CN119291173B - A marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus - Google Patents
A marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus Download PDFInfo
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Abstract
本发明涉及生物诊断技术领域，公开了一种标志物用途及用于诊断神经精神性狼疮的磁微粒化学发光试剂盒，标志物为腺苷高半胱氨酸酶2；试剂盒包括校准品、质控品、酶标二抗、磁微粒试剂、化学发光底物；酶标二抗为碱性磷酸酶标记的腺苷高半胱氨酸酶2抗体，用于检测标记物；磁微粒试剂为磁微粒包被的腺苷高半胱氨酸酶2抗体，用于分离标记物；本发明公开的腺苷高半胱氨酸酶2可以作为用于诊断系统性红斑狼疮并发症神经精神性狼疮的标志物。采用该标志物得到的试剂盒通过检测SAHH2的表达水平，能够有效区分NPSLE与其他类型的神经精神疾病，为早期诊断、病情监测和治疗评估提供帮助。
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 includes 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
The invention relates to the technical field of biological diagnosis, in particular to a marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus.
Background
Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is a diffuse connective tissue disease mediated by autoimmune mechanisms to produce multiple autoantibodies against cellular components and to cause multiple systemic, multiple organ involvement. Neuropsychiatric lupus (Neuropsychiatric Systemic Lupus Erythematosus, NPSLE), which mainly includes 19 types of SLE neuropsychiatric lesions, is a common systemic involvement and a serious prognostic inefficiency of heavy SLE. NPSLE patients can die from various nervous system injuries or mental behavioral abnormalities, including brain tissue necrosis caused by intracranial thrombosis or hemorrhage, encephalitis, status epilepticus and the like, brain herniation caused by serious intracranial pressure rise, and self-injury, suicide and the like of mental injury patients, and cause a heavy socioeconomic burden. Early diagnosis and monitoring is highly challenging due to the complex symptoms of NPSLE and similarity to other neuropsychiatric diseases.
Conventional immunological indicators such as anti-nuclear antibodies (ANA), anti-double stranded DNA antibodies (anti-dsDNA), anti-Sm antibodies, etc. are of great importance in the diagnosis of SLE. However, these immunological markers do not clearly distinguish whether NPSLE is present.
Disclosure of Invention
The invention provides a marker application and a magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus.
The technical scheme adopted by the invention is that the marker is an adenosine homocysteine 2 in the preparation of a reagent or a detection kit for diagnosing the neuropsychiatric lupus.
Further, the detection process comprises the following steps:
obtaining an individual serum sample;
detecting the relative content of the adenylyl homocysteine 2 in the serum sample.
Further, an elevated level of adenylyl homocysteine 2 in the serum sample is an indicator of aiding in the diagnosis of neuropsychiatric lupus.
A magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus, wherein 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 alkaline phosphatase-labeled adenosine homocysteine 2 antibody and is used for detecting a marker;
the magnetic particle reagent is a magnetic particle coated adenosine homocysteine 2 antibody, and is used for separating the marker.
Further, the preparation method of the enzyme-labeled secondary antibody comprises the following steps:
adding sodium periodate solution and ethylene glycol solution into alkaline phosphatase solution, and stopping reaction after full incubation;
and adding the adenosine homocysteine enzyme 2 antibody into the reaction solution, dialyzing, reducing and cleaning to obtain the enzyme-labeled secondary antibody.
Further, the preparation method of the magnetic particle reagent comprises the following steps:
Cleaning and activating the magnetic particles;
Incubating the biotin-labeled adenylyl homocysteine 2 antibody with the activated magnetic particles;
Removing unbound antibody through magnetic separation, adding a closed free radical, and cleaning to obtain the magnetic particle reagent.
Further, the concentration of the enzyme-labeled secondary antibody in the kit is 0.05 mug/mL.
Further, the concentration of the magnetic particle reagent in the kit is 0.05. Mu.g/mL.
The beneficial effects of the invention are as follows:
The adenosine homocysteine enzyme 2 (SAHH 2) disclosed by the invention can be used as a marker for diagnosing the complication of systemic lupus erythematosus and neuropsychiatric lupus. The kit obtained by adopting the marker can effectively distinguish NPSLE from other types of neuropsychiatric diseases by detecting the expression level of SAHH2, and provides assistance for early diagnosis, disease monitoring and treatment evaluation.
Drawings
FIG. 1 is a graph showing the standard of the luminescence signal intensity versus SAHH2 concentration in the kit according to the present invention.
FIG. 2 shows SAHH2 concentrations in different samples according to the present invention.
FIG. 3 shows Western blot analysis of SAHH2 in different samples according to the invention.
Detailed Description
The invention will be further described with reference to the drawings and specific examples.
Use of a marker, namely adenosylhomocysteine 2, in the manufacture of a reagent or test kit for diagnosing neuropsychiatric lupus. The detection process comprises the following steps:
obtaining an individual serum sample;
detecting the relative content of the adenylyl homocysteine 2 in the serum sample.
Elevated levels of adenosylhomocysteine 2 in serum samples are an indicator of aiding in the diagnosis of neuropsychiatric lupus.
A magnetic particle chemiluminescence kit for diagnosing neuropsychiatric lupus, wherein the kit comprises a calibrator, a quality control product, an enzyme-labeled secondary antibody, a magnetic particle reagent and a chemiluminescent substrate;
the calibrator is a constructed SAHH2 standard for calibrating a curve, and the SAHH2 gene is cloned into an expression vector by a gene cloning technology, and is expressed and purified by using escherichia coli or a mammalian cell system.
The quality control product is SAHH2 protein.
The enzyme-labeled secondary antibody is an alkaline phosphatase-labeled adenosine homocysteine 2 antibody and is used for detecting a marker;
the preparation process is as follows:
coupling alkaline phosphatase AP with Anti-SAHH2 antibody by adopting a coupling method to obtain the high-sensitivity enzyme-labeled secondary antibody reagent.
S11, dissolving 1 mg of AP in 0.5 mL of distilled water, and taking 0.125 mL for marking reaction after the AP is fully dissolved.
S12, adding a sodium periodate solution with the concentration of 12.125 mL to mmol/L, uniformly mixing, and incubating at the temperature of 2-8 ℃ for 30 min.
S13, adding an ethylene glycol solution of 0.125 mL with the concentration of 32 mmol/L, uniformly mixing and incubating for 30min at room temperature in a dark place to terminate the reaction of the sodium periodate.
S14, adding 0.25 mg Anti-SAHH2 antibody into a reaction system, removing unbound substances by dialysis, and performing dialysis process by using 0.05 mol/L sodium carbonate buffer with pH of 9.5 at 2-8 ℃ for 16-24 hours.
And S15, adding sodium borohydride solution after the reaction is completed, and further incubating for 2 hours to remove possible unreacted reality. Then, the solution was dialyzed and centrifuged with PBS, and the solution was stored with glycerol.
The magnetic particle reagent is a magnetic particle coated adenosine homocysteine 2 antibody, and is used for separating the marker.
The preparation process is as follows:
S21, using magnetic particles with the particle size of 500 nm, thoroughly cleaning the magnetic particles by using 0.05 mol/L MES buffer solution, adding glutaraldehyde with the concentration of 1.25 wt% and PBS buffer solution with the pH of 6.0 with the concentration of 0.05 mol/L, and reacting for 1 hour to activate the surfaces of the magnetic particles.
S22, incubating the biotin-labeled SAHH2 antibody with the activated magnetic particles at a ratio of 1 mg magnetic particles to 250 μg antibody at 25℃for 1 hour.
S23, removing unbound antibodies through magnetic separation, adding 2% BSA to block free radicals, and further cleaning to obtain the immunomagnetic beads.
The concentration of the magnetic particle reagent is 0.05 mug/mL, and the magnetic particle reagent is suitable for subsequent capturing and analysis.
Assembling the kit after the preparation is completed
And assembling the obtained reagent and the chemiluminescent substrate into a kit according to an optimized proportion.
The standard curve is then constructed.
A series of gradient standards (500 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 20 ng/mL, 0 ng/mL) were prepared using SAHH2 fusion protein standards and diluted with 0.05 mol/L pH7.6 PBS buffer. By reacting the standard solutions with the kit, a standard curve is drawn for quantitative analysis of subsequent samples. The standard curve is shown in figure 1.
The buffer used for the calibrator and quality control agent was prepared by preparing 0.1M Tris buffer (pH 7.6) and adding 0.5% Bovine Serum Albumin (BSA), 0.1% Tween-20 (Tween-20) and 1.0% sucrose to improve the stability of the solution and consistency of the reaction.
The expression level of SAHH2 can be detected by the kit, and whether SLE patient is NPSLE can be detected by combining immunological indexes such as antibodies and the like. The combined detection of the kit and the conventional immune marker can accurately diagnose NPSLE.
The process is as follows:
if a patient is a patient with SLE with definite systemic lupus erythematosus, the patient has clinical manifestations of NPSLE with neuropsychiatric lupus with cognitive dysfunction and mild mental symptoms. However, the manifestation of cognitive and mental symptoms is not obvious and is not specific, and whether the symptom is NPSLE is difficult to be determined, and the concentration of SAHH2 in serum is detected by using the kit.
The process is as follows:
serum samples were obtained and the concentration of SAHH2 in serum was measured using the kit of the present invention according to the instruction manual.
Standard curve preparation data quantification was performed on standard curves constructed from laboratory standard solutions (500 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 20 ng/mL, 0 ng/mL).
SAHH2 concentration in serum of the tested patient is 350 ng/mL, significantly higher than the normal reference range (SAHH 2 concentration in normal healthy individuals. Ltoreq.50 ng/mL), and is consistent with clinical expectations for neuropsychiatric lupus.
Patients with typical SLE associated symptoms such as joint pain, rash and the like, and cognitive disorders (such as memory decline, inattention) and mild psychotic symptoms (such as anxiety and depressed mood) are accompanied, so that in combination with the detection results, the patients can be clearly diagnosed as neuropsychiatric lupus (NPSLE) rather than other types of neuropsychiatric diseases or non-SLE associated psychotic symptoms. The detection of SAHH2 provides an important biomarker for aiding in diagnosis, helping to rule out the possibility of other diseases.
SAHH2 is expressed at significantly higher levels in NPSLE patients than in normal populations and in other types of neuropsychiatric disease patients. SAHH2 concentration detection will play an increasingly important role in early diagnosis, condition monitoring, treatment assessment, etc. of neuropsychiatric lupus. In particular, in the patient population in which SLE is accompanied by neuropsychiatric symptoms, diagnosis can be aided by changes in SAHH2 levels.
For example, after the occurrence of neuropsychiatric symptoms such as cognitive dysfunction and mood swings, the SAHH2 concentration in serum is detected by the kit for auxiliary detection in order to determine whether the neuropsychiatric lupus exists.
Serum samples were obtained and the concentration of SAHH2 in serum was measured using the kit of the present invention according to the instruction manual.
Standard curve preparation data quantification was performed on standard curves constructed from laboratory standard solutions (500 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 20 ng/mL, 0 ng/mL).
The luminescence signal is measured by a chemiluminescent instrument, so that the concentration of SAHH2 in the sample is obtained, and a quantitative result can be obtained for analyzing the SAHH2 level of the patient.
In the serum sample of the patient, the SAHH2 concentration was 42 ng/L, and the SAHH2 detection results showed that the concentration was within the normal range. SAHH2 concentrations were normal indicating that the patient's symptoms may not be caused by NPSLE. NPSLE patients generally exhibit elevated SAHH2 concentrations, reflecting immune response-initiated neuronal damage and inflammatory responses. Thus, the normal range of SAHH2 concentrations precludes the possibility of neuropsychiatric lupus.
The patient clinically exhibited neuropsychiatric symptoms (e.g., cognitive dysfunction, mood swings, depressive symptoms, etc.) similar to NPSLE, but no abnormal elevation in SAHH2 concentration was seen in serum. This situation considers the possibility of other neuropsychiatric diseases.
The kit can effectively eliminate the possibility of neuropsychiatric lupus (NPSLE). SAHH2 is used as a specific biomarker of the neuropsychiatric lupus, has higher specificity, and can distinguish NPSLE from symptoms caused by other neuropsychiatric diseases or medicines.
SAHH2 detection may become an important auxiliary index for clinical decision in the future, helping doctors to distinguish different types of neuropsychiatric diseases and optimizing treatment schemes.
By using the magnetic particle chemiluminescence kit, if the SAHH2 concentration in serum of a patient is obviously increased, an important basis is provided for the diagnosis of NPSLE, and a doctor is helped to make accurate diagnosis and adjust a treatment scheme in time by combining clinical symptoms and other examination results. If the results indicate that SAHH2 concentrations in some SLE patients remain within normal ranges, the possibility of neuropsychiatric lupus (NPSLE) is precluded. In combination with clinical symptoms, imaging examinations and other immunological indicators, the physician can further determine the source of the symptoms and formulate an appropriate treatment regimen for the patient. The detection method not only provides clear diagnosis basis, but also helps clinicians avoid misdiagnosis and promotes the implementation of personalized treatment.
The detection method not only provides a high-efficiency tool for early diagnosis, but also provides powerful support for disease monitoring and treatment effect evaluation, and is beneficial to improving the treatment prognosis of patients with neuropsychiatric lupus.
Different neuropsychiatric disease patients were tested and SAHH2 expression levels in their blood samples were compared and the results are shown in FIGS. 2 and 3.
Four different types of neuropsychiatric diseases, alzheimer's disease patients, depression anxiety patients, neuropsychiatric lupus patients and healthy control groups were selected. SAHH2 concentrations of the four groups of samples were detected using the SAHH2 magnetic particle chemiluminescent kit. As a result, as shown in fig. 2, it can be seen from the figure that SAHH2 exhibited significantly high expression in patients with neuropsychiatric lupus, whereas in patients with alzheimer's disease and depression anxiety, the expression level was relatively low, and no significant change was seen as compared with the healthy control group.
The expression of SAHH2 in each set of samples was verified by western immunoblotting, as shown in fig. 3, from which it can be seen that the results are consistent with the detection trend of the chemiluminescent kit. By detecting the expression level of SAHH2, the kit can effectively distinguish NPSLE from other neuropsychiatric diseases, and provides potential clinical application value for early diagnosis, disease monitoring and treatment evaluation.
To demonstrate the effectiveness of the kit of the invention, the following samples were selected for testing:
Sample serum obtained from patient group 50 patients diagnosed with SLE, 25 of which had neuropsychiatric symptoms (NPSLE group) and 25 of which had no neuropsychiatric symptoms (non-NPSLE group).
Control group 30 healthy volunteers were taken as normal control group.
SAHH2 concentrations in serum samples of the patient group and the control group were determined using the kit of the present invention.
The results were as follows:
In the patient group, the concentration of SAHH2 in the serum sample is obviously increased, and the average concentration of SAHH2 in the serum sample is 250 ng/mL (at 150-350 ng/mL).
Wherein the SAHH2 concentration (18/25) in the serum sample of 72% of the patient groups is higher than 200 ng/mL, and the patient groups show obvious neuropsychiatric symptoms such as cognitive dysfunction, emotional instability and the like.
Non-NPSLE group:
SAHH2 concentration was significantly lower in this group than in the NPSLE group, with an average concentration of 55 ng/mL (range: 30 ng/mL-80 ng/mL).
Most patients (25/25) with SAHH2 concentrations below 100 ng/mL exhibit lighter systemic symptoms without significant neuropsychiatric symptoms.
Control group
SAHH2 concentration in serum samples of healthy volunteers was stabilized at about 50 ng/mL (range: 40: 40 ng/mL-60: 60 ng/mL).
To demonstrate the performance of the kit of the invention, 31 samples clinically diagnosed as systemic lupus erythematosus disease were assayed using the kit of the invention. The result shows that the positive coincidence rate of the kit is 70.97%, which indicates that the clinical positive detection rate of the kit is high.
50 Random healthy human body samples are measured, and the negative coincidence rate is 100%, which indicates that the clinical negative coincidence rate of the kit is high.
Quality control materials with high (400 ng/mL), medium (50 ng/mL) and low (5 ng/mL) values are measured twice a day, and the detection is carried out in the morning and afternoon, 4 times a day, the total detection is carried out for 10 days, each concentration is measured 80 times, the variation coefficient is calculated, and the precision of the reagent in high-value, medium-value and low-value specimens is checked. The results show that the variation coefficient is within 8%, which indicates that the repeatability of the kit is good.
The kit obtained by the invention is placed at 37 ℃ for 7 days, and the signal retention rate of quality control of high, medium and low 3 concentrations is measured. The result is more than 95%, which shows that the reagent kit is stable and meets clinical requirements.
The kit of the invention is characterized in that bilirubin, hemoglobin, rheumatoid factors, fat, RF and HAMA with different concentrations are respectively added into serum containing substances with different concentrations of high, medium and low to carry out detection verification, and the detection results show that the 6 substances have no interference on the detection results of the kit.
The test shows that the kit has good accuracy, good repeatability, good stability and good specificity.
SAHH2 is an enzyme specifically expressed by neurons, and plays a metabolic role mainly in the brain. In NPSLE patients, abnormal activation of the immune system can trigger neuronal damage, leading to overexpression of SAHH 2. This change has a higher specificity in the clinical manifestations of neuropsychiatric lupus. While in other types of neuropsychiatric diseases, the expression level of SAHH2 is relatively low, even without significant changes. By measuring the concentration of SAHH2 in the sample, NPSLE can be significantly distinguished from other disease types.
By detecting the level of SAHH2 expression in serum samples, the disease can be identified at an early stage of NPSLE, particularly where symptoms are not yet apparent, providing the patient with an opportunity for early intervention.
Claims (3)
1. Use of a marker in the preparation of a reagent and test kit for diagnosing neuropsychiatric lupus, wherein the marker is adenylhomocysteine 2.
2. Use according to claim 1, characterized in that the detection process comprises the following steps:
obtaining an individual serum sample;
detecting the relative content of the adenylyl homocysteine 2 in the serum sample.
3. The use according to claim 2, wherein an elevated level of adenosylhomocysteine 2 in the serum sample is indicative for aiding in the diagnosis of neuropsychiatric lupus.
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| CN111707825A (en) * | 2020-07-29 | 2020-09-25 | 四川携光生物技术有限公司 | Kit for combined detection of tumor markers MCT1 and MCT4, and preparation method and application thereof |
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| CN107449914A (en) * | 2017-07-10 | 2017-12-08 | 北京大学人民医院 | The application of UCHL1 autoantibody |
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| CN115598352A (en) * | 2022-10-14 | 2023-01-13 | 南京大学（Cn） | Application of neuropsychiatric lupus cerebrospinal fluid auxiliary diagnosis marker |
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