Immunofluorescence kit for diagnosing primary and secondary IgA nephropathy

Abstract

The invention belongs to the technical field of immunofluorescence detection, and particularly provides an immunofluorescence kit for diagnosing primary and secondary IgA nephropathy, the immunofluorescence kit contains a primary antibody and a secondary antibody, and the primary antibody is an anti-CD175 antibody and / or an anti-CD175s antibody; the second antibody is an antibody capable of being specifically combined with the first antibody, and the second antibody is labeled by using a fluorescent dye. The kit disclosed by the invention is simple and convenient to operate, and low-glycosylation IgA1 deposited in the kidney of an IgA nephropathy patient can be identified in a short time; the method has extremely high sensitivity and specificity, such as 92.7% and 83.9%. The kit disclosed by the invention can be used for accurately identifying primary IgA nephropathy and secondary IgA nephropathy including systemic lupus erythematosus, purpura nephritis, membranous nephropathy and the like, and has extremely high application value.

Description

Technical Field

[0001] The invention belongs to the technical field of immunofluorescence detection, and particularly relates to an immunofluorescence kit for diagnosing primary and secondary IgA nephropathy. Background Art

[0002] IgA, or immunoglobulin A, is second only to IgG in serum content. It accounts for 10-20% of serum immunoglobulins and exists in mucosal tissues, such as the digestive tract, respiratory tract, and urogenital system.

[0003] IgA nephropathy generally refers to primary IgA nephropathy, which is the most common primary glomerular disease. It refers to the deposition of IgA in the glomerular mesangium or mainly IgA deposition, with or without the deposition of other immunoglobulins in the glomerular mesangium. Lesion types include focal segmental lesions, endocapillary proliferative lesions, mesangial proliferative lesions, crescentic lesions and sclerotic lesions. Its clinical manifestations are recurrent macroscopic hematuria or microscopic hematuria, which may be accompanied by varying degrees of proteinuria. Some patients may experience severe hypertension or renal insufficiency.

[0004] The diagnosis of primary IgA nephropathy (IgAN) must rely on renal biopsy, mainly observing the deposition of IgA in the glomerular mesangial area. Immunofluorescence or immunohistochemistry staining is an essential diagnostic method to confirm the presence of IgA deposition. In recent years, the study of IgA nephropathy has made significant progress. It was found that the main IgA deposited in the glomerular mesangial area of ​​IgAN patients is IgA1 lacking O-glycosylated galactose in the hinge region, also known as hypoglycosylated IgA1 (Gd-IgA1). This Gd-IgA triggers an immune response as an autoantigen, leading to the production of anti-Gd-IgA1 antibodies, the formation of large molecular immune complexes and deposition in the glomerular mesangial area, which in turn triggers mesangial cell proliferation and extracellular matrix deposition, causing damage to the kidneys. Secondary IgA nephropathy such as systemic lupus erythematosus (lupus nephritis), purpuric nephritis, and membranous nephropathy also has IgA deposition. The deposited IgA usually does not contain Gd-IgA1, so the pathogenesis is significantly different from that of IgAN.

[0005] In clinical practice, immunofluorescence is usually used to detect IgA deposited in the kidney. This method alone cannot distinguish between primary and secondary IgA nephropathy. Therefore, rapid and effective detection of Gd-IgA1 is of great significance for the diagnosis and differential diagnosis of IgAN in renal pathology. Previous studies used the snail agglutinin (helixaspersa, HAA) detection system to measure the level of Gd-IgA in serum. However, HAA has poor polysaccharide recognition activity and stability, and cannot be directly used to detect IgA deposited in renal tissue. Therefore, it cannot be used for pathological diagnosis and differentiation of IgA nephropathy. In addition, although mass spectrometry analysis based on renal biopsy tissue can be used to conduct in-depth research on the hinge region glycoforms of IgA, thereby revealing the glycoform characteristics of IgA in individual disease states and its key role in pathogenic heterogeneity, this method is very challenging and complicated in technical processes such as the decomposition of sugar chains, making it difficult to be applied in clinical work. In recent years, Japanese scientists have used the peptide chain with glycosylation defects in the hinge region of Gd-IgA to prepare an anti-Gd-IgA antibody (KM55), which is believed to be able to specifically recognize the Gd-IgA1 molecule. However, more and more foreign studies and previous studies by our team have shown that KM55 cannot quickly and effectively distinguish primary IgA nephropathy from secondary IgA nephropathy.

[0006] In summary, the limitations of the detection methods for Gd-IgA1 specific markers hinder their clinical application and affect the differential diagnosis and treatment options for IgA nephropathy. It is necessary to develop new, rapid, specific, and sensitive technologies for diagnosing IgA nephropathy, which has broad clinical application prospects and important scientific significance. In other words, because it is quick and convenient to distinguish whether IgA nephropathy is primary or secondary IgA nephropathy, it can greatly help the patient's subsequent medication and treatment plans. Therefore, a new immunofluorescence kit for diagnosing primary and secondary IgA nephropathy is needed in the art. Summary of the invention

[0007] In view of the above problems in the prior art, the present invention provides an immunofluorescence kit for detecting primary IgA nephropathy and secondary IgA nephropathy, which can identify hypoglycosylated IgA1 deposited in the kidney.

[0008] The present invention provides an immunofluorescence kit for diagnosing primary and secondary IgA nephropathy. The immunofluorescence kit contains a primary antibody and a secondary antibody, wherein the primary antibody is an anti-CD175 antibody and / or an anti-CD175s antibody; the secondary antibody is an antibody that can specifically bind to the primary antibody, and the secondary antibody has been labeled with a fluorescent dye.

[0009] In a specific embodiment, the primary antibody is an anti-human CD175 antibody and / or an anti-human CD175s antibody.

[0010] In a specific embodiment, the primary antibody is mouse anti-human CD175 antibody and / or mouse anti-human CD175s antibody, preferably mouse anti-human CD175 antibody.

[0011] In a specific embodiment, the primary antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0012] In a specific embodiment, the secondary antibody is a goat anti-mouse IgG antibody labeled with Cy3.

[0013] In a specific embodiment, the kit further includes one or more of a diluent, a buffer, a washing solution and a blocking solution, wherein the washing solution and the blocking solution are used to wash and block non-specifically bound antibodies; the secondary IgA nephropathy includes one or more of purpuric nephritis, lupus nephritis and membranous nephropathy.

[0014] The present invention also provides a method for using the immunofluorescence kit as described above, wherein the ratio of the primary antibody to CD175 monoclonal antibody is 1:100-1:200, and the ratio of the secondary antibody to CD175s monoclonal antibody is 1:50-1:100, and finally the fluorescence signal is observed and recorded using a fluorescence microscope.

[0015] In the present invention, the sample used for detection by the immunofluorescence kit is fresh frozen renal biopsy tissue or formaldehyde-fixed paraffin-embedded renal tissue, preferably fresh frozen renal biopsy tissue.

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] The present invention uses an immunofluorescence method to detect for the first time the expression of CD175 and CD175s antigens in IgA and non-IgA mesangial proliferative glomerulonephritis. The results show that CD175 and / or CD175s are specifically and significantly differentially expressed in IgAN with IgA deposition in the mesangial region, while they are not expressed in mesangial proliferative glomerulonephritis with non-IgA deposition. Therefore, CD175 and / or CD175s glycoprotein antigens can be used as a candidate biomarker for the specific detection of Gd-IgA1 deposition for clinical diagnosis. In addition, the ROC analysis results of the present invention on a large sample of primary IgAN and secondary IgA deposition showed that the sensitivity and specificity of CD175 glycoprotein antigen for the diagnosis of primary IgAN were 92.7% and 83.9%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram showing that both CD175 antibody and CD175s antibody recognize hypoglycosylated IgA deposited in the renal mesangium.

[0019] Figure 2 Differentiate primary IgA nephropathy from secondary IgA nephropathy by detecting CD175 antibodies or CD175s antibodies.

[0020] Figure 3 The receiver operating curve was used to evaluate the value of CD175 antibody immunofluorescence detection in the diagnosis of primary and secondary IgA nephropathy. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0022] The following embodiments are only used to illustrate the present invention, but are not limited to limiting the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0023] In the examples of the present invention, unless otherwise specified, all reagent components are commercially available products well known to those skilled in the art. In the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0024] The technical solution of the present invention is as follows:

[0025] The present invention provides an immunofluorescence kit for diagnosing primary and secondary IgA nephropathy, which comprises at least a primary antibody and a secondary antibody, wherein the primary antibody contains an anti-human CD175 antibody and / or an anti-human CD175s antibody; and the secondary antibody is labeled with a fluorescent dye and can specifically bind to the primary antibody.

[0026] In the present invention, the immunofluorescence kit for diagnosing primary and secondary IgA nephropathy is for samples with conventional IgA immunofluorescence positive, especially for but not limited to cases with IgA deposited in the glomerular mesangial area. The collection and processing methods of these samples, as well as immunofluorescence detection and positive sample determination are all conventional methods and techniques.

[0027] The IgA is immunoglobulin A, and the hinge region contains 6 O-glycan sites, specifically T225, T228, S230, S232, T233 and T236 of IgA1.

[0028] The structure of the glycoprotein antigen CD175 is GalNAcα1-O-Ser / Thr; the structure of the CD175s glycoprotein antigen is NeuAcα2-6GalNAcα1-O-Ser / Thr.

[0029] The primary IgA nephropathy refers to the deposition of IgA1 in the glomerular mesangial region, specifically IgA1 lacking O-sugar chain galactose in the hinge region.

[0030] The secondary IgA nephropathy refers to IgA deposited in the renal mesangial region, but IgA1 lacking O-glycosylated galactose in the non-hinge region, including systemic lupus erythematosus, purpuric nephritis, membranous nephropathy, etc.

[0031] As a preferred embodiment, the titer of mouse anti-human CD175 monoclonal antibody is 1:100-1:200, and the titer of mouse anti-human CD175s is 1:50-1:100. The above antibodies are purchased from commercial companies. In addition, these antibodies can also be prepared by conventional antibody preparation methods.

[0032] As a preferred embodiment, the secondary antibody is a goat anti-mouse IgG antibody labeled with Cy3.

[0033] In the above technical solution, the kit also includes diluents, buffers, washing reagents, etc. required for detection. These reagents can be conventional reagents for antigen-antibody immunofluorescence detection in the art, which will not be described in detail here.

[0034] As a preferred embodiment, the method for using the fluorescence kit is as follows:

[0035] The frozen sections were fixed in pre-cooled methanol, washed with PBS buffer, and then immunostaining rapid blocking solution was added. After blocking, the sections were shaken dry, primary antibody was added, incubated at room temperature and rinsed, fluorescent secondary antibody was added, incubated again at room temperature and rinsed, then transferred to a dark place and fluorescent anti-quenching agent was added; after adding sealing agent, coverslips were used to seal the sections, and observations were made under a fluorescence microscope.

[0036] The present invention finds through immunofluorescence experiments that CD175 or CD175s glycoprotein antigen is highly expressed in the glomerular mesangial region of patients with primary IgA nephropathy and is co-expressed with IgA1 deposited in the glomerular mesangial region. Immunofluorescence detection of CD175 or CD175s glycoprotein antigen in renal tissue can be used as a marker of IgA1 lacking O-sugar chain galactose.

[0037] Another aspect of the present invention also provides the use of an immunofluorescence detection kit comprising a mixed primary antibody of CD175 and CD175s for diagnosis and differential diagnosis of primary and secondary IgA nephropathy.

[0038] The present invention has found through experiments that CD175 and / or CD175s are highly expressed in patients with primary IgA nephropathy, while other patients with secondary IgA nephropathy have little expression. The ROC analysis results show that CD175 or CD175s detection has extremely high sensitivity and specificity. Therefore, CD175 and CD175s glycoprotein antigen immunofluorescence detection can be used for the diagnosis and differential diagnosis of renal IgA deposition diseases.

[0039] The inventor believes that the existing method for diagnosing IgA nephropathy is a pathological diagnosis method of renal puncture, and then the patient is determined to be primary IgA nephropathy or secondary IgA nephropathy through medical history investigation and subsequent biochemical examination. In the present invention, the patient is generally still determined to be IgA nephropathy through pathological diagnosis of renal puncture, but it is no longer necessary to do medical history investigation and subsequent biochemical examination. Instead, the kit of the present invention is used to determine whether the patient suffers from primary IgA nephropathy or secondary IgA nephropathy. The detection object of the kit of the present invention is still the section of the kidney pathological specimen.

[0040] The primary antibody used in the kit of the present invention is an antibody to the antigen CD175 (also known as Tn) or an antibody to the antigen CD175s (also known as sTn), both of which can be purchased directly. The secondary antibody in the kit is a conventional antibody. When using the kit of the present invention, the pathological section is first dripped with the primary antibody, then the secondary antibody, and finally placed under a fluorescence microscope for detection. If the test result is a fluorescence intensity of more than one plus sign, it represents a positive result, that is, the patient has primary IgA nephropathy; otherwise, it is negative, representing secondary IgA nephropathy.

[0041] Generally, it is not necessary to set IgA antibody in the kit of the present invention, because at this time, we have already known from the pathological staining results of the renal puncture specimen slices that the patient has IgA nephropathy and has IgA deposition. Before using the kit of the present invention, it is just unknown whether the patient's IgA nephropathy is primary IgA nephropathy or secondary IgA nephropathy.

[0042] In general, those skilled in the art know that what is deposited in the kidneys in primary IgA nephropathy is hypoglycosylated IgA (Gd-IgA), and what is deposited in the kidneys in secondary IgA nephropathy is usually normal IgA, not Gd-IgA. Those skilled in the art also know that there are CD175 antigen and CD175s antigen on Gd-IgA molecules, but relatively small amounts of CD175 antigen and CD175s antigen can also appear on normal IgA molecules; CD175 antigen and CD175s antigen belong to a molecular modification of secretory proteins, and they can also appear on other protein molecules. Therefore, in the prior art of the present invention, there is no scheme for distinguishing between primary and secondary IgA nephropathy by identifying Gd-IgA in tissues by using antibodies to detect CD175 antigen and / or CD175s antigen, and there is even less scheme in the prior art for distinguishing between primary and secondary IgA nephropathy by using antibodies to CD175 antigen and / or CD175s antigen to identify Gd-IgA in kidney sections of patients.

[0043] In addition, the kit of the present invention can only be used for immunofluorescence and for detecting kidney sections. When immunohistochemistry is used, because CD175 antigen and CD175s antigen are unstable and easily destroyed during tissue processing, the immunohistochemical effect is poor, and immunofluorescence is required. Gd-IgA itself is deposited in large quantities in the kidneys of patients with primary IgA nephropathy, and when CD175 antigen or CD175s antigen is detected in serum, it is difficult to distinguish between primary and secondary IgA nephropathy.

[0044] Example 1

[0045] This embodiment provides an immunofluorescence kit for identifying hypoglycosylated IgA.

[0046] 1. Tissue collection: Frozen sections of renal biopsies from 5 cases of primary IgA nephropathy. Immunofluorescence confirmed the deposition of IgA in the glomerular mesangium in these cases, with a fluorescence intensity of no less than 1+, and other diseases with secondary IgA deposition were excluded in the cases and clinically.

[0047] 2. Renal tissue IgA immunofluorescence detection and positive sample determination are routine methods and techniques.

[0048] 3. Sample fixation and blocking: Place the frozen sections in methanol pre-cooled at 4°C for 10 minutes, wash with PBS buffer 3 times, 3 minutes each time. Add immunostaining rapid blocking solution (4°C refrigerator, Bio-Tech) and block at room temperature for 20 minutes.

[0049] 4. Add primary antibody: Shake off the blocking solution, add primary antibody, add rabbit anti-human IgA antibody (Abcam, USA) and mouse anti-human CD175 antibody (Invitrogen, USA) diluted in PBS, or add rabbit anti-human IgA antibody and mouse anti-human CD175s antibody (Invitrogen, USA). The antibody titer of rabbit anti-human IgA monoclonal antibody is 1:100-1:200, the titer of mouse anti-human CD175 monoclonal antibody is 1:100-1:200, and the titer of mouse anti-human CD175s is 1:50-1:100. Place the slide in a light-proof humid box, incubate at 4°C overnight, and wash 3 times with PBS buffer, 3 minutes each time.

[0050] 5. Add fluorescent secondary antibodies: FITC-labeled goat anti-rabbit IgG antibody and Cy3-labeled goat anti-mouse IgG antibody, incubate at 37°C for 2 hours; wash with PBS in the dark for 3 times, 3 minutes each time. To reduce nonspecific staining, centrifuge the secondary antibody at 3000-5000rpm before use;

[0051] 6. Sealing: Add anti-fluorescence quenching sealing agent, cover with coverslip, and incubate in the dark at room temperature for 10 minutes.

[0052] 7. Film reading and result interpretation: The images were observed and collected under a fluorescence microscope, and the results were interpreted by two renal pathologists using a semi-quantitative method (intensity level 0-4).

[0053] 8. The results showed that Gd-IgA identified by immunofluorescence using monoclonal antibodies against CD175 and CD175s glycoprotein antigens was deposited in the mesangial area of ​​IgA nephropathy, and this deposition was highly consistent with renal deposition of IgA.

[0054] In this example, rabbit anti-human IgA antibody was added when the primary antibody was added, and FITC-labeled goat anti-rabbit IgG antibody was added when the secondary antibody was added, the purpose of which was to prove that the specimen itself was a kidney section specimen of an IgA nephropathy-positive patient.

[0055] The fluorescence microscope images in Example 1 are shown in Figure 1 . Figure 1 The first column is a control stained with IgA antibody, which shows green fluorescence. Figure 1 The second column is Gd-IgA, which represents low glycosylated IgA and shows red fluorescence. The upper picture is stained with CD175 antibody, and the lower picture is stained with CD175s antibody. The third column is merge, which overlaps the two pictures on the left and shows yellow fluorescence. It is equivalent to staining with IgA antibody and CD175 antibody or CD175s antibody. As long as it is a kidney section of a patient with primary IgA nephropathy, its fluorescence detection spectrum will appear as follows Figure 1 Fluorescence shown.

[0056] Example 2

[0057] This embodiment provides CD175 antibody or CD175s antibody for detecting primary IgA nephropathy and secondary IgA nephropathy.

[0058] 1. Tissue collection: 40 cases of primary IgA nephropathy renal biopsy frozen sections, all of which have been confirmed by immunofluorescence to have IgA deposited in the glomerular mesangial area, with a fluorescence intensity of not less than 1+. Twelve cases of purpuric nephritis, 16 cases of lupus nephritis, and 14 cases of membranous nephropathy with immunofluorescence and a fluorescence intensity of not less than 1+ were collected. Ten cases of renal microlesion specimens without IgA deposition were collected as negative controls.

[0059] 2. Sample fixation and blocking are the same as in Example 1. Rabbit anti-human IgA antibody and mouse anti-human CD175 antibody diluted in PBS are added dropwise, or rabbit anti-human IgA antibody and mouse anti-human CD175s antibody are added dropwise, and the primary antibody titer is the same as in Example 1; the remaining steps are the same as in Example 1.

[0060] 3. The results show: Figure 2 As shown, immunofluorescence using monoclonal antibodies against CD175 or CD175s glycoprotein antigens is shown in Figure 2 The results of the second row of pictures show that the immunofluorescence staining of CD175 or CD175s in primary IgA nephropathy (IgAN) is positive, while the staining of CD175 or CD175s in HSPN, idiopathic membranous nephropathy (MN), and lupus nephritis (SLE) are all negative.

[0061] Specifically, Figure 2 The first row shows the pictures of IgA antibodies stained for four diseases: IgAN, HSPN, MN, and SLE. All four show green fluorescence. Figure 2 The second row is the result of staining with CD175 antibody or CD175s antibody. Figure 2 IgA antibodies are not used in the second row; Figure 2 From the photos in the second row, we can see that the first picture on the left is primary IgA nephropathy, which shows red fluorescence; while the three pictures on the right are secondary IgA nephropathy, none of which show any fluorescence. Figure 2 The results of fluorescence staining of only one case section from each of 40 cases of primary IgA nephropathy, 12 cases of purpuric nephritis, 16 cases of lupus nephritis, and 14 cases of membranous nephropathy are shown.

[0062] Example 3

[0063] This example uses a receiver operating curve to evaluate the value of CD175 antibody immunofluorescence detection in the diagnosis of primary and secondary IgA nephropathy.

[0064] like Figure 3 As shown, we used the receiver operating characteristic curve (ROC) to evaluate the diagnosis and differential diagnosis of CD175 antibody for primary and secondary IgA nephropathy, and the result was AUC = 0.932, which shows that the kit of the present invention has a good diagnostic value. Specifically, the ROC analysis results of the large sample of 40 cases of primary IgA nephropathy and a total of 42 cases of secondary IgA nephropathy as shown in Example 2 in this example show that the sensitivity and specificity of the kit of the present invention for primary and secondary IgA nephropathy detection are 92.7% and 87.8%, respectively, indicating that the kit of the present invention can achieve effective diagnosis and differentiation of primary and secondary IgA nephropathy.

[0065] In general, the present invention belongs to the field of immunohistology technology. The marker of the present invention is O-glycan antigen CD175 or its sialylated form CD175s, and the reagent includes at least one of anti-CD175 antibody and anti-CD175s antibody. The kit of the present invention is simple and convenient to operate, and can identify low-glycosylated IgA1 deposited in the kidneys of patients with IgA nephropathy in a short time; the method has extremely high sensitivity and specificity, for example, reaching 92.7% and 83.9%. The kit of the present invention can accurately identify primary IgA nephropathy and secondary IgA nephropathy including systemic lupus erythematosus, purpuric nephritis, membranous nephropathy, etc., and has extremely high application value.

[0066] The embodiments described above are only preferred implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any equivalent replacement or modification made by any technician familiar with the field within the technical scope disclosed by the present invention based on the technical scheme and inventive concept of the present invention should be covered within the protection scope of the present invention.

Claims

1. An immunofluorescence kit for diagnosing primary and secondary IgA nephropathy, characterized in that: The immunofluorescence kit contains a primary antibody and a secondary antibody, and the primary antibody is an anti-CD175 antibody and / or an anti-CD175s antibody; the secondary antibody is an antibody that can specifically bind to the primary antibody, and the secondary antibody has been labeled with a fluorescent dye.

2. The immunofluorescence kit for diagnosing primary and secondary IgA nephropathy according to claim 1, characterized in that: The primary antibody is an anti-human CD175 antibody and / or an anti-human CD175s antibody.

3. The immunofluorescence kit for diagnosing primary and secondary IgA nephropathy according to claim 2, characterized in that: The primary antibody is mouse anti-human CD175 antibody and / or mouse anti-human CD175s antibody, preferably mouse anti-human CD175 antibody.

4. The immunofluorescence kit for diagnosing primary and secondary IgA nephropathy according to claim 1, characterized in that: The primary antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

5. The immunofluorescence kit for diagnosing primary and secondary IgA nephropathy according to claim 1, characterized in that: The secondary antibody is a goat anti-mouse IgG antibody labeled with Cy3.

6. The immunofluorescence kit for diagnosing primary and secondary IgA nephropathy according to claim 1, characterized in that: The kit also includes one or more of a diluent, a buffer, a washing solution and a blocking solution, wherein the washing solution and the blocking solution are used to wash and block non-specifically bound antibodies; the secondary IgA nephropathy includes one or more of purpuric nephritis, lupus nephritis and membranous nephropathy.

7. A method for using the immunofluorescence kit according to any one of claims 1 to 6, characterized in that: The ratio of CD175 monoclonal antibody used as the primary antibody was 1:100-1:200, while the ratio of CD175s monoclonal antibody used as the primary antibody was 1:50-1:

100. Finally, the fluorescence signal was observed and recorded using a fluorescence microscope.

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