD79B are not diagnostic markers of NoLN.

[0129] In order to further verify the diagnostic effect of the marker, a statistical analysis was performed on a data cohort of 67 NoLN samples and 22 control samples. The results are shown in Figure 6. In the validation set, COBLL1 ( Fig. 6A ) and CD79B( Figure 6B ) The expression difference between PBMCs of NoLN patients and normal controls did not reach 1.5 times, and p>0.05, which fully proved that COBLL1 and CD79B are not diagnostic markers of NoLN.

[0130] 4.2 Differences in gene expression between NoLN patients and LN patients

[0131] In this case, 189 NoLN samples and 541 LN sample data cohorts were used for expression difference statistical analysis. The results are shown in Figure 7. In the disease group of the discovery set, COBLL1 ( Fig. 7A ) and CD79B( Figure 7B ) were differentially expressed between NoLN patients and LN patients (p<0.05), demonstrating that COBLL1 and CD79B can distinguish LN from NoLN patients.

[0132] In order to further verify the diagnostic effect of the marker, the expression difference statistical analysis was performed on the data cohorts of 67 NoLN samples and 154 LN samples. The results are shown in Figure 8. In the disease group of the validation set, COBLL1( Fig. 8A ) and CD79B( Figure 8B ) were differentially expressed between NoLN patients and LN patients (p<0.05), which fully demonstrated that COBLL1 and CD79B could distinguish LN and NoLN patients.

[0133] Example 5: Multi-marker combined detection

[0134] In this example, 541 LN samples and 49 control sample data cohorts were used for statistical analysis, and ROC curves of four combined detection combinations (1) COBLL1+CD79B, (2) CD79B+EGLN1, (3) COBLL1+FAM129C, and (4) COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 were plotted in the discovery set. The results are shown in FIG6 . In this cohort, the AUC value of COBLL1+CD79B combined detection was found to be 0.834 ( Fig. 9A ), the AUC value of CD79B+EGLN1 combined detection can reach 0.823 ( Fig. 9B ), the AUC value of COBLL1+FAM129C combined detection can reach 0.802 ( Fig. 9C ), the AUC value of COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 combined detection can reach 0.864 ( Fig.9D ); It was demonstrated that COBLL1 and CD79B can be combined with each other as markers for the joint detection of lupus nephritis, and can also be combined with other existing known diagnostic markers.

[0135] In order to further verify the effect of combined detection, statistical analysis was performed on the data cohort of 154 LN samples and 22 control samples, and the ROC curves of the four combined detection combinations of (1) COBLL1+CD79B, (2) CD79B+EGLN1, (3) COBLL1+FAM129C, and (4) COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 in the validation set were plotted. The results are shown in Figure 10. In this cohort, the AUC value of COBLL1+CD79B combined detection was found to be 0.831 ( Fig. 10A ), the AUC value of CD79B+EGLN1 combined detection can reach 0.817 ( Fig. 10B ), the AUC value of COBLL1+FAM129C combined detection can reach 0.825 ( Fig. 10C), the AUC value of COBLL1+CD79B+VPREB3+E2F5+FBXL13+C3orf62 combined detection can reach 0.854 ( Fig. 10D ); It is fully proved that COBLL1 and CD79B can be combined with each other as markers for the combined detection of lupus nephritis, and can also be combined with other existing known diagnostic markers for combined detection. When combined detection is performed, a higher AUC value can be obtained, which significantly improves the diagnostic efficiency.

[0136] Example 6: Clinical sample testing

[0137] In this case, 60 whole blood samples were collected from people, including 20 samples from patients with systemic lupus erythematosus and lupus nephritis, and 20 samples from patients with systemic lupus erythematosus and without lupus nephritis; at the same time, 20 whole blood samples from healthy people were collected as controls. The inclusion criteria for lupus nephritis patients in this case were: patients with a SLE disease activity index (SLEDAI) greater than 5.

[0138] 6.1 Detection of gene expression levels

[0139] After collecting all whole blood specimens, peripheral blood mononuclear cells were isolated, dissolved with Trizol (Beyotime, R-0016), and stored at -80°C. Total RNA was isolated from peripheral blood mononuclear cells according to the manufacturer's instructions. mRNA was reverse transcribed using the transScript all-in-one-first-strand cDNA synthesis superMix for qPCR (One-step gDNAremoval) kit (TransGen Biotech, AT341-01). qPCR detection was performed using the PerfectStart Green qPCRSuperMix kit (TransGen Biotech, AQ601-02). The relative expression of genes was calculated using the 2-ΔCT method. p value < 0.05 was considered statistically significant.

[0140] The test results showed that the expression levels of COBLL1 and CD79B in 20 samples of patients with systemic lupus erythematosus and lupus nephritis were significantly lower than those in 20 samples used as control samples ( Fig.11A , Fig. 11B ); however, the expression levels of COBLL1 and CD79B in the 20 samples of SLE without lupus nephritis were not significantly different from those in the 20 control samples ( Fig. 11C , Fig.11D ). The results showed that there were statistical differences in the expression levels of COBLL1 and CD79B genes between lupus nephritis patients and healthy subjects, and they could be used as biomarkers for lupus nephritis.

[0141] 6.2 Protein expression level detection

[0142] To detect CD79B protein in plasma samples, the Bradford method was first used to configure the standard curve protein sample, and CD79B protein was used as the standard. A standard curve was usually drawn between 20 and 150 μg / 100 μL, and the concentration of the sample to be tested was calculated based on the standard curve. Next, SDS-PAGE electrophoresis was performed and transfected onto a PVDF membrane. Finally, the transfected membrane was blocked and incubated overnight with a monoclonal CD79B-specific antibody, washed and incubated with an HRP-labeled secondary antibody for 1 hour, and DAB was used to develop the brown protein color band. The results showed that there was a statistical difference in the expression level of CD79B protein between patients with lupus nephritis and healthy people, and it can be used as a biomarker for lupus nephritis.

[0143] It can be understood that although this example is a study on the expression level of the CD79B gene; however, it is foreseeable that there is a statistical difference in the expression level of the CD79B gene between lupus nephritis patients and healthy people, and there must be a statistical difference in the expression level of the CD79B protein between lupus nephritis patients and healthy people; therefore, CD79B, as a biomarker for lupus nephritis, can be the CD79B gene or the CD79B protein, and lupus nephritis can be diagnosed based on the expression level of the CD79B gene or the CD79B protein. On this basis, detection primers and / or probes can be designed based on the CD79B gene, or specific detection polypeptides can be designed based on the CD79B protein, and these primers, probes or polypeptides can be used as reagents for diagnosing lupus nephritis.

[0144] The protein expression level of another marker of the present application, COBLL1, can be detected by referring to the above method.

[0145] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

[0146] Table 1. Gene-encoded proteins and functions

[0147]

[0148]

Claims

1. Use of a reagent for detecting the level of one or more of the following biomarkers in the preparation of a lupus nephritis diagnostic product, wherein the biomarkers include COBLL1 and CD79B.

2. A product for the diagnosis of lupus nephritis, wherein: The product contains reagents for detecting the levels of one or more of the following biomarkers, including COBLL1 and CD79B.

3. The use according to claim 1 or the product according to claim 2, wherein: The biomarker level includes nucleic acid expression level or protein expression level.

4. The use according to claim 1 or the product according to claim 3, wherein: The reagents for detecting the nucleic acid expression level include specific primers and / or probes designed for the nucleic acid of the biomarker; or The reagent for detecting the protein expression level includes a polypeptide that specifically binds to or recognizes the biomarker protein.

5. The product according to claim 4, wherein: The nucleic acid includes the gene of the biomarker, a DNA fragment extracted from the biomarker gene, or RNA, mRNA, miRNA or LcRNA corresponding to the biomarker gene or its DNA fragment; or The polypeptide includes at least one of an antibody, an antigen binding fragment, a single-chain antibody, and a ligand that specifically binds to the biomarker protein.

6. The use according to claim 1 or the product according to any one of claims 2 to 5, wherein: The product may also include reagents or components for detecting other markers.

7. A method for diagnosing lupus nephritis, wherein: The following steps are involved: (i) obtaining the level of a biomarker in a sample to be tested, wherein the biomarker is selected from one or more of the following: COBLL1 and CD79B; (ii) comparing the biomarker level in the test sample with the corresponding biomarker level in the control sample; if there is a significant difference, the subject from which the test sample is derived has an indication of lupus nephritis; or, comparing the biomarker level in the test sample with a set threshold; if the biomarker level in the test sample is lower than the threshold, the subject from which the test sample is derived has an indication of lupus nephritis.

8. A method for evaluating or screening lupus nephritis markers, wherein: This includes correlation or concordance analysis of candidate biomarkers with COBLL1 or CD79B levels.

9. A method for evaluating or screening a therapeutic agent for lupus nephritis, wherein: The method comprises evaluating the level of a biomarker in a sample after treatment with a therapeutic agent, wherein the biomarker is selected from one or more of the following: COBLL1 and CD79B.

10. The method according to any one of claims 7 to 9, wherein: The biomarker level includes nucleic acid expression level or protein expression level; Preferably, the nucleic acid expression level is the expression level of DNA, RNA or mRNA; or the protein expression level is the level of binding between the protein as a marker and its specific polypeptide.

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