Application of urine IgA-C3 in IgA nephropathy diagnosis and treatment effect prediction marker

Abstract

The invention belongs to the technical field of medical detection and diagnosis, and relates to application of urine IgA-C3 in IgA nephropathy diagnosis and treatment effect prediction markers, the urine IgA-C3 is applied to products for auxiliary diagnosis of the IgA nephropathy, and a new breakthrough is brought to diagnosis and treatment of the IgA nephropathy. The reliable and stable urine IgA-C3 compound ELISA detection method established by the invention can effectively detect the level of the compound in the urine of a patient, and provides a powerful tool for early diagnosis of diseases; through detection and analysis of a large number of patients and control people, the advantage of urine IgA-C3 in the aspect of differential diagnosis of IgA nephropathy and other nephropathy is defined, and the area under the ROC curve shows good diagnosis efficiency.

Description

Technical Field

[0001] This invention belongs to the field of medical testing and diagnostic technology, specifically relating to the application of urinary IgA-C3 as a biomarker for the diagnosis and treatment efficacy prediction of IgA nephropathy. Background Technology

[0002] IgA nephropathy is the most common primary glomerular disease worldwide, with a particularly high incidence in Asian populations, accounting for 30%-40% of primary glomerular diseases. The core pathological feature of this disease is the deposition of IgA immune complexes in the glomerular mesangial area. Clinical phenotypes are highly heterogeneous; patients may present with asymptomatic hematuria and proteinuria. Some patients may rapidly progress to renal insufficiency, and ultimately, approximately 30%-40% of patients progress to end-stage renal disease within 20 years of diagnosis, requiring dialysis or kidney transplantation to sustain life, placing a heavy burden on patients' families and the social healthcare system.

[0003] Currently, the gold standard for diagnosing IgA nephropathy remains renal biopsy. While this method can clearly identify the pathological type and extent of the lesion, it has limitations such as invasiveness, potential complications (e.g., bleeding, infection), and the inability to repeat testing, making it unsuitable for early disease screening, dynamic monitoring of the disease, and evaluation of treatment effectiveness. Furthermore, patients respond significantly to treatment, some exhibit treatment resistance, and traditional monitoring indicators (such as serum creatinine and quantified urine protein) have low sensitivity, often only showing significant changes when substantial kidney damage occurs. This makes it difficult to identify patients who are unresponsive to treatment early and adjust treatment plans in a timely manner, leading some patients to miss the optimal intervention window.

[0004] Given this situation, current domestic and international clinical guidelines clearly state that there is still a lack of specific biomarkers that can be used for early diagnosis, dynamic assessment of disease progression, guidance of treatment plans, and accurate prediction of prognosis in IgA nephropathy.

[0005] Biomarkers, as detectable indicators that can objectively reflect pathological processes and assess treatment response, play an irreplaceable role in the early diagnosis, prognosis, and personalized treatment of kidney diseases. Therefore, the development of non-invasive biomarkers has always been a research hotspot in the field of IgA nephropathy. Ideal IgA nephropathy biomarkers should possess characteristics such as non-invasiveness, high sensitivity, and high specificity, compensating for the limitations of renal biopsy and enabling early diagnosis, assessment of disease severity, prediction of treatment response, and prognosis. In recent years, galactose deficiency IgA1 (Gd-IgA1) has been recognized as a core biomarker in the field. However, its clinical translation and research application still face many bottlenecks. From a detection perspective, there is a lack of standardized, universal detection reagents. Different research teams often use self-developed reagents or modified methods, making it difficult to ensure consistency in the detection system. Furthermore, existing detection methods have poor sensitivity and specificity, and the results show significant inter-center variability. Existing data shows that the diagnostic efficacy of Gd-IgA1 for IgAN varies considerably among different experimental centers, with the area under the receiver operating characteristic (AUC) as low as 0.4, far below the ideal diagnostic threshold. From a clinical application perspective, the association between Gd-IgA1 and disease prognosis remains inconsistent in existing studies. Moreover, changes in the expression level of this indicator after treatment intervention show only a weak correlation with the degree of improvement in proteinuria, making it difficult to use as a reliable basis for assessing treatment response. In addition, while other candidate biomarkers such as urinary CD163 and urinary mononuclear cell chemotactic protein-1 (MCP-1) show elevated expression during the active phase of IgAN, this upregulation is not specific to IgAN and similar changes can occur in the active phases of various kidney diseases, lacking sufficient disease specificity. This further limits the promotion and application of IgAN biomarkers in clinical management. Other potential biomarkers, such as IgA-IgG complexes, anti-GdIgA1 antiglycemic antibodies, secretory IgA, and dimeric IgA, have been reported, but most suffer from insufficient specificity, limited sensitivity, or inability to simultaneously meet the needs of diagnostic and therapeutic monitoring, and therefore have not yet been widely applied in clinical practice.

[0006] Therefore, there is a need for an ideal biomarker that can accurately assist in the early diagnosis of IgA nephropathy, dynamically monitor treatment response, and guide individualized treatment to solve the above-mentioned technical problems. Summary of the Invention

[0007] This invention discloses the following technical solution: the application of urinary IgA-C3 as a biomarker for the diagnosis and treatment efficacy prediction of IgA nephropathy.

[0008] The present invention also discloses an enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, wherein the ELISA kit is an ELISA kit for the detection of urine IgA-C3.

[0009] Preferably, the enzyme-linked immunosorbent assay kit includes: anti-human C3 antibody and HRP-labeled IgA antibody.

[0010] Preferably, the enzyme-linked immunosorbent assay kit includes: IgA standard, washing solution, chromogenic substrate and stop solution.

[0011] This invention also discloses a method for detecting IgA nephropathy. This method uses the aforementioned enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy and includes the following steps: Step 1: Take an ELISA plate coated with functional protein anti-human C3 antibody, add the supernatant of the urine sample to be tested, and incubate. Step 2: After completing the incubation in Step 1, wash the plate with washing buffer and then add HRP-labeled IgA antibody for incubation. Step 3: After completing the incubation in step 2, perform color development, termination, and value reading; Step 4: Perform steps 1 to 3 using IgA standards of different concentrations to create a standard curve; Step 5: Based on the standard curve prepared in Step 4, calculate the level of IgA-C3 complex in the urine sample that can bind to the functional protein anti-human C3 antibody. Step 6: Compare the levels of IgA-C3 complexes that can bind to the anti-human C3 antibody of the functional protein in the urine of the test patient and the disease control. If the IgA-C3 / UTP level in the urine of the test patient is greater than the 80th percentile of the IgA-C3 / UTP level in the urine of the disease control (sensitivity 64.3%, specificity 80%), then the test patient has IgA nephropathy.

[0012] The beneficial effects of this invention are: 1. This invention, based on the glycosylation mass spectrometry analysis of the cyclic polyIgA complex, reveals that the polyIgA complex contains components such as complement C3, providing a theoretical basis for the IgA-C3 complex as a diagnostic tool for IgA nephropathy.

[0013] 2. This invention establishes a reliable and stable ELISA method for the urine IgA-C3 complex.

[0014] 3. This invention improves the diagnostic efficacy of IgA nephropathy. The urinary IgA-C3 / UTP level of this invention has better diagnostic efficacy in differentiating IgA nephropathy from other kidney diseases.

[0015] 4. This invention can guide the treatment of IgA nephropathy. A decrease in urinary IgA-C3 / UTP indicates that the clinical treatment is effective. Therefore, this invention can guide the treatment of IgA nephropathy.

[0016] 5. The urinary IgA-C3 / UTP of this invention not only has the efficacy of disease differential diagnosis, but also has the potential value of disease assessment and prognosis prediction. It is expected to become a core biomarker that connects the entire process of "diagnosis-monitoring-prognosis" of IgA nephropathy, and provide a new breakthrough for solving the current clinical problem of lacking accurate non-invasive diagnostic tools. Attached Figure Description

[0017] Figure 1This is a comparison diagram of O-glycosylation mass spectrometry analysis of purified polyIgA according to the present invention; Figure 2 This is a comparison chart showing the correlation between urine samples from IgA nephropathy patients and IgA-C3 and urinary protein in other kidney diseases according to the present invention; Figure 3 This is a comparison diagram of urinary IgA-C3 / UTP and urinary IgA-C3 / UPCR levels in patients with IgA nephropathy and controls with other kidney diseases according to the present invention. Figure 4 This is a plot of the area under the ROC curve for IgA nephropathy and other disease controls in this invention. Figure 5 This is a graph showing the correlation between urinary IgA-C3 / UTP levels and the severity of IgA nephropathy. Figure 6 This is a graph showing the urinary IgA-C3 / UTP level detection in IgA nephropathy patients according to the present invention.

[0018] In the figure, IgAN represents IgA nephropathy, IgAMM represents IgA myeloma, HC represents healthy controls, and DC represents controls for other kidney diseases. Detailed Implementation

[0019] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] like Figures 1-6 As shown in this embodiment, the core pathophysiological mechanism of IgA nephropathy lies in the abnormal deposition of pathogenic IgA1 (especially galactose-deficient IgA1) in the glomerular mesangial area, thereby activating the complement replacement pathway and triggering local inflammatory responses and kidney damage. This embodiment, through glycosylation mass spectrometry analysis of circulating polyIgA complexes from IgA nephropathy patients and healthy individuals, suggests that the polyIgA1 complexes from IgA nephropathy patients contain a higher proportion of complement C3, significantly higher than in healthy individuals, indicating that the IgA-C3 complex may be a potential characteristic biomarker. Compared to circulating IgA-related biomarkers, urinary IgA-C3 complexes, directly derived from the shedding of immune complexes or local synthesis at the site of glomerular injury, more directly reflect the local pathological activity of the kidney. Its detection has significant advantages of being non-invasive and allowing for repeated sampling, meeting the clinical needs for early diagnosis and dynamic monitoring of IgA nephropathy.

[0021] This embodiment, through mass spectrometry analysis of purified polyIgA molecules' O-glycans, determined that the circulating polyIgA1 complex derived from IgA nephropathy patients contained a high proportion of complement C3, significantly higher than that in healthy individuals. Figure 1 As shown.

[0022] This implementation method establishes an in vitro ELISA method for detecting IgA-C3 complexes in urine. Currently, using this established in vitro ELISA method, 70 patients diagnosed with IgA nephropathy by renal biopsy and 35 controls with other kidney diseases (including membranous nephropathy, minimal change disease, lupus nephritis, focal segmental glomerulosclerosis, ANCA-associated vasculitis, and diabetic nephropathy) were tested. The results showed that the level of IgA-C3 complexes in urine was positively correlated with urinary protein in IgA nephropathy patients, while no significant correlation was found between the level of IgA-C3 complexes and urinary protein in the disease control group. Figure 2 As shown, urinary IgA-C3 and urinary protein are significantly positively correlated in patients with IgA nephropathy. Figure 2 A); In other kidney disease control groups, urinary IgA-C3 was not significantly correlated with urinary protein ( Figure 2 B).

[0023] After adjusting for urinary protein, urinary IgA-C3 complex levels were significantly higher in patients with IgA nephropathy compared to controls with other diseases, with increases in both urinary IgA-C3 / UTP and urinary IgA-C3 / UPCR. Figure 3 As shown, the urinary IgA-C3 level in the urine of patients with IgA nephropathy and controls with other kidney diseases was detected by ELISA. The urinary IgA-C3 / UTP and urinary IgA-C3 / UPCR levels in patients with IgA nephropathy were significantly higher than those in controls with other kidney diseases.

[0024] Regarding differential diagnostic efficacy, compared with other nephropathy controls, the areas under the ROC curve for urinary IgA-C3 / UTP and urinary IgA-C3 / UPCR were: urinary IgA-C3 / UTP: AUC = 0.787, 95% CI: 0.698-0.875; urinary IgA-C3 / UPCR: AUC = 0.808, 95% CI: 0.725-0.892. Figure 4 As shown, the area under the ROC curve analysis of the control group that distinguishes between IgA nephropathy and other diseases suggests that urinary IgA-C3 / UTP and urinary IgA-C3 / UPCR have good differential diagnostic efficacy.

[0025] This study further analyzed the correlation between urinary IgA-C3 / UTP and clinical indicators in patients with IgA nephropathy. The results showed that patients in the acute phase of gross hematuria following infection had significantly higher urinary IgA-C3 / UTP levels than those without gross hematuria. Patients were further divided into two groups based on their urinary IgA-C3 / UTP levels; the high-level group showed an increasing trend in 24-hour urinary protein quantification. Simultaneously, urinary IgA-C3 / UTP levels were positively correlated with serum creatinine levels (r=0.39, p<0.001) and negatively correlated with eGFR (r=-0.33, p=0.006). These results suggest a significant correlation between urinary IgA-C3 / UTP levels and the severity of IgA nephropathy. Figure 5 As shown, patients with gross hematuria following infection had significantly higher urinary IgA-C3 / UTP levels than patients without gross hematuria. Figure 5 A); Based on urinary IgA-C3 / UTP levels, patients were divided into two groups. The high urinary IgA-C3 / UTP level group showed an increasing trend in 24-hour urinary protein quantification. Figure 5 B); Urinary IgA-C3 / UTP levels are positively correlated with serum creatinine levels (B); Figure 5 C); urinary IgA-C3 / UTP levels are negatively correlated with eGFR ( Figure 5 D).

[0026] Therefore, urinary IgA-C3 / UTP levels are correlated with treatment response. A study of urinary IgA-C3 / UTP levels in serum samples from 19 patients with IgA nephropathy treated with budesonide enteric-coated capsules showed that the ratio decreased with decreasing proteinuria after treatment, suggesting a predictive role in treatment response. Figure 6 As shown, the longitudinal cohort of IgA nephropathy patients indicates that urinary IgA-C3 / UTP levels decrease as IgA levels improve with treatment. This suggests that urinary IgA-C3 / UTP not only possesses diagnostic efficacy for disease differentiation but also has potential value in disease assessment and prognosis prediction. It is expected to become a core biomarker connecting the entire process of "diagnosis-monitoring-prognosis" in IgA nephropathy, providing a new breakthrough in addressing the current clinical predicament of lacking accurate, non-invasive diagnostic tools.

[0027] In summary, this invention successfully applies urinary IgA-C3 / UTP to products used in the auxiliary diagnosis of IgA nephropathy, bringing a new breakthrough to the diagnosis and treatment of IgA nephropathy. The established reliable and stable ELISA method for detecting the urinary IgA-C3 complex can effectively detect the level of this complex in patient urine, providing a powerful tool for the early diagnosis of the disease. Through analysis of a large number of patients and control groups, the advantages of urinary IgA-C3 / UTP in the differential diagnosis of IgA nephropathy and other kidney diseases have been clarified, and its area under the ROC curve shows good diagnostic efficacy.

[0028] Moreover, the urinary IgA-C3 / UTP ratio not only performs exceptionally well in diagnosis but also holds significant guiding value for the treatment of IgA nephropathy. Changes in urinary IgA-C3 / UTP levels can indicate the effectiveness of clinical treatment, allowing physicians to adjust treatment plans promptly and prevent patients from missing the optimal intervention window. Simultaneously, urinary IgA-C3 / UTP levels are closely correlated with the severity of IgA nephropathy, directly reflecting the local pathological activity of the kidneys. Furthermore, it holds potential value in disease assessment and prognosis prediction, and is expected to connect the entire process of "diagnosis-monitoring-prognosis" for IgA nephropathy.

[0029] In future clinical applications, the findings of this invention are expected to be widely adopted, becoming an important auxiliary means for the diagnosis and treatment of IgA nephropathy, providing patients with more precise and efficient medical services, and reducing the burden on patients' families and the social healthcare system. At the same time, it also provides new directions and ideas for subsequent related research, promoting the further development of the field of IgA nephropathy.

[0030] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. Application of urinary IgA-C3 as a biomarker for the diagnosis and predictive effect of IgA nephropathy.

2. An enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, characterized in that, The enzyme-linked immunosorbent assay kit is an ELISA kit for detecting urine IgA-C3.

3. The enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy according to claim 2, characterized in that, The enzyme-linked immunosorbent assay kit includes: anti-human C3 antibody and HRP-labeled IgA antibody.

4. The enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy according to claim 2, characterized in that, The enzyme-linked immunosorbent assay kit includes: IgA standard, washing solution, chromogenic substrate and stop solution.

5. A method for detecting IgA nephropathy, characterized in that, The detection method uses the enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy as described in any one of claims 2 to 4, and the detection method includes the following steps: Step 1: Take an ELISA plate coated with functional protein anti-human C3 antibody, add the supernatant of the urine sample to be tested, and incubate. Step 2: After completing the incubation in Step 1, wash the plate with washing buffer and then add HRP-labeled IgA antibody for incubation. Step 3: After completing the incubation in step 2, perform color development, termination, and value reading; Step 4: Perform steps 1 to 3 using IgA standards of different concentrations to create a standard curve; Step 5: Based on the standard curve prepared in Step 4, calculate the level of IgA-C3 complex in the urine sample that can bind to the functional protein anti-human C3 antibody. Step 6: Compare the levels of IgA-C3 complexes that can bind to the functional protein anti-human C3 antibody in the urine of the test patient and the disease control. If the IgA-C3 / UTP level in the urine of the test patient is greater than the 80th percentile of the IgA-C3 / UTP level in the urine of the disease control, then the test patient has IgA nephropathy.

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