A marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus

Abstract

The present invention relates to the field of biological diagnosis technology, and discloses a marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus, wherein the marker is adenosine homocysteinease 2; the kit comprises a calibrator, a quality control product, an enzyme-labeled secondary antibody, a magnetic particle reagent, and a chemiluminescent substrate; the enzyme-labeled secondary antibody is an adenosine homocysteinease 2 antibody labeled with alkaline phosphatase, which is used to detect the marker; the magnetic particle reagent is an adenosine homocysteinease 2 antibody coated with magnetic particles, which is used to separate the marker; the adenosine homocysteinease 2 disclosed in the present invention can be used as a marker for diagnosing neuropsychiatric lupus erythematosus complications. The kit obtained by using the marker can effectively distinguish NPSLE from other types of neuropsychiatric diseases by detecting the expression level of SAHH2, and provide help for early diagnosis, disease monitoring and treatment evaluation.

Description

Technical Field

[0001] The present invention relates to the technical field of biological diagnosis, and in particular to a marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a diffuse connective tissue disease mediated by autoimmune mechanisms, characterized by the production of multiple autoantibodies against cellular components and leading to multi-system and multi-organ involvement. Neuropsychiatric Systemic Lupus Erythematosus (NPSLE) mainly includes 19 types of SLE neuropsychiatric damage, which is a common systemic manifestation of severe SLE and a serious poor prognostic factor. NPSLE patients can die from various neurological damage or mental and behavioral abnormalities, including brain tissue necrosis caused by intracranial thrombosis or hemorrhage, encephalitis, status epilepticus, etc.; brain herniation caused by severe increase in intracranial pressure; and self-harm and suicidal behavior of patients with mental damage, etc., resulting in a heavy socioeconomic burden. Because the symptoms of NPSLE are complex and similar to other neuropsychiatric diseases, early diagnosis and monitoring are highly challenging.

[0003] Conventional immunological indicators such as antinuclear antibodies (ANA), anti-double-stranded DNA antibodies (anti-dsDNA), and anti-Sm antibodies are of great significance in the diagnosis of SLE. However, these immunological markers cannot clearly distinguish whether NPSLE exists. Summary of the invention

[0004] The present invention aims at the problems existing in the prior art and provides a marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus.

[0005] The technical solution adopted by the present invention is: use of a marker in preparing a reagent or a detection kit for diagnosing neuropsychiatric lupus, wherein the marker is adenosine homocysteinease 2.

[0006] Furthermore, the detection process includes the following steps:

[0007] Obtain individual serum samples;

[0008] Detect the relative content of adenosine homocysteinease 2 in serum samples.

[0009] Furthermore, an elevated level of adenosine homocysteinease 2 in the serum sample is an indication for auxiliary diagnosis of neuropsychiatric lupus.

[0010] A magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus, the kit comprising a calibrator, a quality control product, an enzyme-labeled secondary antibody, a magnetic particle reagent, and a chemiluminescence substrate;

[0011] The enzyme-labeled secondary antibody is an alkaline phosphatase-labeled adenosine homocysteinease 2 antibody, which is used to detect the marker;

[0012] The magnetic particle reagent is a magnetic particle-coated adenosine homocysteinease 2 antibody, which is used to separate the marker.

[0013] Furthermore, the preparation method of the enzyme-labeled secondary antibody is as follows:

[0014] Sodium periodate solution and ethylene glycol solution were added to the alkaline phosphatase solution, and the reaction was terminated after sufficient incubation;

[0015] The adenosine homocysteinease 2 antibody is added to the reaction solution, dialyzed, reduced and then washed to obtain the enzyme-labeled secondary antibody.

[0016] Furthermore, the preparation method of the magnetic particle reagent is as follows:

[0017] Cleaning and activating the magnetic particles;

[0018] Incubate the activated magnetic particles with biotin-labeled AMP2 antibody;

[0019] The unbound antibodies are removed by magnetic separation, and the blocked free groups are added and washed to obtain the magnetic particle reagent.

[0020] Furthermore, the concentration of the enzyme-labeled secondary antibody in the kit is 0.05 μg / mL.

[0021] Furthermore, the concentration of the magnetic particle reagent in the kit is 0.05 μg / mL.

[0022] The beneficial effects of the present invention are:

[0023] The adenosine homocysteinease 2 (SAHH2) disclosed in the present invention can be used as a marker for diagnosing neuropsychiatric lupus erythematosus complications. The kit obtained by using the marker can effectively distinguish NPSLE from other types of neuropsychiatric diseases by detecting the expression level of SAHH2, providing assistance for early diagnosis, disease monitoring and treatment evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a standard curve diagram of the luminescent signal intensity and SAHH2 concentration in the kit obtained in the present invention.

[0025] Figure 2 : is the SAHH2 concentration in different samples in the examples of the present invention.

[0026] Figure 3 This is a protein immunoblot analysis of SAHH2 in different samples in the examples of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] A marker is used in the preparation of a reagent or a detection kit for diagnosing neuropsychiatric lupus, wherein the marker is adenosine homocysteinease 2. The detection process comprises the following steps:

[0029] Obtain individual serum samples;

[0030] Detect the relative content of adenosine homocysteinease 2 in serum samples.

[0031] Elevated levels of adenosine homocysteinease 2 in serum samples are an indication for auxiliary diagnosis of neuropsychiatric lupus.

[0032] A magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus, the kit comprising a calibrator, a quality control product, an enzyme-labeled secondary antibody, a magnetic particle reagent, and a chemiluminescence substrate;

[0033] The calibrator is a constructed SAHH2 standard, which is used for the calibration curve; the SAHH2 gene is cloned into an expression vector through gene cloning technology, and expressed and purified using Escherichia coli or mammalian cell systems.

[0034] The quality control product was SAHH2 protein.

[0035] The enzyme-labeled secondary antibody is an alkaline phosphatase-labeled adenosine homocysteinease 2 antibody, which is used to detect the marker;

[0036] The preparation process is as follows:

[0037] Alkaline phosphatase AP was coupled with Anti-SAHH2 antibody by a coupling method to obtain a highly sensitive enzyme-labeled secondary antibody reagent.

[0038] S11: Dissolve 1 mg of AP in 0.5 mL of distilled water. After it is fully dissolved, take 0.125 mL for labeling reaction.

[0039] S12: Add 0.125 mL of 12 mmol / L sodium periodate solution, mix well, and incubate at 2-8 °C for 30 min.

[0040] S13: Add 0.125 mL of 32 mmol / L ethylene glycol solution, mix well and incubate at room temperature in the dark for 30 min to terminate the sodium periodate reaction.

[0041] S14: 0.25 mg of Anti-SAHH2 antibody was added to the reaction system, and unbound substances were removed by dialysis. The dialysis process used 0.05 mol / L sodium carbonate buffer with a pH of 9.5 at 2 to 8 °C for 16 to 24 hours.

[0042] S15: After the reaction is completed, sodium borohydride solution is added and incubated for another 2 hours to remove possible unreacted substances. The solution is then dialyzed and centrifuged using PBS solution and stored using glycerol.

[0043] The magnetic particle reagent is a magnetic particle-coated adenosine homocysteinease 2 antibody, which is used to separate the marker.

[0044] The preparation process is as follows:

[0045] S21: Use magnetic microparticles with a particle size of 500 nm, wash them thoroughly with 0.05 mol / L MES buffer, add 1.25 wt.% glutaraldehyde and 0.05 mol / L PBS buffer solution with a pH of 6.0, and react for 1 hour to activate the surface of the magnetic microparticles.

[0046] S22: Incubate biotinylated SAHH2 antibody with activated magnetic particles at a ratio of 1 mg magnetic particles: 250 μg antibody at 25 °C for 1 hour.

[0047] S23: Remove unbound antibodies by magnetic separation, add 2% BSA to block free groups, and obtain immunomagnetic beads after further washing.

[0048] The concentration of magnetic particle reagent is 0.05 μg / mL, which is suitable for subsequent capture and analysis.

[0049] After preparation, assemble the kit

[0050] The reagents obtained above and the chemiluminescent substrate are assembled into a kit according to an optimized ratio.

[0051] Then the standard curve was constructed.

[0052] Using the SAHH2 fusion protein standard, dilute it with 0.05 mol / L pH7.6 PBS buffer to prepare a series of gradient standard solutions (500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 0 ng / mL). These standard solutions react with the kit to draw a standard curve for subsequent quantitative analysis of samples. The standard curve is shown in Figure 1. Figure 1 shown.

[0053] Preparation of buffer for calibrators and quality control agents: Prepare 0.1 M Tris buffer (pH 7.6) and add 0.5% bovine serum albumin (BSA), 0.1% Tween-20, and 1.0% sucrose to improve the stability of the solution and the consistency of the reaction.

[0054] The expression level of SAHH2 can be detected by the above kit, and combined with immunological indicators such as antibodies, it can be detected whether SLE patients have NPSLE. The combined detection of the kit of the present invention and conventional immune markers can accurately diagnose NPSLE.

[0055] The process is as follows:

[0056] For example, a patient is diagnosed with systemic lupus erythematosus (SLE), and is accompanied by cognitive impairment and mild psychiatric symptoms, and has the clinical manifestations of neuropsychiatric lupus (NPSLE). However, the manifestations of cognitive and psychiatric symptoms are not obvious and non-specific, and it is difficult to determine whether it is NPSLE. The kit of the present invention is used to detect the concentration of SAHH2 in serum.

[0057] The process is as follows:

[0058] A serum sample was obtained, and the concentration of SAHH2 in the serum was detected using the kit of the present invention according to the instruction manual.

[0059] Standard curve preparation: Data quantification was performed based on the standard curve constructed using laboratory standard solutions (500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 0 ng / mL).

[0060] The SAHH2 concentration in the patient's serum was detected to be 350 ng / mL, which was significantly higher than the normal reference range (usually the SAHH2 concentration in healthy individuals is ≤50 ng / mL) and was consistent with the expected clinical manifestations of neuropsychiatric lupus.

[0061] The patient has typical SLE-related symptoms, such as joint pain, rash, etc. At the same time, it is accompanied by cognitive impairment (such as memory loss, lack of concentration) and mild mental symptoms (such as anxiety and depression). Therefore, combined with the test results, it can be clearly diagnosed as neuropsychiatric lupus erythematosus (NPSLE), rather than other types of neuropsychiatric diseases or non-SLE-related mental symptoms. The detection of SAHH2 provides an important biomarker for auxiliary diagnosis and helps to rule out the possibility of other diseases.

[0062] The expression level of SAHH2 in NPSLE patients is significantly higher than that in normal people and patients with other types of neuropsychiatric diseases. SAHH2 concentration detection will play an increasingly important role in the early diagnosis, disease monitoring, and treatment evaluation of neuropsychiatric lupus. Especially in the group of patients with SLE accompanied by neuropsychiatric symptoms, changes in SAHH2 levels can assist in diagnosis.

[0063] For example, when a patient with systemic lupus erythematosus develops neuropsychiatric symptoms such as cognitive impairment, mood swings, etc., in order to determine whether there is neuropsychiatric lupus, the kit of the present invention is used to detect the concentration of SAHH2 in the serum for auxiliary detection.

[0064] A serum sample was obtained, and the concentration of SAHH2 in the serum was detected using the kit of the present invention according to the instruction manual.

[0065] Standard curve preparation: Data quantification was performed based on the standard curve constructed using laboratory standard solutions (500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 0 ng / mL).

[0066] By measuring the luminescent signal with a chemiluminescence instrument, the concentration of SAHH2 in the sample can be obtained, and quantitative results can be obtained to analyze the patient's SAHH2 level.

[0067] The patient's serum sample showed a SAHH2 concentration of 42 ng / L, and the SAHH2 test results all showed that the concentration was within the normal range. The normal SAHH2 concentration suggests that the patient's symptoms may not be caused by NPSLE. NPSLE patients usually show elevated SAHH2 concentrations, reflecting neuronal damage and inflammatory responses caused by immune responses. Therefore, the normal range of SAHH2 concentrations excludes the possibility of neuropsychiatric lupus.

[0068] The patient showed clinical neuropsychiatric symptoms similar to NPSLE (such as cognitive impairment, mood swings, depression, etc.), but the serum SAHH2 concentration was not abnormally elevated. In this case, other neuropsychiatric diseases may be considered.

[0069] This kit can effectively rule out the possibility of neuropsychiatric lupus erythematosus (NPSLE). SAHH2, as a specific biomarker for neuropsychiatric lupus erythematosus, has high specificity and can distinguish NPSLE from other neuropsychiatric diseases or drug-induced symptoms.

[0070] SAHH2 testing may become an important auxiliary indicator for clinical decision-making in the future, helping doctors distinguish different types of neuropsychiatric diseases and optimize treatment plans.

[0071] By using the magnetic particle chemiluminescence kit of the present invention, if the patient's serum SAHH2 concentration is significantly increased, it provides an important basis for the diagnosis of NPSLE. Combined with clinical symptoms and other test results, it helps doctors make accurate diagnoses and adjust treatment plans in a timely manner. If the test results show that the SAHH2 concentration of some SLE patients is maintained within the normal range, the possibility of neuropsychiatric lupus erythematosus (NPSLE) is ruled out. Combined with clinical symptoms, imaging examinations and other immunological indicators, doctors can further determine the source of the symptoms and formulate appropriate treatment plans for patients. This detection method not only provides a clear basis for diagnosis, but also helps clinicians avoid misdiagnosis and promote the implementation of personalized treatment.

[0072] This detection method not only provides an efficient tool for early diagnosis, but also provides strong support for disease monitoring and treatment effect evaluation, helping to improve the treatment prognosis of patients with neuropsychiatric lupus.

[0073] Patients with different neuropsychiatric diseases were tested and the expression levels of SAHH2 in their blood samples were compared. The results are as follows: Figure 2 and Figure 3 shown.

[0074] Four different types of neuropsychiatric diseases were selected, including patients with Alzheimer's disease, patients with depression and anxiety, patients with neuropsychiatric lupus, and a healthy control group. The SAHH2 magnetic particle chemiluminescence kit was used to detect the SAHH2 concentration of these four groups of samples. The results are as follows Figure 2 As shown in the figure, it can be seen that SAHH2 shows significantly high expression in patients with neuropsychiatric lupus, while in patients with Alzheimer's disease and depression and anxiety, its expression level is relatively low, and no significant change is observed compared with the healthy control group.

[0075] The expression of SAHH2 in each group of samples was verified by Western blotting. Figure 3 As shown in the figure, it can be seen that the results are consistent with the detection trend of the chemiluminescence kit. By detecting the expression level of SAHH2, the kit can effectively distinguish NPSLE from other neuropsychiatric diseases, providing potential clinical application value for early diagnosis, disease monitoring and treatment evaluation.

[0076] In order to illustrate the effect of the kit of the present invention, the following samples were selected for testing:

[0077] Serum samples obtained from the patient group: 50 patients diagnosed with SLE, including 25 with neuropsychiatric symptoms (NPSLE group) and 25 without neuropsychiatric symptoms (non-NPSLE group).

[0078] Sample serum obtained from the control group: 30 healthy volunteers served as the normal control group.

[0079] The kit of the present invention was used to measure the SAHH2 concentration in the serum samples of the patient group and the control group.

[0080] The results are as follows:

[0081] In the NPSLE patient group, the SAHH2 concentration in serum samples was significantly increased, and the average concentration of SAHH2 in serum samples was 250 ng / mL (range 150-350 ng / mL).

[0082] Among them, 72% of the patient groups (18 / 25) had SAHH2 concentrations in serum samples higher than 200 ng / mL, showing obvious neuropsychiatric symptoms such as cognitive impairment and emotional instability.

[0083] Non-NPSLE group:

[0084] The SAHH2 concentration in this group of patients was significantly lower than that in the NPSLE group, with a mean concentration of 55 ng / mL (range: 30 ng / mL-80 ng / mL).

[0085] The SAHH2 concentration of the vast majority of patients (25 / 25) was lower than 100 ng / mL, and these patients presented with milder systemic symptoms without significant neuropsychiatric symptoms.

[0086] Control group

[0087] The SAHH2 concentration in serum samples of healthy volunteers was stable at around 50 ng / mL (range: 40 ng / mL to 60 ng / mL).

[0088] To illustrate the performance of the kit of the present invention, 31 samples of patients clinically diagnosed with systemic lupus erythematosus were tested using the kit of the present invention. The results showed that the positive coincidence rate of the kit of the present invention was 70.97%, indicating that the clinical positive detection rate of the kit of the present invention was high.

[0089] 50 random healthy human samples were tested, and the negative coincidence rate was 100%, indicating that the clinical negative coincidence rate of the kit of the present invention is high.

[0090] The high (400 ng / mL), medium (50 ng / mL), and low (5 ng / mL) value quality control products were measured twice a day, divided into morning and afternoon tests, with 4 replicates each time, for a total of 10 days, and each concentration was measured 80 times in total. The coefficient of variation was calculated to check the precision of the reagents in high, medium, and low value specimens. The results showed that the coefficient of variation was within 8%, indicating that the kit of the present invention has good repeatability.

[0091] The kit obtained by the present invention was placed at 37°C for 7 days, and the signal retention rates of three quality control concentrations, high, medium and low, were measured. The result was >95%, indicating that the kit was stable and met clinical requirements.

[0092] The kit of the present invention adds different concentrations of bilirubin, hemoglobin, rheumatoid factor, lipid, RF, and HAMA to serum containing high, medium, and low concentrations of the test object for detection verification. The test results show that the above 6 substances have no interference with the detection results of the kit.

[0093] The above tests show that the kit of the present invention has good accuracy, good repeatability, good stability and good specificity.

[0094] SAHH2 is an enzyme specifically expressed in neurons, which mainly plays a metabolic role in the brain. In NPSLE patients, abnormal activation of the immune system can trigger neuronal damage, leading to overexpression of SAHH2. This change has a high specificity in the clinical manifestations of neuropsychiatric lupus. In other types of neuropsychiatric diseases, the expression level of SAHH2 is relatively low, or even without significant changes. By measuring the concentration of SAHH2 in the sample, NPSLE can be significantly distinguished from other types of diseases.

[0095] By detecting the expression level of SAHH2 in serum samples, the disease can be identified in the early stages of NPSLE, especially when symptoms are not yet obvious, providing patients with the opportunity for early intervention.

Claims

1. Use of a marker in the preparation of a reagent and a detection kit for diagnosing neuropsychiatric lupus, characterized in that: The marker is adenosine homocysteinease 2.

2. The use according to claim 1, characterized in that The detection process includes the following steps: Obtain individual serum samples; The relative content of adenosine homocysteinease 2 in serum samples was detected.

3. The use according to claim 2, characterized in that: The elevated level of adenosine homocysteinease 2 in the serum sample is an indication for auxiliary diagnosis of neuropsychiatric lupus.

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