CN110045125B - Biomarker for diagnosing retroperitoneal fibrosis and application thereof - Google Patents

Abstract

The invention discloses application of Roman snail lectin in preparing an agent for diagnosing retroperitoneal fibrosis. The invention detects the glycan spectrum of serum IgG4 molecular surface of IgG4-RD patient with agglutinin specificity by using agglutinin microarray, and the result shows that the content of HPA agglutinin binding glycan is reduced in IgG4-RD patient. Since HPA lectin is specifically binding to N-acetylgalactosamine, this indicates that the expression of N-acetylgalactosamine glycosylation level is reduced in IgG4-RD patients. Three subgroups were further studied for IgG4-RD glycosylation expression, and the results showed that HPA lectin-binding glycan levels were the lowest expressed in retroperitoneal fibrotic patients. Reduced levels of HPA lectin-binding glycans can be used as a biological marker for diagnosing retroperitoneal fibrosis.

Description

A biomarker for diagnosing retroperitoneal fibrosis and use thereof

technical field

The invention belongs to the field of biological detection, and in particular relates to a biomarker for diagnosing retroperitoneal fibrosis and use thereof.

Background technique

IgG4-related disease (IgG4RD) is a new immune-mediated autoinflammatory disease recognized in recent years. The disease is characterized by hyperplasia and enlargement of affected organs or tissues, significantly increased serum IgG4 level (>1350 mg/L), and infiltration of IgG4-positive lymphocytes (IgG4-positive plasma cells account for more than 50% of total plasma cells) in the affected tissues. The disease can involve lacrimal glands, salivary glands, pancreas, retroperitoneal tissue, bile ducts, lungs, kidneys, prostate and other organs or tissues. The clinical manifestations include Mikulitz disease, autoimmune pancreatitis, retroperitoneal fibrosis, Cholangitis, interstitial pneumonia, periorbital inflammatory pseudotumor, etc. In 2010, the journal Autoimmunity Reviews, entitled "A New Syndrome Is Born: The Clinical Spectrum of IgG4-Associated Diseases", announced the recognition of this new disease. In 2012, the comprehensive diagnostic criteria of the disease were published for the first time in the world, making its diagnosis standardized. Pathological features of IgG4-RD are lymphocytic infiltration, striate fibrosis, and obliterative phlebitis. Early diagnosis and early treatment of IgG4-RD can prevent severe organ damage, tissue fibrosis, and even death.

The glycoprotein concentration in human serum is approximately 40 g/L and is an excellent source for finding biomarkers of human disease. Unlike RNA and proteins, the synthesis of glycans attached to glycoproteins does not require a template. Glycosylation is a process influenced by a variety of factors, including: cell type and its activation state; environmental factors, such as the presence of available metabolites; the age of cells, as some glycans may be lost over time; inflammation mediators such as cytokines and chemokines. All of these factors may be altered in an autoimmune environment. For example, some autoimmune diseases have characteristic cytokines. These cytokines affect the expression of glycosidases, sialidases and glycosyltransferases, which directly affect glycan synthesis. Theoretically, the characteristic immune state can be manifested in the glycosylation of serum glycoproteins.

In view of the important role of glycosylation in diseases, the high-throughput glycosylation analysis technology-lectin microarray was used to screen the expression of serum IgG4 glycosylation in IgG4-RD patients, in order to explore the role of glycosylation in IgG4- Clinical application value in RD.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a biomarker for diagnosing retroperitoneal fibrosis and use thereof.

First, the present invention provides a biomarker for diagnosing retroperitoneal fibrosis, which is a complex formed by the combination of Roman snail lectin and IgG4.

Wherein, the IgG4 contains N-acetylgalactosamine.

Secondly, the present invention also provides the use of the biomarker in the preparation of a reagent for diagnosing retroperitoneal fibrosis.

Specifically, the diagnosis comprises: determining the level of a complex formed by the binding of Roman snail agglutinin to IgG4 in a biological sample obtained from a patient presenting with an IgG4-related disease; optionally,

The level of the complex formed by the binding of Roman snail lectin and IgG4 in the biological sample is compared with control data, wherein, relative to the control data, the level of the complex formed by the binding of Roman snail lectin and IgG4 in the sample is compared. A detectably lower level indicates the possibility of retroperitoneal fibrosis.

Wherein, the biological sample is a serum sample.

Preferably, the level of the complex formed by the binding of Roman snail lectin to IgG4 is measured by the following steps, including:

a. contacting a biological sample from a patient with Roman snail lectin;

b. Formation of a lectin-glycan complex between IgG4 present in the biological sample and Roman snail lectin;

c. Washing to remove any unbound IgG4;

d. adding a detection antibody that is labeled and reactive with the antibody from the biological sample;

e. washing to remove any unbound labeled detection antibody; and

f. Converting the label of the detection antibody to a detectable signal. Secondly, the present invention also provides the use of Roman snail agglutinin in the preparation of a reagent for diagnosing retroperitoneal fibrosis.

Wherein, the Roman snail lectin is deposited or immobilized on a solid-phase surface carrier.

Wherein, the solid phase surface carrier is preferably in the form of latex beads, porous flat plates or membrane strips, nanotubes, sheets with two-dimensional codes, and the like.

Wherein, the detection antibody is labeled by being covalently linked to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.

In another aspect, the present invention also provides the use of Roman snail lectin in the preparation of a reagent for diagnosing retroperitoneal fibrosis.

Wherein, the diagnosing comprises: contacting the Roman snail lectin with a biological sample obtained from a patient exhibiting an IgG4-related disease, and measuring the level of the complex formed by the binding of the roman snail lectin to IgG4; optionally,

The level of the complex formed by the binding of Roman snail lectin and IgG4 in the biological sample is compared with control data, wherein, relative to the control data, the level of the complex formed by the binding of Roman snail lectin and IgG4 in the sample is compared. A detectably lower level indicates the possibility of retroperitoneal fibrosis.

Wherein, the biological sample is a serum sample.

Preferably, the level of the complex formed by the binding of Roman snail lectin to IgG4 is measured by the following steps, including:

a. contacting a biological sample from a patient with Roman snail lectin;

b. Formation of a lectin-glycan complex between IgG4 present in the biological sample and Roman snail lectin;

c. Washing to remove any unbound IgG4;

d. adding a detection antibody that is labeled and reactive with the antibody from the biological sample;

e. washing to remove any unbound labeled detection antibody; and

f. Converting the label of the detection antibody to a detectable signal.

Secondly, the present invention also provides the use of Roman snail agglutinin in the preparation of a reagent for diagnosing retroperitoneal fibrosis.

Wherein, the Roman snail lectin is deposited or immobilized on a solid-phase surface carrier.

Wherein, the solid phase surface carrier is preferably in the form of latex beads, porous flat plates or membrane strips, nanotubes, sheets with two-dimensional codes, and the like.

Wherein, the detection antibody is labeled by being covalently linked to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.

In another aspect, the present invention also provides a diagnostic kit for detecting and/or quantifying IgG4 that can bind to Roman snail lectin in a biological sample, comprising: a solid-phase surface carrier, wherein the Roman snail The lectin was deposited or immobilized on the solid surface carrier, and the complex formed by the binding of Roman snail lectin and IgG4 was used as a biomarker of retroperitoneal fibrosis.

In a preferred embodiment of the invention, the kit further comprises a detection antibody that is labeled and reactive with the antibody from the biological sample.

Preferably, the solid surface carrier is in the form of latex beads, porous plates or membrane strips, nanotubes, sheets with two-dimensional codes, and the like.

In this study, lectin microarray was used to detect the glycan profile of serum IgG4 molecules in IgG4-RD patients specifically bound to lectins. The results showed that the content of HPA lectin-bound glycans was reduced in IgG4-RD patients. Since HPA lectin specifically binds N-acetylgalactosamine, this suggests that the level of N-acetylgalactosamine glycosylation is reduced in IgG4-RD patients. Further investigation of IgG4-RD glycosylation expression in the three subgroups revealed that HPA lectin-binding glycan levels were lowest in patients with retroperitoneal fibrosis.

The results of the study showed that IgG4-RD patients, especially those with retroperitoneal fibrosis, had reduced levels of HPA lectin-binding glycans, which could be used as biomarkers for the diagnosis of retroperitoneal fibrosis.

Description of drawings

Figure 1 shows the layout of lectin microarrays of 56 lectins (triple wells) on an array slide.

Figure 2 shows a schematic diagram of the lectin microarray in IgG4-RD patients.

Figure 3 shows the comparison of HPA lectin signal values in IgG4-RD group, DC group and HC group (**: P<0.01).

Figure 4 shows the comparison of HPA lectin signal values and ROC curves in patients with Mikulitz disease and retroperitoneal fibrosis.

Figure 5 shows the comparison of HPA lectin signal values and ROC curves in patients with autoimmune pancreatitis and retroperitoneal fibrosis.

Figure 6 shows the correlation of IgG4 concentration before and after purification.

Figure 7 shows the correlation of HPA lectin signal values between serum IgG4 and purified IgG4 in lectin microarrays.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Experimental specimens: The three groups of people included in this study included: IgG4-RD group (167 cases of IgG4-RD patients, including 59 cases of Mikulitz disease in group A1, 50 cases of autoimmune pancreatitis in group A2, and retroperitoneal fibrosis in group A3). 58 cases), DC group (130 cases of AID disease control), HC group (86 cases of healthy subjects). The diagnosis of IgG4-RD group and DC group all met the diagnostic criteria of the corresponding diseases. Fresh blood was collected from all the enrolled people, and the serum was immediately separated and frozen at -80°C for future use.

Example 1 Analysis of serum IgG4 glycosylation by lectin microarray

A lectin microarray consisting of 56 lectin microchips. Fifty-six lectins were immobilized into the chip in triplicate, and the total serum of each patient was diluted 1:1000, added to the array, and incubated overnight at 4°C. Then, the anti-IgG4-Cy3 conjugate was hybridized to the microchip for 45 min in the dark. The fluorescence intensities of all proteins and the fluorescence intensities of low signals were analyzed independently. Chip images were converted to digital format for analysis.

The signal-to-noise ratio (S/N) of each lectin spot was calculated using the signal-to-noise ratio of each lectin spot (medium intensity of spot foreground versus background). To prevent inter-array bias of lectin microarrays, we normalized the S/N data using inter-array normalization. Significant differences in lectin-binding activity were determined by the distribution of data within groups according to the following rules: (1) the mean S/N of lectins in the IgG4-RD group should not be less than the maximum S/N in the control group (50% ( IgG4-RD group) ≥ the maximum value (control group); (2) the lower quartile of S/N of the IgG4-RD group should not be less than the upper quartile of the control group (25% (IgG4-RD group) ≥ 75% (control group); (3) the minimum S/N of the IgG4-RD group should not be less than the median value of the control group [minimum (IgG4-RD group) ≥ 50% (control group)].

A lectin microarray containing 56 lectins was used to detect the glycosylation status in the experimental specimens (Figure 1). Lectins can specifically bind to glycan molecules at the end of glycoproteins to form complexes, and the type and content of glycans on the surface of target proteins can be studied through the specific binding of different lectins to glycans. Due to its high efficiency, lectin microarrays have been more and more widely used in the study of glycosylation. After the frozen specimens are equilibrated at room temperature, they are added to the lectin microarray and reacted with it. After washing, blocking, fluorescent secondary antibody reaction and fluorescence detection, the signal value of each lectin specifically bound to the glycan can be obtained. , the signal value correlates with binding affinity and binding strength (Figure 2).

To ensure that the collected fluorescent signal originates from the specific binding of IgG4, a Cy3-labeled IgG4 antibody was used. Lectin S/N data that met any of the three aforementioned rules were considered significantly different, with a total of 6 lectins (Table 1).

Table 1 Lectins with significant differences in lectin microarrays

The affinity signal values of the six lectins showed significant differences among the three groups of samples. Through lectin-glycan binding signal analysis, we found that the Roman snail agglutinin (HPA) signal value was generally decreased in the IgG4-RD group compared with the DC group and the HC group (Fig. 3). Further analysis of the IgG4-RD subgroup The distribution of HPA lectin signal values found that the A3 retroperitoneal fibrosis group had the lowest value among the three subgroups (data and ROC diagnostic curves are shown in Figures 4-5). Given that HPA specifically binds N-acetylgalactosamine (GalNAc) glycans, it is inferred that patients with IgG4-RD, especially those with retroperitoneal fibrosis, have higher levels of N-acetylgalactosamine glycosylation of IgG4 in serum Compared with other groups, it showed a decreasing trend, which can be used as a biomarker for the diagnosis and differential diagnosis of retroperitoneal fibrosis.

Embodiment 2 Serum IgG4 purification and identification

To further determine whether changes in glycosylation in patients with IgG-RD were due to an increase in serum IgG4 concentration, or actual changes in glycosylation, a second lectin microarray and lectin blot Dotblot were used for validation. The second lectin consists of six lectins, including HPA, DSL, LTL, VVA mannose, MNA-M and ConA. The operation is the same as before.

IgG4 was isolated from serum by immunoprecipitation. The samples included 12 IgG-RD patients, 3 DC patients and 1 HC patient. 20 μl of mouse anti-IgG4 antibody (SouthernBiotech, Birmingham, USA) was coupled to 20 μl of beads (NHS-activated Sepharose ^™ 4 Fast Flow, GE healthcare Life Sciences, Pittsburgh, USA), followed by the addition of 0.1 M Tris-HCl to seal excess sites . The mouse anti-IgG4 antibody beads were washed 3 times with an acidic solution and an alkaline solution. Use 5 μl of serum per column. The column was incubated overnight. After washing 8 times with PBST and 2 times with water, IgG4 was eluted into the vacuum tube with 20 μl of 0.1 M glycine. Protein purity was identified by Dotblot, protein concentration was determined by protein silver staining kit (Beyotiome, Shanghai, China), and all IgG4 samples were stored at -80°C for subsequent processing.

By comparing the purified IgG4 concentration and the relative serum IgG4 content in 16 patients, it was found that the purified IgG4 concentration results had a good correlation with the serum IgG4 level (r=0.593, P=0.015) (Figure 6). The results showed that for HPA lectin, the signal value of serum IgG4 microarray was proportional to the signal value of purified IgG4 microarray (Figure 7). This suggests that the level of glycosylation of the HPA lectin-bound glycan, N-acetylgalactosamine, in the serum of patients with IgG4-RD is abnormal.

Example 3 Correlation analysis of IgG4 glycosylation and laboratory characteristics in 167 IgG-RD patients

Significant differences were observed between lectin signals in IgG-RD patients, and we further evaluated the relationship between lectin signals and clinical laboratory indicators. The results of correlation analysis showed that the content of HPA lectin-binding glycans in IgG4-RD patients did not correlate with laboratory indicators (Table 2).

Table 2 Correlation of lectin-binding glycan content and laboratory characteristics in 167 IgG4-RD patients

*NS: No significant difference

Example 4 The relationship between IgG4 glycosylation and organ involvement in 167 IgG-RD patients

Different levels of lectins specifically bound to glycans were compared in patients presenting with various organ involvements. The results are shown in Table 3. HPA lectin-binding glycan levels were associated with multiple pancreas, bile duct involvement, and retroperitoneal fibrosis in patients with IgG4-RD (Table 3). This suggests that reductions in HPA lectin-binding glycans in IgG4-RD patients are associated with pancreatic, bile duct involvement, and retroperitoneal fibrosis. Involvement of these organs is a common clinical manifestation in patients with retroperitoneal fibrosis, and our results also show that HPA is different and statistically significant in patients with and without involvement of these organs, which can be used as this indicator to diagnose peritoneal fibrosis. Evidence for post-fibrosis.

Table 3 Comparison of organ involvement and IgG4 glycosylation in patients with IgG4-RD

NS* was not significantly different.

Claims (9)

1. Use of a biomarker which is a complex formed by binding of roman snail lectin to IgG4 in the manufacture of a reagent for the diagnosis of retroperitoneal fibrosis.

2. The use of claim 1, wherein said diagnosing comprises: determining the level of a complex formed by binding of roman snail lectin to IgG4 in a biological sample obtained from a patient presenting an IgG 4-related disease; comparing the level of complex formed by binding of Roman snail lectin to IgG4 in the biological sample to control data, wherein the level of complex formed by binding of Roman snail lectin to IgG4 in the sample is detectably reduced relative to the control data, indicating a likelihood of developing retroperitoneal fibrosis.

3. Use of Roman snail lectin in the manufacture of an agent for the diagnosis of retroperitoneal fibrosis.

4. The use of claim 3, wherein said diagnosing comprises: contacting roman snail lectin with a biological sample obtained from a patient who exhibits IgG 4-related disease, and determining the level of complexes formed by binding of roman snail lectin to IgG 4; comparing the level of complex formed by binding of Roman snail lectin to IgG4 in the biological sample to control data, wherein the level of complex formed by binding of Roman snail lectin to IgG4 in the sample is detectably reduced relative to the control data, indicating a likelihood of developing retroperitoneal fibrosis.

5. The use of claim 2 or 4, wherein the biological sample is a serum sample.

6. Use as claimed in claim 2 or 4, wherein the level of complexes formed by binding of Roman snail lectin to IgG4 is measured by steps comprising: a. contacting a biological sample from a patient with roman snail lectin; b. forming lectin-glycan complexes between IgG4 present in the biological sample and the roman snail lectin; c. washing to remove any unbound IgG 4; d. adding a detection antibody that is labeled and reactive with an antibody from the biological sample; e. washing to remove any unbound labeled detection antibody; converting the label of the detection antibody to a detectable signal.

7. Use as claimed in claim 6, wherein the Roman snail lectin is deposited or immobilised on a solid surface support.

8. The use according to claim 7, wherein the solid surface support is in the form of latex beads, porous plates or membrane strips, nanotubes, flakes with two-dimensional codes.

9. The use of claim 6, wherein the detection antibody is covalently linked to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.

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