Biomarkers for antiphospholipid syndrome for discriminating between pathological and non-pathological pregnancy events and uses thereof

Abstract

The present application belongs to the field of biological detection, and particularly relates to a biological marker for anti-phospholipid syndrome for identifying pathological pregnancy events and non-pathological pregnancy events and use thereof. In the present study, the differences in glycan specific binding of serum IgG and lectin of anti-phospholipid syndrome patients (simple arterial events, simple venous events, pathological pregnancy events) and healthy controls and disease controls were detected by using a lectin chip containing 56 kinds of lectins. The results showed that the content of Sambucus nigra (SNA-I, black elderberry-I) lectin binding glycan was reduced in anti-phospholipid syndrome patients with pathological pregnancy events. The results of lectin immunoblotting verification showed that the content of Sambucus nigra (SNA-I) lectin binding glycan was still reduced in anti-phospholipid syndrome patients with pathological pregnancy events. It can be seen that the complex formed by Sambucus nigra lectin and IgG can be used as a biological marker for evaluating whether anti-phospholipid syndrome patients have pathological pregnancy events.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a biomarker for antiphospholipid syndrome for identifying pathological pregnancy events and non-pathological pregnancy events and use thereof. BACKGROUND

[0002] Antiphospholipid syndrome (APS) is a systemic autoimmune disease, and the presence of antiphospholipid antibodies (aPL) in serum leads to clinical manifestations such as arterial and venous thrombosis, thrombocytopenia, pathological pregnancy (repeated miscarriage, stillbirth, placental insufficiency, preeclampsia), etc. APS is relatively rare, affecting all age groups, and female patients account for a larger proportion. The disease can be divided into primary APS and secondary APS, and secondary APS is more common in systemic lupus erythematosus or rheumatoid arthritis and other autoimmune diseases. At present, the main basis for diagnosing APS is the presence of three kinds of antiphospholipid antibodies in serum, including anticardiolipin antibody (aCL), anti-β2-glycoprotein I antibody (aβ2GPI antibody) and lupus anticoagulant (LA). However, individuals with aPL positivity do not necessarily have APS, and clinicians need more biomarkers to help identify and diagnose APS, so as to intervene in the disease as early as possible and actively treat. For patients who have been diagnosed with APS, predicting the likelihood of different clinical symptoms in the early stages of disease progression through biomarkers is beneficial to achieve better efficacy, especially the prediction and early monitoring of pathological pregnancy events, which is of great significance to the protection of the mother and the healthy birth of the fetus.

[0003] Immunoglobulin G (IgG) is the most abundant antibody in human serum, and is involved in multiple processes of humoral immune response: antibody-dependent cell-mediated cytotoxicity (ADCC), antigen neutralization, complement activation and complement-dependent cytotoxicity (CDC) and hypersensitivity. The structure and function of IgG are regulated by various post-translational modifications, such as glycosylation, which affects the structural stability, conformation, half-life and effector function of IgG. Existing studies have found that the glycosylation of IgG is related to the progression and severity of various autoimmune diseases, proving that abnormal glycosylation expression has great potential as a diagnostic marker for diseases.

[0004] Traditional glycosylation detection techniques (such as mass spectrometry and liquid chromatography) are time-consuming and labor-intensive, with complex sample preparation processes. In comparison, lectin microarrays are more convenient and rapid, ensuring that proteins maintain their native conformation while maintaining extremely high detection throughput. They are more suitable for comparing different patients or different states within the same patient, meeting the requirements of clinical research and facilitating widespread application. Current diagnostic methods for antiphospholipid syndrome rely on a combination of serological testing for aPL antibodies and corresponding clinical symptoms, including early onset embolism, recurrent thrombosis at abnormal sites, late miscarriage, and HELLP syndrome. This method lacks a clear quantitative relationship and relies heavily on disease symptoms. Diagnosis is often made only after corresponding symptoms are present, making it difficult to predict and monitor early disease progression.

[0005] This study intends to use high-throughput glycosylation analysis technology - lectin microarray technology to detect the glycosylation profile of peripheral IgG in APS patients, compare its differences with healthy controls and disease controls, as well as the differences between APS patients with different clinical manifestations, in order to explore potential biomarkers of antiphospholipid syndrome and its clinical manifestations. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides biomarkers of antiphospholipid syndrome for distinguishing pathological pregnancy events from non-pathological pregnancy events and uses thereof.

[0007] First, the present invention provides a biomarker for antiphospholipid syndrome for distinguishing pathological pregnancy events from non-pathological pregnancy events, which is a complex formed by the binding of black elderberry agglutinin-I, SNA-I lectin and IgG.

[0008] Wherein, the IgG contains sialic acid.

[0009] Secondly, the present invention also provides use of the biomarker in preparing a reagent for distinguishing antiphospholipid syndrome from pathological pregnancy events and non-pathological pregnancy events.

[0010] Specifically, the diagnosis comprises: determining the complexes formed by the binding of SNA-I lectin and IgG in the biological sample to be tested;

[0011] The level of the complex formed by the binding of SNA-I lectin and IgG in the biological sample is compared with control data, wherein a detectable decrease in the level of the complex formed by the binding of SNA-I lectin and IgG in the sample relative to the control data indicates the possibility of a pathological pregnancy event in the patient with antiphospholipid syndrome providing the serum.

[0012] Specifically, the SNA-I binding level of the patient to be tested is compared with the data of eligible patients in the database (ie, APS patients with pure arterial events or pure venous events) to predict the possibility of pathological pregnancy events.

[0013] Wherein, the biological sample is a serum sample.

[0014] Preferably, the levels of complexes formed by the binding of SNA-I lectin and IgG are measured by the following steps, comprising:

[0015] a. contacting a biological sample from a patient with a lectin of interest;

[0016] b. IgG present in the biological sample forms a lectin-glycan complex with the target lectin;

[0017] c. Wash to remove any unbound IgG;

[0018] d. adding a labeled detection antibody reactive with the antibody from the biological sample;

[0019] e. washing to remove any unbound labeled detection antibody; and

[0020] f. converting the label of the detection antibody into a detectable signal.

[0021] Wherein, the target lectin is deposited or fixed on a solid surface carrier. Preferably, the solid surface carrier is in the form of latex beads, porous plates or membrane strips, nanotubes, sheets with QR codes, etc. Preferably, the detection antibody is labeled by covalently linking to an enzyme, a marker with a fluorescent compound or metal, or a marker with a chemiluminescent compound.

[0022] This study used a lectin microarray containing 56 lectins to examine the glycan profiles of serum IgG and lectin-specific binding in patients with antiphospholipid syndrome. The results showed that the content of SNA-I lectin-bound glycans was significantly reduced in patients with APS who experienced a postpartum pregnancy. Since SNA-I lectin specifically binds sialic acid, this suggests that sialic acid levels are reduced in patients with APS who experienced a postpartum pregnancy. To confirm the reliability of these findings, lectin immunoblotting was also performed. The results showed that the content of SNA-I lectin-bound glycans was still reduced in patients with APS who experienced a postpartum pregnancy, consistent with the lectin microarray results.

[0023] After diagnosing APS, the present invention can predict the possibility of pathological pregnancy events by detecting the level of binding between the patient's serum IgG and SNA-I, so as to detect the disease as early as possible in the early stage of progression and take timely measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A is the entire lectin chip; 1B-1C are the locations of 56 lectins on the chip microarray.

[0025] Figure 2 Shown is the signal-to-noise ratio of specific binding of lectin SNA-I in patients with APS who presented with pathological pregnancy events, pure arterial events, and pure venous events detected by lectin chip (*p<0.05).

[0026] Figure 3 Shown is a schematic representation of an immunoblot for SNA-I lectin.

[0027] Figure 4 Shown are the numerical results of SNA-I lectin immunoblotting fluorescence. Pathological pregnancy events were significantly reduced compared with simple arterial and venous events (p<0.05).

[0028] Figure 5 Shown is the ROC curve analysis of the lectin SNA-I chip results, pathological pregnancy events vs. (pure arterial events vs. pure venous events). DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0030] Example 1 Lectin microarray analysis of serum IgG glycosylation

[0031] Experimental Specimens: This study included 178 patients with antiphospholipid syndrome, 45 with Takayasu arteritis, 45 with rheumatoid arthritis, and 45 with cardiovascular disease, as well as 100 healthy controls (see Table 1). Patients with antiphospholipid syndrome, Takayasu arteritis, rheumatoid arthritis, and cardiovascular disease met the diagnostic criteria for the respective diseases. Fresh blood was collected from all participants, and serum was immediately separated and stored at -80°C for future use.

[0032] Table 1 Basic information of the subjects in the lectin chip experiment

[0033]

[0034] A lectin microarray containing 56 lectins was used to detect the glycosylation status in the experimental specimens. Lectins can specifically bind to the glycan molecules at the end of glycoproteins to form complexes. The types and contents of glycans on the surface of the target protein are studied through the specific binding of different lectins to glycans. Lectin microarrays have been increasingly widely used in glycosylation research due to their high efficiency. Each lectin chip used in this study has 14 lectin microarrays, and the lectin microarray contains 56 types of lectins, and each lectin is fixed to the array in triplicate. Then, the diluted serum sample is added to the lectin microarray, reacted with it, and then Cy3-labeled IgG antibody is added to obtain the signal value of each lectin specifically binding to the IgG glycan. The signal value is related to the binding affinity and binding strength, see. Figure 1 .

[0035] After the frozen specimens were equilibrated at room temperature, each specimen serum was diluted 1:200 and added to the microarray and incubated at 4°C overnight. Then, Cy3-labeled IgG antibodies were hybridized with the lectins on the microarray for 1 hour in a dark environment. The fluorescence intensity of all lectins was analyzed independently. The chip images were converted into digital format for analysis. The signal-to-noise ratio (S / N) of each lectin spot was calculated using the difference between the foreground value and the background value of each lectin spot. To prevent bias in the lectin microarray between arrays, we used inter-array normalization to normalize the S / N data. The significant difference in lectin binding ability was determined by the data distribution between groups according to the following rules: (1) inter-group comparison >1.3 or <0.77; (2) inter-group comparison test type: if normality was met, T test was selected; otherwise, non-parametric test was selected, and p value <0.05.

[0036] The results are shown in Table 2-3. Figures 2-3 The S / N data showed that the IgG glycosylation in the pathological pregnancy event group was significantly reduced compared with the group with pure arterial events and pure venous events.

[0037] Conclusion: Compared with (pure arterial events + pure venous events), the specific binding of serum IgG to SNA-I in pathological pregnancy samples of APS patients was significantly reduced, indicating that the expression of sialic acid level of IgG was significantly reduced in pathological pregnancy events.

[0038] Table 2 Lectin and corresponding glycan binding specificity

[0039]

[0040] Table 3 Lectin chip results

[0041]

[0042] *p<0.05

[0043] **p<0.01

[0044] Example 2 Serum lectin imprint verification experiment

[0045] Experimental specimens and methods: To further clarify the reliability of the above-mentioned lectin microarray detection conclusions, lectin microblotting was performed on randomly selected samples of APS patients, healthy controls, and disease controls (12 cases each) from the same batch. At the same time, lectin microblotting was performed on samples of APS patients from the same batch with isolated arterial events (12 cases for each lectin), isolated venous events (24 cases for each lectin), and pathological pregnancy events (36 cases for each lectin) .

[0046] Serum samples were diluted 1:100, mixed with sample buffer, and boiled at 100°C for 10 minutes. The samples were then electrophoresed on a 10% precast gel using SDS-PAGE. The proteins were then electrophoretically transferred to a PVDF membrane. The membrane was blocked and hybridized with Cy3-labeled lectin. Finally, the fluorescence signal was detected using a fluorescence imaging device. The intensity of the fluorescence signal is proportional to the binding affinity of the lectin to the glycoprotein sugars.

[0047] Results: Compared with (pure arterial events + pure venous events), serum IgG binding to SNA-I lectin was decreased in APS patients with pathological pregnancy events (p < 0.05) ( Figure 4 ). This indicates that the level of sialic acid, a polysaccharide bound by SNA-I lectin, in the serum of patients with pathological pregnancy events is abnormal. ROC curve analysis showed that when the cutoff value was 3.107, the AUC value of SNA-I in distinguishing pathological pregnancy events from (pure arterial events + pure venous events) in APS patients was 0.6318, which has good diagnostic efficacy ( Figure 5 ).

[0048] Conclusion: The results of lectin blot validation were consistent with those of lectin chip detection. Compared with pure arterial events and pure venous events, the serum IgG of APS patients with pathological pregnancy events expressed low levels of sialic acid, which can be used as a biomarker between different clinical events to predict the possibility of pathological pregnancy events in APS patients, so as to detect them as early as possible in the early stage of disease progression and take timely measures.

[0049] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Use of a complex formed by the binding of SNA-I lectin to IgG containing sialic acid in the preparation of a reagent for antiphospholipid syndrome for distinguishing pathological pregnancy events from non-pathological pregnancy events.

2. The use according to claim 1, characterized in that The diagnosis comprises: determining the complex formed by the binding of SNA-I lectin and IgG in a biological sample to be tested, wherein the biological sample is a serum sample; The level of the complex formed by the binding of SNA-I lectin and IgG in the biological sample is compared with control data, wherein a detectable decrease in the level of the complex formed by the binding of SNA-I lectin and IgG in the sample relative to the control data indicates the possibility of a pathological pregnancy event in the patient with antiphospholipid syndrome providing the serum.

3. The use according to claim 2, wherein The level of complexes formed by SNA-I lectin binding to IgG was measured by the following steps: a. contacting a biological sample from a patient with a lectin of interest; b. IgG present in the biological sample forms a lectin-glycan complex with the target lectin; c. Wash to remove any unbound IgG; d. adding a labeled detection antibody 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 into a detectable signal.

4. The use according to claim 3, characterized in that The target lectin is immobilized on a solid surface carrier.

5. The use according to claim 4, characterized in that The solid surface carrier is in the form of latex beads, porous plates, membrane strips, nanotubes or sheets with QR codes.

6. The use according to claim 3, characterized in that The detection antibody is labeled by covalently linking to an enzyme, a label with a fluorescent compound, a label with a metal, or a label with a chemiluminescent compound.

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