CN112924672A - Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof - Google Patents
Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof Download PDFInfo
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Abstract
The invention discloses an application of LCA agglutinin in preparing a reagent for diagnosing rheumatoid arthritis and pulmonary interstitial fibrosis. The invention detects the glycan profile of the serum IgG molecule of a Rheumatoid Arthritis (RA) patient specifically combined with the agglutinin by adopting an agglutinin microarray, and the result shows that the content of LCA agglutinin combined glycan is increased in the RA patient. Since LCA lectin is specifically binding to glucose, this indicates that the expression of glucose levels is elevated in RA patients. Further studies found that LCA lectin-binding glycan levels were expressed higher in patients with RA-associated interstitial lung disease (RA-ILD) than in patients with non-ILD, and LCA lectin-binding glycan levels could serve as a biomarker for RA-ILD.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof.
Background
Rheumatoid Arthritis (RA) is a common rheumatic immune disease, a chronic systemic immune disease with joint synovial lesions as the main characteristic, and the main clinical characteristics are chronic symmetrical joint swelling and pain, synovitis, joint tenderness with morning stiffness, joint function loss and extraarticular organ involvement. Research shows that about 500 million RA patients exist at present in China, and more than 80 percent of the patients who do not reach clinical remission bring heavy burden to patients, families and society. The disease is a typical '5D' disease which causes disability, death and pain, has great side effects on treatment medicines and causes huge economic loss, and has attracted high attention of clinical workers. Currently, clinical biomarkers for diagnosing RA and even RA-related pulmonary interstitial disease (RA-ILD) have certain limitations, and sensitivity and specificity caused by different methodologies are different, so that new biomarkers are urgently needed for early diagnosis and early treatment of RA-ILD.
Immunoglobulin-linked glycans affect the effector functions of antibodies, depending on the branching structure of the N-glycans and/or terminal sugars of the N-glycans or O-glycans, including galactose and sialic acid. IgG is the highest serum content of Ig and is structurally classified into a receptor-binding Fc fragment and a Fab arm that binds to an antigen. Research shows that autoimmune diseases have unique glycosylation expression profiles and play an important role in the occurrence and development of the autoimmune diseases. For example, abnormal levels of glycosylation of the Fc portion of serum IgG can result in changes in immune effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, or complement-dependent cytotoxicity of the body. In addition, the abnormal level of glycosylation of the Fab fragment of IgG affects antibody half-life in vivo, alters antibody stability and aggregation, leads to survival of malignant B cells through glycan signaling pathways, and is associated with intravenous immunoglobulin anti-inflammatory activity. Therefore, the research of glycosylation becomes a potential tool for deeply understanding the pathogenesis, disease classification, disease evaluation, treatment effect, prognosis judgment and disease outcome of autoimmune diseases.
In view of the important role of glycosylation in diseases, we intend to screen the expression of serum IgG glycosylation of RA-ILD patients by a high-throughput glycosylation analysis technology, namely a lectin microarray technology, so as to investigate the clinical application value of glycosylation in RA-ILD.
Disclosure of Invention
In order to solve the problems, the invention provides a biomarker for diagnosing rheumatoid arthritis and pulmonary interstitial fibrosis and application thereof.
First, the present invention provides a biomarker for diagnosing rheumatoid arthritis with pulmonary interstitial fibrosis, which is a complex formed by binding LCA lectin to IgG.
Wherein said IgG comprises glucose.
Secondly, the invention also provides the application of the biomarker in preparing a reagent for diagnosing rheumatoid arthritis.
Specifically, the diagnosis includes: determining the level of a complex formed by binding of LCA lectin to IgG in a biological sample obtained from a patient exhibiting rheumatoid arthritis; optionally, the step of (a) is carried out,
comparing the level of the complex formed by binding of LCA lectin to IgG in the biological sample to control data, wherein a detectably increased level of the complex formed by binding of LCA lectin to IgG in the sample relative to the control data is indicative of a likelihood of rheumatoid arthritis.
Further, detectably elevated levels of complexes formed by binding of LCA lectin to IgG are indicative of a likelihood of rheumatoid joint-associated pulmonary interstitial fibrosis relative to data from rheumatoid arthritis patients without associated pulmonary interstitial fibrosis.
Wherein the biological sample is a serum sample.
The invention also provides the use of LCA lectin in the manufacture of a reagent for the diagnosis of rheumatoid arthritis.
Wherein the diagnosing comprises: contacting LCA lectin with a biological sample obtained from a patient exhibiting rheumatoid arthritis and determining the level of complexes formed by binding of LCA lectin to IgG; optionally, the step of (a) is carried out,
comparing the level of the complex formed by binding of LCA lectin to IgG in the biological sample to control data, wherein a detectably increased level of the complex formed by binding of LCA lectin to IgG in the sample relative to the control data is indicative of a likelihood of rheumatoid arthritis.
Further, detectably elevated levels of complexes formed by binding of LCA lectin to IgG are indicative of a likelihood of rheumatoid joint-associated pulmonary interstitial fibrosis relative to data from rheumatoid arthritis patients without associated pulmonary interstitial fibrosis.
Wherein the biological sample is a serum sample.
In a particular embodiment of the invention, the level of the complex formed by the binding of LCA lectin to IgG is measured by the steps comprising:
a. contacting a biological sample from a patient with LCA lectin;
b. forming a lectin-glycan complex between IgG and LCA lectin present in the biological sample;
c. washing to remove any unbound IgG;
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; and
f. converting the label of the detection antibody to a detectable signal.
Wherein said LCA lectin is deposited or immobilized on a solid surface support.
The solid phase surface carrier is preferably in the form of latex beads, porous plates or membrane strips, nano-tubes, flakes with two-dimensional codes and the like.
Wherein the detection antibody is labeled by covalent attachment 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 IgG capable of binding to LCA lectin in a biological sample, comprising: a solid surface support, wherein said LCA lectin is deposited on or immobilized on the solid surface support, wherein a complex formed by the binding of the LCA lectin to IgG serves as a biomarker for rheumatoid arthritis and interstitial pulmonary fibrosis.
In a preferred embodiment of the invention, the kit further comprises a detection antibody which is labeled and reactive with an antibody from the biological sample.
Preferably, the solid surface support is in the form of latex beads, porous plates or membrane strips, nanotubes, flakes with two-dimensional codes, or the like.
The invention detects the glycan profile of the serum IgG molecule of a Rheumatoid Arthritis (RA) patient specifically combined with the agglutinin by adopting an agglutinin microarray, and the result shows that the content of LCA agglutinin combined glycan is increased in the RA patient. Since LCA lectin is specifically binding to glucose, this indicates that the expression of glucose levels is elevated in RA patients. Further studies found that LCA lectin-binding glycan levels were expressed higher in patients with RA-associated interstitial lung disease (RA-ILD) than in patients with non-ILD, and LCA lectin-binding glycan levels could serve as a biomarker for RA-ILD.
Drawings
FIG. 1 shows the layout of a lectin microarray of 56 lectins (three wells) on an array slide.
FIG. 2 shows comparison of LCA lectin signal values for the RA-ILD group, RA-non ILD group, DC group and HC group.
FIG. 3 shows the ConA lectin stamp verification chart of the same lot of the lectin microarray.
FIG. 4 shows comparison of LCA lectin blot results of the same lot of the lectin microarray.
Fig. 5 shows the validation of LCA lectin stamp for the new batch of specimens.
Fig. 6 comparison of LCA lectin blot results grey scale values for new batch samples.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Experimental specimen: subjects included in this study were divided into three groups: RA group (35, of which 15 RA-ILDs), disease control group (DC, including 40 autoimmune diseases, of which 20 systemic lupus erythematosus, 20 idiopathic inflammatory myopathy), healthy control group (HC, including 43 healthy examiners). The subject data are shown in Table 1. Wherein the diagnosis of both the RA group and the DC group meets the diagnosis criteria of the corresponding disease. All the people who entered the group collected fresh blood, and immediately separated serum, and frozen at-80 ℃ for later use.
TABLE 1 lectin microarray assay basic data for 118 subjects
Example 1 lectin microarray analysis of serum IgG glycosylation
A lectin microarray consisting of 56 kinds of lectin microparticles. 56 lectins were fixed in triplicate on a chip, and serum from each patient was diluted 1:200, added to the microarray and incubated overnight at 4 ℃. Then, the anti-IgG-Cy 5 conjugate was hybridized to the microarray chip for 1 hour in a dark environment. The fluorescence intensity of all proteins was analyzed independently. And converts the chip image to a digital format for analysis.
The signal-to-noise ratio (S/N) for each lectin spot was calculated using the signal-to-noise ratio (difference between foreground and background values for the spot) for each lectin spot. To prevent bias of lectin microarrays between arrays, we normalized the S/N data using normalization between arrays. Significant differences in lectin binding were determined by data distribution among groups according to the following rules (1) comparison between groups >1.7 or < 0.588; (2) the test types are compared between groups, if normality is met, T test is selected, otherwise non-parametric test is selected, if P value <0.05 or fisher test P value <0.05 or [ ROC _ AUC > -0.6 and ROC _ P value <0.05) ].
A lectin microarray containing 56 lectins was used to detect the glycosylation state in the experimental samples (FIG. 1). The lectin can be specifically combined with glycan molecules at the tail end of the glycoprotein to form a complex, and the type and content of the glycan on the surface of the target protein can be researched through the specific combination of different lectins and the glycan. Lectin microarrays are now being widely used in glycosylation research due to their high efficiency. After the frozen samples are balanced at room temperature, the samples are added into a lectin microarray to react with the lectin microarray, and then the signal value of each lectin and the specific binding glycan of each lectin can be obtained through the steps of washing, sealing, fluorescent secondary antibody reaction, fluorescence detection and the like, wherein the signal value is related to binding affinity and binding strength.
To ensure that the collected fluorescent signal is derived from a specific binding of IgG, we labeled IgG antibodies with Cy 5. The S/N data for the two lectins satisfying the above two conditions were considered to have significant differences, and 7 lectins were selected in total (Table 2).
Table 2 lectins with significant differences in lectin microarrays
Man: mannose; GlcNAc: n-acetylglucosamine; glc: glucose; GalNAc: n-acetylgalactosamine; gal: galactose; DC: a disease control group; HC: healthy control group. *: p < 0.05; **: p < 0.01.
The affinity signal values for the 6 lectins showed significant differences between the four groups of samples. S/N data show IgG glycosylation changes in the RA-ILD patient group relative to the RA-non ILD, DC and HC groups: (1) increased mannose sugar chains of RA-ILD patients were shown by detecting increased binding to concanavalin A (ConA) (P < 0.05); (2) an increase in N-acetylglucosamine (GlcNAc) in RA-ILD patients was indicated by detecting an increase in binding to Datura agglutinin (DSL) (< 0.05 for all P); (3) an increase in glucose (Glc) in RA-ILD patients was indicated by detecting increased binding to lentil Lectin (LCA) and pea lectin (PSA) (both P < 0.05); (4) an increase in galactose (Gal) in RA-ILD patients (both P <0.05) was indicated by detecting an increase in binding to Moringa oleifera lectin (black elderberry) (MNA-M); (5) by detecting a decrease in binding to soybean lectin (SBA), a decrease in N-acetylgalactosamine (GalNAc) in RA-ILD patients was indicated (P both < 0.05). FIG. 2 shows LCA lectin signal comparison for the RA-ILD, RA-non ILD, DC and HC groups.
Example 2 serolectin imprinting validation experiment
To further clarify the differential expression glycosylation detected by the lectin microarray, lectin imprinting was performed using 12 samples of the same lot (6 RA-ILD and 6 RA-non ILD) and a new lot (12 samples of the same lot (5 RA-ILD and 7 RA-non ILD, for the basic data shown in Table 3) of the lectin microarray. Serum specimen 1: 100, mixing with a loading buffer solution, boiling for 5 minutes at 100 ℃, performing SDS-PAGE electrophoresis in 10% preformed gel, and electrically transferring proteins in the preformed gel to a PVDF membrane. And (3) sealing the PVDF film which is successfully transferred, hybridizing the PVDF film with Cy 3-labeled lectin, and finally detecting a fluorescence signal by a fluorescence imager. The intensity of the fluorescence signal is proportional to the binding force of the lectin-bound glycoprotein glycosyl groups.
By comparing the results of lectin blotting on 12 micro-aligned batches of patient specimens (fig. 3), the binding strength of serum IgG to LCA lectin was increased in RA-non ILD patients compared to RA-non ILD patients (fig. 4). Furthermore, comparison of the results of lectin blotting on 12 new batches of patient specimens (fig. 5) revealed that the binding strength of serum IgG to LCA lectin was also significantly increased in RA-ILD patients (P <0.05 for all) compared to RA-non ILD patients (fig. 6).
The above results indicate that LCA lectin-bound glycan-glucose levels are abnormal in the serum of RA-ILD patients, and RA-ILD patients have higher LCA lectin-bound glycan levels than RA-non ILD patients.
TABLE 3 lectin blot validation of disease data for New batch RA patients
The results indicate that LCA lectin-binding glycan levels are expressed higher in RA-ILD patients than in RA-non ILD patients, indicating that LCA lectin-binding glycan levels can serve as a biological marker for RA-ILD.
Claims (10)
1. A biomarker for diagnosing rheumatoid arthritis with pulmonary interstitial fibrosis, which is a complex formed by binding of LCA lectin and IgG.
2. Use of the biomarker of claim 1 for the preparation of a reagent for the diagnosis of rheumatoid arthritis.
3. The use of claim 2, wherein said diagnosing comprises: determining the level of a complex formed by binding of LCA lectin to IgG in a biological sample obtained from a patient exhibiting rheumatoid arthritis; optionally, the step of (a) is carried out,
comparing the level of the complex formed by binding of LCA lectin to IgG in the biological sample to control data, wherein a detectably increased level of the complex formed by binding of LCA lectin to IgG in the sample relative to the control data is indicative of a likelihood of rheumatoid arthritis.
4. The use of claim 3, wherein a detectably increased level of a complex formed by binding of LCA lectin to IgG is indicative of a likelihood of rheumatoid joint-associated pulmonary interstitial fibrosis, relative to data from a patient with rheumatoid arthritis not associated with pulmonary interstitial fibrosis.
Use of LCA lectin in the manufacture of a reagent for the diagnosis of rheumatoid arthritis.
6. The use of claim 5, wherein said diagnosing comprises: contacting LCA lectin with a biological sample obtained from a patient exhibiting rheumatoid arthritis and determining the level of complexes formed by binding of LCA lectin to IgG; optionally, the step of (a) is carried out,
comparing the level of the complex formed by binding of LCA lectin to IgG in the biological sample to control data, wherein a detectably increased level of the complex formed by binding of LCA lectin to IgG in the sample relative to the control data is indicative of a likelihood of rheumatoid arthritis.
7. The use of claim 6, wherein a detectably increased level of a complex formed by binding of LCA lectin to IgG is indicative of a likelihood of rheumatoid joint-associated pulmonary interstitial fibrosis relative to data from a rheumatoid arthritis patient not associated with pulmonary interstitial fibrosis.
8. The use according to claim 3 or 6, wherein the biological sample is a serum sample.
9. The use of claim 3 or 6, wherein the level of a complex formed by binding of LCA lectin to IgG is measured by the steps comprising:
a. contacting a biological sample from a patient with LCA lectin;
b. forming a lectin-glycan complex between IgG and LCA lectin present in the biological sample;
c. washing to remove any unbound IgG;
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; and
f. converting the label of the detection antibody to a detectable signal.
10. Use according to claim 9, wherein the LCA lectin is deposited or immobilized on a solid surface support, preferably in the form of a latex bead, a multi-well plate or membrane strip, a nanotube, a two-dimensional coded sheet or the like, preferably the detection antibody is labeled by covalent attachment to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.
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