Application of erythrocyte as M protein carrier in diagnosis of monoclonal immunoglobulin disease

Abstract

The invention provides application of red blood cells as an M protein carrier in diagnosis of monoclonal immunoglobulin diseases, and proves that the monoclonal immunoglobulin combined with the red blood cells exists in peripheral blood for the first time, and not only the complete monoclonal immunoglobulin but also a monoclonal light chain or a monoclonal heavy chain exists. Polyclonal immune globulin in normal serum is rarely combined with erythrocytes, so that electrophoresis by using erythrocyte protein is more sensitive and recognizable than electrophoresis by using plasma to identify M protein and isotype thereof. The invention provides the application of the reagent for detecting the erythrocyte M protein in diagnosis or auxiliary diagnosis of the monoclonal immunoglobulin disease, and a favorable tool is provided for positioning the M protein isotype disease of a patient and guiding the monoclonal guide treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of red blood cells as M protein carriers in the diagnosis of monoclonal immunoglobulin disease. Background Art

[0002] Monoclonal gammopathy is characterized by the presence of a monoclonal immunoglobulin (M-protein) in plasma, urine, or both. M-protein is produced by clonal proliferation of plasma cells or B lymphocytes and can be an indicator of an underlying malignancy or a precursor to malignancy, such as monoclonal gammopathy of undetermined significance (MGUS) and monoclonal gammopathy of renal significance (MGRS). Detection of M-protein is a fundamental part of the diagnosis and management of patients with monoclonal gammopathy.

[0003] A range of laboratory tests have been developed to identify M-protein, including serum and urine protein electrophoresis (SPEP and UPEP), serum and urine immunofixation (sIFE and uIFE), capillary immunophenotyping electrophoresis (CE / IS), serum light chain (sTLC) and free light chain (sFLC), mass spectrometry, and quantitative measurement of heavy / light chain (HLC) isotypes. Although SPEP, IFE, and CE / IS are the most widely used clinical tests, there is a lack of standardization in reporting practices between laboratories in the setting of high polyclonal immunoglobulins associated with plasma M-protein, and there is considerable variability, especially in the setting of low M-protein concentrations. The introduction of quantitative serum tests for immunoglobulin sFLC has improved the sensitivity and objective quantitative detection of laboratory testing strategies, however, sFLC testing does not accurately localize the M-protein isotype and guide clonality-directed therapy. Therefore, the development of a test method that can accurately detect M-protein and accurately localize the isotype is of great significance for clinically directed treatment of monoclonal immunoglobulin diseases. Summary of the invention

[0004] In response to the above problems, the present invention has proved for the first time through research that there are monoclonal immunoglobulins bound to red blood cells in peripheral blood, and that M protein exists on the surface of peripheral blood red blood cells. Detection of red blood cell M protein can be used as an indicator for the diagnosis or auxiliary diagnosis of monoclonal immunoglobulin disease.

[0005] To achieve the above purpose, the specific technical solutions provided by the present invention are as follows: A first aspect of the present invention provides use of a reagent for detecting red blood cell M protein in a sample in the preparation of a diagnostic product for monoclonal immunoglobulin disease, wherein the sample comprises red blood cells derived from a subject.

[0006] Monoclonal gammopathy, also known as abnormal globulinemia and paraglobulinemia, most commonly affects the kidneys. The incidence has been on the rise in recent years.

[0007] M protein is an abnormal immunoglobulin produced by the massive proliferation of plasma cells or B lymphocytes. Its essence is an immunoglobulin or a fragment of immunoglobulin (light chain, heavy chain, etc.). M protein positivity can be seen in multiple myeloma, malignant lymphoma, chronic lymphocytic leukemia, macroglobulinemia, non-lymphoreticular tumors, mononuclear cell leukemia, cryoglobulinemia, heavy chain disease, monoclonal gammopathy with renal significance, monoclonal gammopathy of undetermined significance, etc. Monoclonal gammopathy of undetermined significance refers to a type of disease in which monoclonal immunoglobulin (M protein <3g / dl) is present in the blood, there is a small amount or no M protein in the urine, the proportion of bone marrow plasma cells is less than 10%, and there is no terminal organ damage. According to the type of M protein, it is mainly divided into non-IgM type (IgG or IgA type), IgM type and light chain type MGUS, among which IgG type is the most common.

[0008] Furthermore, the diagnosis refers to detecting M protein in red blood cells to obtain the result of whether the subject suffers from monoclonal gammopathy or has a risk of suffering from monoclonal gammopathy.

[0009] In the present invention, the term sample refers to a composition obtained or derived from a subject (eg, an individual of interest), and the sample may be from the subject's blood and other fluid samples and tissue samples of biological origin.

[0010] Furthermore, the sample is a peripheral blood sample from a subject.

[0011] Furthermore, the reagents for detecting red blood cell M protein in the sample include reagents used in one or more of the following methods: electrophoresis, hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry and protein chip method.

[0012] Furthermore, the reagents may also include auxiliary detection reagents for protein expression, which include but are not limited to: blocking solution; antibody diluent; washing buffer; color development stop solution; and standard substances used to prepare standard curves.

[0013] Furthermore, the monoclonal gammopathy includes one or more of monoclonal gammopathy of undetermined significance, multiple myeloma, light-chain amyloidosis, light-chain deposition disease, heavy-chain deposition disease, macroglobulinemia, cryoglobulinemia, monoclonal gammopathy of renal significance, cast nephropathy, membranous nephropathy, minimal change nephropathy, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, and non-lymphoreticular tumors.

[0014] Further, the subject refers to any individual of interest. In some embodiments, the subject refers to a living organism suffering from or suspected of suffering from a monoclonal gammopathy, including humans and other mammals.

[0015] Furthermore, the subject includes humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, and monkeys.

[0016] Furthermore, the product includes a kit, a chip, a test strip, or a device, an equipment, or a computer-readable storage medium.

[0017] In the present invention, the product may include a solid substrate such as a chip, a slide, an array, etc., which has reagents capable of detecting and / or quantifying one or more blood or other sample-derived M proteins immobilized at predetermined locations on the substrate. As an illustrative example, a chip may be provided with reagents immobilized at discrete predetermined locations for detecting and quantifying M proteins in a blood sample.

[0018] In the present invention, the computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in combination with an instruction execution system, apparatus, or device. Specifically, the computer-readable storage medium is a method for distinguishing different components, elements, parts, portions, or assemblies at different levels. However, if other words can achieve the same purpose, the words may be replaced by other expressions. It is well known to those skilled in the art that the present invention may be implemented as an apparatus, method, or computer program product.

[0019] Another aspect of the present invention provides a kit for diagnosing monoclonal gammopathy, the kit comprising a reagent for detecting red blood cell M protein in a sample, wherein the sample is a peripheral blood sample from a subject.

[0020] Furthermore, the monoclonal gammopathy includes one or more of monoclonal gammopathy of undetermined significance, multiple myeloma, light-chain amyloidosis, light-chain deposition disease, heavy-chain deposition disease, macroglobulinemia, cryoglobulinemia, monoclonal gammopathy of renal significance, cast nephropathy, membranous nephropathy, minimal change nephropathy, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, and non-lymphoreticular tumors.

[0021] Furthermore, the reagents for detecting red blood cell M protein in the sample include reagents used in one or more of the following methods: electrophoresis, hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry and protein chip method.

[0022] Furthermore, the reagents may also include auxiliary detection reagents for protein expression, which include but are not limited to: blocking solution; antibody diluent; washing buffer; color development stop solution; and standard substances used to prepare standard curves.

[0023] In the present invention, the step of pre-treating the above-mentioned sample before it is used for the detection of the present invention can be included. The pre-treatment may include the treatment required for releasing or separating the compound, or removing excess material or waste. Suitable techniques include centrifugation, extraction, fractionation, purification and / or enrichment of the compound. In addition, other pre-treatments are performed to provide the compound in a form or concentration suitable for compound analysis. For example, if gas chromatography coupled to mass spectrometry is used for the method of the present invention, it will be necessary to derivatize the compound before the gas chromatography. Suitable and necessary pre-treatments depend on the tools for carrying out the method of the present invention and are well known to those skilled in the art. The pre-treated sample as described above is also included in the term "sample" as used in the present invention.

[0024] Furthermore, the kit also includes reagents for processing samples.

[0025] Advantages and beneficial effects of the present invention: The present invention demonstrates for the first time the presence of monoclonal immunoglobulins bound to erythrocytes in peripheral blood, and not only complete monoclonal immunoglobulins but also monoclonal light or heavy chains. The present invention provides evidence that electrophoresis using erythrocyte proteins is more sensitive and recognizable than using serum immunophenotyping electrophoresis to identify M proteins and their isotypes, especially in the case of low M protein concentrations and high polyclonal immunoglobulin backgrounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The following are representative results of red blood cell electrophoresis, from left to right, rabbit monoclonal antibodies against human λ, κ, IgG, IgM and IgA. ab is a representative Western blot result of a normal control; c is a representative Western blot result of a patient with lupus nephritis, showing different bands for IgG, IgM, IgA, κ and λ; d is a representative Western blot result of a patient with primary membranous nephropathy, whose serum anti-PLA2R antibody test was positive, but no immunoglobulin binding was detected on red blood cells; eh is a representative Western blot result of a patient with monoclonal gammopathy.

[0027] Figure 2 Statistical chart of serum immunoglobulin levels in patients with or without erythrocyte M protein (including all samples), where a is the serum λ free light chain (FLC) level, b is the serum κ free light chain (FLC) level, c is the serum IgM level, d is the serum IgA level, and e is the serum IgG level. The dotted line indicates the upper limit of the normal free light chain or immunoglobulin serum level.

[0028] Figure 3 Figure 1 shows the results of red blood cell electrophoresis (part 1), where ax is the red blood cell electrophoresis results of 24 normal controls.

[0029] Figure 4 Figure 2 shows the results of red blood cell electrophoresis (Part 2), where y-an is the results of red blood cell electrophoresis of 16 normal controls.

[0030] Figure 5 Figure 2 is the result of red blood cell electrophoresis (the third part), where ao-as are the results of red blood cell electrophoresis of 5 patients with lupus nephritis, and at-ax are the results of red blood cell electrophoresis of 5 patients with primary membranous nephropathy.

[0031] Figure 6 ROC curves for validation set samples (a) and screening set samples (b).

[0032] Figure 7 The following are representative results of the comparison between red blood cell electrophoresis and serum immunophenotyping electrophoresis. The left side of each figure shows the protein blot result of red blood cell electrophoresis, and the right side shows the corresponding serum immunophenotyping electrophoresis result. Among them, a is a representative protein blot result of the red blood cell electrophoresis and serum immunophenotyping electrophoresis results being consistent; b is a representative protein blot result of serum immunophenotyping electrophoresis negative and red blood cell electrophoresis positive; c is a representative protein blot result of serum immunophenotyping electrophoresis inconsistent with red blood cell electrophoresis; d is a representative protein blot result of serum immunophenotyping electrophoresis positive and red blood cell electrophoresis negative. A and B are representative protein blot results of patients with monoclonal gammopathy.

[0033] Figure 8 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy cases 1 to 4).

[0034] Fig. 9 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy cases 5-8).

[0035] Fig.10 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy cases 9-12).

[0036] Fig.11 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy from case 13 to case 16).

[0037] Fig.12 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy No. 17-20).

[0038] Fig.13 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy No. 21-24).

[0039] Fig.14 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy No. 25-28).

[0040] Fig.15 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy cases 29-32).

[0041] Fig.16 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal immunoglobulin disease No. 33-36).

[0042] Fig.17 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal immunoglobulin disease No. 37-40).

[0043] Fig.18 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal gammopathy No. 41-44).

[0044] Fig.19 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal immunoglobulin disease No. 45-47).

[0045] Fig. 20 The results of red blood cell electrophoresis and serum immunophenotyping electrophoresis (including samples of patients with monoclonal immunoglobulin disease No. 48-49). DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0047] Example

[0048] 1. Sample Source This study included 19 patients newly diagnosed with monoclonal gammopathy in the Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong First Medical University from April 2023 to January 2024 as the screening set, and 30 patients newly diagnosed with monoclonal gammopathy in the Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong First Medical University from February 2024 to December 2024 as the validation set (age ≥ 18 years). Twenty age- and sex-matched healthy individuals with normal immunophenotyping electrophoresis results and normal sTLC and sFLC ratios were recruited in the screening set and validation set as normal controls, as well as 5 patients with lupus nephritis (LN) and 5 patients with primary membranous nephropathy (PMN) with positive serum antiphospholipase A2 receptor (PLA2R) antibodies as disease controls. Written informed consent was obtained from each patient. This study was conducted in accordance with the Declaration of Helsinki. This study was approved by the Local Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

[0049] 2. Materials and Methods 1. Serum M protein detection: In local clinical practice, patients with kidney disease admitted to the hospital for kidney disease routinely undergo serum sTLCs detection, sFLCs detection, and capillary immunophenotyping electrophoresis (CE / IS) for monoclonal gammopathy screening. sTLC was analyzed by turbidity analyzer Dade Behring BN II (Siemens, Germany) using the N anti-human immunoglobulin / L-chain kit. The concentrations of κFLC and λFLC were evaluated using the Siemens N-Latex FLCκ (κFLC) and λ (λFLC) assays (Siemens Medical, Germany) according to the manufacturer's instructions. CE / IS was performed using CAPILLARYS 2 (capillary system) according to the manufacturer's instructions. According to the data from LSI Medience, the reference ranges of κ and λ were 2.42-18.92 and 4.44-26.18 mg / L, respectively, and the κ / λ ratio was 0.248-1.804. The detection limits of FLC assay were 50.5 mg / L and 43800 mg / L, respectively.

[0050] 2. Extraction of erythrocyte membrane proteins: Immediately after blood collection, erythrocytes were separated by centrifugation at 3000 rpm and washed with phosphate-buffered saline (PBS). Pre-cooled 10 mmol / L hypotonic Tris-HCl buffer (pH 7.4) was then added to the washed erythrocytes at a volume ratio of 1:40, followed by incubation at 4 °C for 2 h. After that, the erythrocyte membranes were precipitated by centrifugation at 4 °C and 12000 rpm for 15 min. RIPA lysis buffer (Solarbio, Beijing, China) was then introduced, and the mixture was incubated on ice for 30 min. Protein samples were extracted from the supernatant after centrifugation. Protein concentration was determined using a BCA protein assay kit (Epizyme, Shanghai, China).

[0051] 3. Western blot analysis: Red blood cell membrane proteins were denatured in protein sample loading buffer (denaturation, reduction, 5×) and boiled for 10 minutes. Subsequently, the proteins were electrophoresed by 10% SDS-PAGE and then transferred to a polyvinyl fluoride membrane (PVDF Millipore). Dilutions of the following antibodies were prepared and incubated with the membrane overnight at 4°C: rabbit monoclonal anti-human λ antibody (dilution 1:5000, ab124719, Abcam), rabbit monoclonal anti-human κ antibody (dilution 1:1000, ab12.4727, Abcam), rabbit monoclonal anti-human IgG antibody (dilution 1:5000, ab133470, Abcam), rabbit monoclonal anti-human IgM antibody (dilution 1:1000, ab212201, Abcam), rabbit monoclonal anti-human IgA antibody (dilution 1:4000, ab133660, Abcam). The membrane was then incubated with HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000, SA00001-2, Proteintech) for 1 hour at room temperature. After adding the developer, the immunoreactive area was visualized using the ECL system and the Bio-Rad electrophoresis image analyzer.

[0052] 4. Statistical analysis: SPSS version 22.0 statistical software (SPSS, Chicago, IL) was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation, median and 25th-75th percentile, median and range or number (%). ROC curve analysis was used to detect the sensitivity and specificity of red blood cell electrophoresis for the diagnosis of monoclonal gammopathy. Quantitative data were expressed as mean ± standard deviation, median and 25th-75th percentile, median and range or number (%).

[0053] 3. Experimental Results 1. Patient characteristics: Based on the results of CE / IS, sTLC or sFLC ratio and renal biopsy, 49 patients were enrolled in this study, including 48 patients with abnormal CE / IS and / or sTLC and / or sFLC ratios and 1 MGRS patient with negative circulating M protein test. The general characteristics of the patients are shown in Table 1.

[0054] Table 1. General characteristics of the patient cohort

[0055] Of the 49 patients, 31 were diagnosed with multiple myeloma (MM), of whom 9 underwent renal biopsy. Renal pathology results included 3 cases of light chain amyloidosis, 2 cases of cast nephropathy, 3 cases of light chain deposition disease (LCDD), and 1 case of cryoglobulinemia. 2 of the 49 patients were diagnosed with macroglobulinemia (WM) without renal biopsy. 12 of the 49 patients were diagnosed with MGRS, including 7 cases of light chain amyloidosis, 2 cases of LCDD, and 1 case of light / heavy chain deposition disease, proliferative glomerulonephritis with monoclonal IgG deposition (PGNMID) could not be clearly excluded, and 2 cases of confirmed PGNMID. The remaining 4 patients were considered to have MGUS. Among them, 1 patient had abnormal CE / IS results (IgG-κ) and sTLC ratio without any signs of organ involvement. 1 patient had abnormal CE / IS results (IgG-λ) and sTLC ratio, and was diagnosed with minimal change nephropathy (MCD) by renal biopsy. One patient had an abnormal CE / IS result (IgM-λ) but a normal ratio of sTLC and sFLC, and was diagnosed with focal segmental glomerulosclerosis (FSGS) by renal biopsy. One patient had an abnormal CE / IS result (IgG-λ) and an abnormal ratio of sTLC and sFLC. Due to the patient's advanced age, a renal biopsy was not performed. However, the patient was positive for anti-PLA2R antibodies, and his proteinuria responded well to rituximab treatment, so the diagnosis of MGUS combined with PLA2R-related membranous nephropathy was considered.

[0056] Table 2. Comparison of the results of M protein identification by red blood cell electrophoresis and serum detection (samples above the dotted line are screening samples, and samples below the dotted line are validation samples)

[0057] 2. Immune proteins on the surface of erythrocytes: Western blot analysis was used in this study to identify immune proteins present on the surface of erythrocytes, including IgG, IgM, IgA, κ, and λ. Among the 40 healthy controls, some people did have very weak λ, κ, IgM, or IgG bands. In 5 patients with lupus nephritis (LN), all tested immune proteins appeared on the surface of erythrocytes. Immunoglobulins bound to erythrocytes in LN patients showed polyclonal characteristics rather than monoclonal origin. In 5 patients with primary membranous nephropathy who were positive for anti-PLA2R antibodies, no immunoglobulins were detected on erythrocytes. Representativeness of erythrocyte electrophoresis ( Figure 1 ) and detailed results ( Figure 3-5 ) respectively as Figure 1 , 3-5. Among the 49 patients with monoclonal gammopathy participating in this study, 45 patients (91.8%) had a significant increase in the amount of erythrocyte membrane-bound immunoglobulins, and they were characterized by monoclonal properties. Not only complete monoclonal immunoglobulins but also only monoclonal immunoglobulin components could be detected on erythrocytes. Among the remaining 4 patients, 1 patient with light chain amyloidosis had all detected immunoglobulins detected on erythrocytes. No immunoglobulins were detected on erythrocytes in 1 LCDD, 1 MGUS (renal pathology type was MCD), and 1 MM patient. Representativeness of erythrocyte electrophoresis ( Figure 7 ) and detailed results ( Figure 8-20 ) respectively as Figure 7-20 shown.

[0058] Figure 6 The ROC curves of the screening set and validation set for the diagnosis of monoclonal gammopathy by red blood cell electrophoresis were shown. The sensitivity of the screening set was 94.7%, the specificity was 90.0%, and the area under the ROC curve (AUC) was 0.924 (CI: 0.83-1.00, P<0.001); the sensitivity of the validation set was 90.0%, the specificity was 90.0%, and the area under the ROC curve (AUC) was 0.900 (CI: 0.80-1.00, P<0.001).

[0059] 3. Correlation between serum immunoglobulin and free light chain levels and erythrocyte M protein deposition: This study investigated whether the deposition of M protein on erythrocytes is determined by the amount of serum immunoglobulin. Figure 2 As shown, in patients with the presence of M protein on erythrocytes, the serum level of the corresponding immunoglobulin is usually found to be elevated. However, it was observed that in a subgroup of patients with normal serum levels of immunoglobulin, the corresponding immunoglobulin was detected on erythrocytes, and conversely, in patients with undetectable erythrocyte M protein, the corresponding immunoglobulin serum level was elevated.

[0060] 4. Comparison of erythrocyte electrophoresis and serum testing for identification of M protein: Of all 49 patients with monoclonal gammopathy included in this study, 45 (91.8%) had M protein detected on erythrocytes, 38 (77.5%) had abnormal CE / IS results, 36 (73.5%) had abnormal sFLC ratios, and 32 (65.3%) had abnormal sTLC ratios. In this study, when considering combined detection of M protein, the combination of serum testing including CE / IS, sTLC, and sFLC ratios could diagnose 98.0% of cases, while the combination of serum testing and erythrocyte electrophoresis could diagnose 100.0% of cases. In addition, because normal serum polyclonal immunoglobulins rarely bind to erythrocytes and the background is relatively clean, electrophoresis using erythrocyte proteins seems to be more sensitive and recognizable than electrophoresis using plasma to identify M protein and its isoforms. Figure 8-20 The results of red blood cell electrophoresis and serum CE / IS for all patients in the study are shown. Figure 7 Representative examples are shown.

[0061] As shown in Table 2 , the results of erythrocyte western blot, serum CE / IS, sFLC, renal biopsy, and bone marrow flow cytometry were concordant in 26 of the 49 patients (53.1%). Eleven of the 49 patients (22.4%) had negative results in CE / IS testing, while erythrocyte western blot analysis showed the presence of exogenous monoclonal immunoprotein bands that were consistent with the results of sFLC, bone marrow flow cytometry, or renal histopathology. Patient 41 deserves special attention. She had a normal sFLC ratio and a negative CE / IS result. Immunofluorescence analysis of the renal biopsy specimen showed IgG 3+, kappa 3+, lambda weakly positive (+ / −), C3 2+, and C1q 3+ deposits, and IgG3 1+ and IgG1 / 2 / 4 were negative. Light microscopic examination showed histologic features consistent with membranoproliferative and nodular glomerular changes. Electron microscopic analysis revealed fine-grained electron-dense deposits on the endothelial side of the glomerular basement membrane. The final diagnosis was considered MGRS (light / heavy chain deposition disease), without definitively excluding the possibility of PGNMID. Western blot analysis of erythrocytes showed the presence of exogenous IgG bands in the absence of κ or λ chains, suggesting that IgG heavy chains were deposited on erythrocytes. Serum CE / IS and erythrocyte electrophoresis results were abnormal but discordant in 8 of the 49 patients (16.3%). Patient 44 deserves special attention. Her sFLC ratio was normal, and serum CE / IS results indicated the presence of IgMλ, but erythrocyte electrophoresis analysis showed a distinct and intense band for IgM in the absence of neither κ nor λ chains. Immunofluorescence analysis of the renal biopsy specimen showed IgG 1+, IgM 1+-2+, κ1+, λ1+ deposition. On light microscopy, the histological features suggested FSGS. The lack of glomeruli available for electron microscopy ruled out a definitive diagnosis of heavy chain deposition disease.

[0062] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting red blood cell M protein in a sample in the preparation of a diagnostic product for monoclonal immunoglobulin disease, characterized in that: The sample contains red blood cells from a subject; The sample is a peripheral blood sample from a subject.

2. The use according to claim 1, characterized in that: The reagents for detecting red blood cell M protein in the sample include reagents used in one or more of the following methods: electrophoresis, hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry and protein chip method.

3. The use according to claim 1, characterized in that: The monoclonal gammopathy includes one or more of monoclonal gammopathy of undetermined significance, multiple myeloma, light-chain amyloidosis, light-chain deposition disease, heavy-chain deposition disease, macroglobulinemia, cryoglobulinemia, monoclonal gammopathy of renal significance, cast nephropathy, membranous nephropathy, minimal change nephropathy, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, and non-lymphoreticular tumors.

4. The use according to claim 1, characterized in that: The subjects include humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, and monkeys.

5. The use according to claim 1, characterized in that: The product includes a test kit, a chip, a test strip, or a device, an equipment, or a computer-readable storage medium.

6. A kit for diagnosing monoclonal gammopathy, characterized in that: The kit comprises a reagent for extracting red blood cell M protein from a sample and a reagent for detecting red blood cell M protein in the sample, and the sample is a peripheral blood sample from a subject.

7. The kit according to claim 6, characterized in that The monoclonal gammopathy includes one or more of monoclonal gammopathy of undetermined significance, multiple myeloma, light-chain amyloidosis, light-chain deposition disease, heavy-chain deposition disease, macroglobulinemia, cryoglobulinemia, monoclonal gammopathy of renal significance, cast nephropathy, membranous nephropathy, minimal change nephropathy, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, and non-lymphoreticular tumors.

8. The kit according to claim 6, characterized in that The reagents for detecting red blood cell M protein in the sample include reagents used in one or more of the following methods: electrophoresis, hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry and protein chip method.

9. The kit according to claim 6, characterized in that The kit also includes reagents for processing the sample.

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