CN110850096B

CN110850096B - Biomarker group and application thereof, protein chip kit and ELISA kit

Info

Publication number: CN110850096B
Application number: CN201910936809.8A
Authority: CN
Inventors: 符聪聪; 吕瑞; 黄若磐
Original assignee: Reboo Guangzhou Biotechnology Co ltd
Current assignee: Reboo Guangzhou Biotechnology Co ltd
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2023-02-17
Anticipated expiration: 2039-09-29
Also published as: CN110850096A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker group and application thereof, a protein chip kit and an ELISA kit. The application provides application of a biomarker group in preparing a product for diagnosing demyelinating diseases of the central nervous system, wherein the biomarker group comprises stromal cell derived factor-1, granulocyte chemotactic protein 2, monocyte chemotactic protein-1, interleukin-2, gamma-interferon, insulin-like growth factor binding protein-3, E-calcium adhesin and macrophage inflammatory protein-1 delta; the central nervous system demyelinating diseases are neuromyelitis spectrum diseases negative for aquaporin 4 antibody, and multiple sclerosis negative for aquaporin 4 antibody. The application fills the gap that no reliable product and method for diagnosing and identifying the multiple sclerosis and the neuromyelitis pedigree diseases under the negative condition of the aquaporin 4 antibody exist in the clinical.

Description

Biomarker group and application thereof, protein chip kit and ELISA kit

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker group and application thereof, a protein chip kit and an ELISA kit.

Background

The demyelinating disease of the central nervous system is an autoimmune system disease mainly based on multifocal and inflammatory demyelination of the central nervous system, and the clinical characteristics of the demyelinating disease mainly comprise repeated attack, repeated remission and relapse. The central nervous system demyelinating diseases which are relatively common clinically comprise Multiple Sclerosis (MS), neuromyelitis optica (NMO), acute disseminated encephalomyelitis and the like, in recent years, the incidence rate of the central nervous system demyelinating diseases is on the rise year by year, the life quality of patients is seriously influenced, and the physical and psychological health of the patients is seriously threatened.

MS and NMO are two demyelinating diseases of the nervous system with a clinically high incidence of disease.

Multiple sclerosis is an autoimmune disease which is characterized mainly by white matter inflammatory demyelinating diseases of the central nervous system and is an incurable progressive disease of the nervous system. The pathological changes of the multiple sclerosis are diffused, so that the symptoms and physical signs are complicated, and neuritis, retrobulbar neuritis, ophthalmoplegia, acroparalysis, pyramidal fasciculation and mental symptoms can appear. The multiple sclerosis lesions are located in the cerebellum and appear as ataxia, tremor of limbs and nystagmus. Its pathological changes invade the medial longitudinal bundle and produce ocular myoclonus which is involuntary and has various patterns of eyeball persistence and irregularity. If dizziness and vertical shock occur, which are difficult to explain, especially the acute vertigo and vertical shock in young patients should be felt after the vertigo stops. The most frequently involved parts of multiple sclerosis are the periventricular white matter, optic nerve, spinal cord, brainstem and cerebellum, and the main symptoms are: paresthesia, quadriplegia, dyskinesia, unilateral visual disorder, diplopia, vertigo, etc.; other symptoms include fatigue, micturition disorders, cognitive disorders, depression, spasticity, ataxia, pain, and the like. Most patients clinically manifest spatial and temporal polytropy. Spatial multiplicity refers to the multiple occurrence of the diseased site, and temporal multiplicity refers to the course of remission-recurrence. A small number of patients present with a single focal indication throughout the course of the disease. The disease can be divided into subtypes according to the onset and the speed of progression, wherein 85% of patients have symptoms manifested as relapsing-remitting types, which are more typical types of MS. Other types of MS include: secondary-progressive, primary-progressive, progressive-recurrent. MS is a rare disease of the immune system that occurs well in the middle and young age groups of 20-40 years, with female patients approximately 1.5-2 times as many as male patients. The nerve system of the patient is gradually disabled, loses self-care ability, blindness and even loses life because the damage and the peeling of the myelin sheath of the nerve are caused by the pathological changes of the autoimmune system, so that the functions of the spinal cord, the brain and the optic nerve are damaged. The pathogenesis of MS is complex, and the research in recent years provides a multi-factor etiology with the comprehensive effects of autoimmunity, virus infection, genetic tendency, environmental factors and individual susceptibility factors, but at present, no one very unified statement exists. Multiple sclerosis is difficult and complicated to diagnose clinically, and further evidence necessary to diagnose MS varies depending on clinical manifestations. In addition to clinical manifestations, the clinical findings mainly include the results of skull CT and Magnetic Resonance Imaging (MRI) combined with patient symptoms and evoked potential methods as indicators, while MRI is the most effective auxiliary diagnostic means for detecting multiple sclerosis, and has a positive rate of 62-94%. At present, the clinical diagnosis index of MS lacks a reliable serological index.

Neuromyelitis optica (NMO) is an immune-mediated inflammatory demyelinating disease of the Central Nervous System (CNS) that is predominantly involved in the optic nerve and spinal cord. The main clinical symptoms of NMO are: severe optic neuritis and transverse myelitis. The age of good onset of NMO is 35-45 years old, and the NMO has obvious difference among different sexes and is good for middle-aged and young women. Neuromyelitis optica, also known as Devic's disease, was proposed in 1894 by Devic and has long been recognized as a clinical subtype of Multiple Sclerosis (MS); in 2004, one particular serum biomarker: the discovery of an aquaporin 4 antibody, AQP4-IgG, helped to distinguish NMO from MS and expanded the diagnostic range to neuromyelitis spectrum disease (NMOSD). There are some nonspecific inflammatory demyelinating diseases with similar pathogenesis to NMO, and NMO-IgG positive rate is also higher. Wingerchuk generalizes and proposes the concept of neuromyelitis optica (NMOSD) Spectrum disease. NMOSD was well-defined by the european neurological consortium (EFNS) in 2010, referring to a group of related diseases whose underlying pathogenesis is similar to that of NMO, but clinical involvement is limited, not fully consistent with NMO diagnosis. NMOSD is a disease with high recurrence and high disability, more than 90 percent of patients have multi-temporal course, asia is relatively common, about 60 percent of patients recur within 1 year, 90 percent of patients recur within 3 years, and most patients remain serious visual disorder andor limb dysfunction, dysuria and the like. The prognosis of NMOSD is worse than MS, about half of patients have serious single-eye visual impairment and even blindness within 5 years, and about 50% of recurrent NMOSD patients cannot walk independently after 5 years of disease occurrence. The AQP4-IgG mediated abnormal immune response is a key factor of NMOSD occurrence and is one of important bases for diagnosing NMOSD, but nearly 30% of NMOSD patients have negative serum AQP4-IgG, most of MS patients have negative serum AQP4-IgG, which brings great challenges for clinically identifying the two diseases, and in clinical diagnosis, the clear diagnosis of the two diseases is particularly important because the treatment schemes of the two diseases are completely different: drugs with good therapeutic effects on MS (such as IFN-. Beta., natalizumab, oral fingolimod, etc.) not only do not have efficacy on NMOSD but also aggravate the condition of NMOSD.

There are many similarities in both MS and NMOSD pathogenesis and clinical presentation. MS is common in young and middle-aged women, with the following main symptoms: paresthesia, quadriplegia, dyskinesia, unilateral visual disorder, diplopia, vertigo, etc. NMOSD patients often have symptoms of mononuclear cell infiltration, demyelination, axonal loss, and microglial syndrome in the central nervous system. NMOSD is mainly characterized by optic neuritis and myelitis, and usually the demyelination of bilateral optic nerves or unilateral optic nerves leads to the single-sided or bilateral visual impairment, eye pain and even blindness of patients. The brain MRI result of the NMOSD patient shows that the injury degree is smaller than that of MS, the injury range of the spinal cord segment is longer than that of MS, the clinical course of the NMOSD is faster, and the early diagnosis can obviously improve the prognosis of the patient. Currently, the clear water channel protein 4 antibody detection is clinically applied as an auxiliary diagnosis for NMOSD. However, the antibody of the aquaporin 4 in the serum of part of NMOSD patients is negative, and the NMOSD diagnosis is difficult to make.

Therefore, systematically studying the expression profiles of proteins in the serum of NMOSD patients and MS patients and identifying novel disease-specific molecular markers are of great significance to our understanding of the causes, changes in the course of disease, and diagnosis and personalized treatment of these two diseases.

Both MS and NMOSD are autoimmune diseases in the central nervous system, the clinical manifestations are similar, the differential diagnosis is more complex, and the diagnosis standards of the two diseases are described below.

Further evidence necessary for the diagnosis of MS varies from clinical presentation to several cases:

1. first kind of situation

The clinical manifestations are as follows: a is more than or equal to 2 clinical attacks; objective clinical evidence of ≧ 2 lesions or objective clinical evidence of 1 lesion with reasonable evidence of 1 prior episode b.

2. The second case

The clinical manifestations are as follows: a is more than or equal to 2 clinical attacks; objective clinical evidence of 1 lesion; further evidence necessary to diagnose MS: the spatial diversity needs to have any of the following 2: (1) At least 2 of the 4 typical lesion areas (paraventricular, near cortex, infratentorial and spinal cord) d of the CNS have more than or equal to 1T 2 lesion; (2) wait for a second clinical episode a involving a different site of the CNS.

3. Situation of the third kind

The clinical manifestations are as follows: 1 clinical episode a; objective clinical evidence for more than or equal to 2 lesions; further evidence necessary to diagnose MS: the time multiplicity is required to have any of the following 3 items: (1) MRI examination at any time with asymptomatic gadolinium enhancement and non-enhancement lesions; (2) Follow-up MRI examination has new T2 lesions and/or gadolinium enhanced lesions regardless of the length of the interval from baseline MRI scan; and (3) waiting for another clinical attack a.

4. Situation of the fourth kind

The clinical manifestations are as follows: 1 clinical episode a; objective clinical evidence for 1 lesion (clinically isolated syndrome); further evidence necessary to diagnose MS: the spatial diversity needs to have any of the following 2: (1) At least 2 of the 4 typical lesion areas (paraventricular, near cortex, infratentorial and spinal cord) d of the CNS have more than or equal to 1T 2 lesion; (2) waiting for a second clinical episode a involving a different part of the CNS. The time diversity is required to meet any one of the following 3: (1) MRI examination at any time with asymptomatic gadolinium enhancement and non-enhancement lesions; (2) Follow-up MRI examination has new T2 lesions and/or gadolinium enhanced lesions regardless of the length of the interval from baseline MRI scan; (2) Follow-up MRI examination has new T2 lesions and/or gadolinium enhanced lesions regardless of the length of the interval from baseline MRI scan; (3) wait for a further clinical episode a.

5. Situation of the fifth kind

The clinical manifestations are as follows: implicit progressive neurological dysfunction (PPMS) suggesting MS; further evidence necessary to diagnose MS: retrospective or prospective investigations showed that the disease progression lasted 1 year with 2 d of the following 3: (1) The MS characteristic focus area (beside ventricles, near cortex or under the tentacle) has more than or equal to 1T 2 focus to prove the space multiple of the focus in the brain; (2) More than or equal to 2T 2 focuses exist in the spinal cord to prove the spatial diversity of the spinal cord focuses; (3) CSF positive results (evidence of isoelectric focusing indicating oligoclonal zones and/or increased IgG index).

When the clinical manifestations meet the above diagnostic criteria and no other more reasonable explanations exist, MS can be diagnosed definitely; suspected MS, but not completely meeting the above diagnostic criteria, is diagnosed as "likely MS"; when clinical manifestations can be more reasonably explained with other diagnoses, the diagnosis is "non-MS".

Note: one episode of "a" (relapse, exacerbation) is defined as: (1) a current or past event characteristic of an acute inflammatory demyelinating lesion of the CNS; (2) subjective narration or objective examination findings by the patient; (3) for at least 24 hours; and (4) no fever or signs of infection. Clinical onset needs to be confirmed by contemporaneous objective examination; even in the absence of objective evidence of the CNS, certain past events with typical symptoms and progression of MS may provide reasonable support for previous demyelinating lesions. The subjective narrative episodic symptoms (past or present) of the patient should be multiple episodes lasting at least 24 hours. Before MS diagnosis is confirmed, the following steps are required: (1) at least 1 episode must be confirmed by objective examination; (2) positive visual evoked potential in patients with previous visual impairment; or (3) the MRI examination reveals demyelinating changes in areas of the CNS that coincide with past nervous system symptoms.

Positive; or (3) the MRI examination reveals demyelinating changes in areas of the CNS consistent with past neurological symptoms.

"b" is the most reliable clinical diagnosis based on objective evidence of 2 episodes. In the absence of objective evidence of nervous system involvement, reasonable evidence for 1 prior episode included: (1) past events with typical symptoms and progression of inflammatory demyelinating lesions; (2) there were at least 1 clinical episode supported by objective evidence.

"c" requires no further evidence. However, the diagnosis of MS still needs to be made by imaging data and according to the above-mentioned diagnosis criteria. When the imaging or other examination (e.g., CSF) is negative, care should be taken to diagnose MS or other possible diagnoses. Before MS diagnosis must be satisfied: (1) all clinical manifestations have no other more rational explanations; and (2) there is objective evidence to support MS.

"d" does not require gadolinium to enhance the lesion. For patients with brainstem or spinal cord syndrome, the focus of responsibility is not listed in the MS focus number statistics.

Due to the constant human exploration of unknown domains, the concept of nmods and diagnostic criteria are constantly evolving: NMO concept (Devic) was first proposed in 1894; first NMO diagnostic criteria determination 1999 (Wingerchuk); in 2004, the aquaporin 4 antibody (AQP 4-IgG) was discovered, NMOSD is an independent disease distinct from MS (Lennon); the concept of NMOSD was proposed in 2007 (Wingerchuk); the latest NMOSD diagnostic expression agreed in 2015.

The NMOSD adult diagnostic criteria are as follows:

aquaporin 4 antibody positive diagnostic criteria: 1. meets at least 1 core clinical symptom; 2. AQP4-IgG positive (cell-based detection method is strongly recommended); 3. excluding other possible diagnoses.

Aquaporin 4 antibody negative or undetected diagnostic criteria

1. In 1 or more clinical episodes, at least 2 core clinical features are met, and all of the following requirements must be met: A. at least 1 core clinical feature must be optic neuritis, acute myelitis (LETM on MRI), or postpolar syndrome. B. Spatial polytropy (2 or more core clinical features appeared). C. Meet additional MRI requirements (as the case may be); 2. AQP4-IgG negative or no detection using the most sensitive detection means available; 3. excluding other possible diagnoses.

Core clinical features

1. Optic neuritis; 2. acute myelitis; 3. postpolar syndrome: hiccup or nausea, vomiting, unexplained from other causes; 4. acute brainstem symptom cluster; 5. symptomatic narcolepsy or acute diencephalon syndrome with typical NMOSD diencephalon foci on MRI; 6. cerebral symptoms group with typical NMOSD brain lesions; large, fused, unilateral or bilateral, subcortical or deep white matter lesions; long (more than or equal to 1/2 of the length of the corpus callosum), diffuse, uneven or edematous corpus callosum lesions; long cortical spinal cord foci, unilateral or bilateral, continuously affecting the inner capsule and cerebral crura; extensive periventricular septal lesions, often with reinforcement.

Aquaporin 4 antibody negative or undetected accessory MRI requirements

1. Acute optic neuritis: requiring head MRI to (a) have normal or only non-specific white matter lesions, or (b) optic nerve MRI to have T2 high signal lesions or T1 enhanced lesions, the length of optic nerve lesions > 1/2 of the total length of optic nerve, or to compromise optic chiasm; 2. acute myelitis: spinal cord MRI suggests that focal length is more than or equal to 3 adjacent segments (LETM) or focal spinal atrophy of more than or equal to 3 adjacent segments exists for a patient with a history of myelitis; 3. postpolar syndrome: the corresponding medullary dorsal/postpolar lesions are required; 4. acute brainstem symptom group: there is a need for corresponding brainstem foci around the ependyma.

In conclusion, clinical diagnosis procedures of MS and NMOSD are complicated and tedious, and can be confirmed by combining with clinical symptoms of patients, disease progression, attack times, MRI, laboratory examinations and other indexes. In the aspect of serological examination, the aquaporin 4 antibody is an immunological marker of NMOSD, is a more specific molecular marker of the disease at present, is one of important reference bases for identifying NMOSD and MS, and needs to be repeatedly detected. In addition, NMOSD patient NMO-IgG is strong positive and has high recurrence possibility, and the antibody titer of NMO-IgG can be used as the evaluation index of recurrence and treatment efficacy. However, nearly 30% of NMOSD patients are negative for serum aquaporin 4 antibody, while most of MS patients are also negative for serum aquaporin 4 antibody. At present, clinically, MS has no specific serological indexes, and clinical symptoms of MS and NMOSD have many similarities, which brings great troubles and challenges to clinical differential diagnosis.

Biomarkers (biomarkers) are biochemical markers that can mark changes or changes that may occur in the structure or function of systems, organs, tissues, cells, and subcellular structures and functions, and have a wide range of uses. The kit not only can discuss pathogenesis from molecular level, but also has unique advantages in accurately and sensitively evaluating early and low-level damage, can provide early warning, prognosis curative effect analysis, accurate staging and typing basis of diseases and the like, and provides basis for auxiliary diagnosis for clinicians to a great extent. Biomarker is used as the most direct, rapid and effective diagnostic means, and the screening and acquisition of the Biomarker can play an important role in multiple aspects of disease diagnosis, development, treatment, curative effect monitoring and the like. The high-throughput protein chip technology appears at the beginning of the century, is a novel scientific research technology for researching proteomics, can realize the quantitative detection of the expression level of protein in a disease sample with high throughput, high sensitivity, high specificity and low cost, and is a favorable tool for screening disease biomarkers.

Currently clinically, there are no reliable biomarkers, protein chips and kits for diagnosing Multiple Sclerosis (MS) and neuromyelitis spectrum disease (NMOSD) under the negative of aquaporin 4 antibody.

Disclosure of Invention

The application provides application of a biomarker panel and a protein chip, a protein chip kit and an ELISA kit thereof.

Aiming at the defects that no reliable biomarker capable of diagnosing and identifying Multiple Sclerosis (MS) and neuromyelitis pedigree disease (NMOSD) under the condition of aquaporin 4 antibody negative exists in the current clinic, and a protein chip and a kit, the invention provides a high-throughput, high-sensitivity, high-specificity and low-cost biomarker capable of diagnosing and identifying two central nervous system demyelinating diseases under the aquaporin 4 antibody negative condition, wherein the two central nervous system demyelinating diseases are respectively Multiple Sclerosis (MS) and neuromyelitis pedigree disease (NMOSD).

In view of this, the present application provides in a first aspect a biomarker panel comprising: granulocyte chemotactic protein 2 (GCP-2), monocyte chemotactic protein-1 (MCP-1), interleukin-2 (IL-2), gamma-interferon (INF gamma), insulin-like growth factor binding protein-3 (IGFBP-3) and E-Cadherin (E-Cadherin).

Preferably, the biomarker panel of the present application further comprises stromal cell derived factor-1 (SDF-1 a), macrophage inflammatory protein-1 delta (MIP-1 delta).

In a second aspect, the present application provides the use of a biomarker panel comprising granulocyte chemotactic protein 2 (GCP-2), monocyte chemotactic protein-1 (MCP-1), interleukin-2 (IL-2), gamma-interferon (INF gamma), insulin-like growth factor binding protein-3 (IGFBP-3) and E-Cadherin (E-Cadherin) for the preparation of a product for the diagnosis of a demyelinating disease of the central nervous system; the central nervous system demyelinating diseases are neuromyelitis spectrum diseases negative for aquaporin 4 antibody, and multiple sclerosis negative for aquaporin 4 antibody.

It should be noted that stromal cell derived factor-1 (SDF-1), also known as chemokine CXCL12, is a small molecule cytokine belonging to the chemokine protein family; granulocyte chemotactic protein 2 (granulocyte chemotactic protein-2, GCP-2) also known as CXCL6, belonging to the ELR + CXC chemokine superfamily; monocyte chemoattractant protein-1 (MCP-1) is a representative of the β subfamily and is chemotactic for monocytes; interleukin-2 (Interleukin-2, IL-2) is a cytokine of the chemokine family, IL-2 has a molecular weight of 15KD and is a glycoprotein containing 113 amino acid residues; INF gamma (gamma-Interferon, interferon-gamma, IFN-gamma) is a water-soluble dimeric cytokine, and the monomer of Interferon gamma protein is a core formed by six alpha helices and a fragment sequence extending and spreading in a C terminal region; insulin-like growth factor binding proteins are regulatory proteins that bind to insulin-like growth factors (IGFs), insulin-like growth factor binding protein-3 (IGFBP-3) belongs to the high affinity class of the superfamily, and the polypeptide chain consists of 264 amino acids (containing 18 Cys); E-Cadherin (E-Cadherin), also known as Uvomorulin, L-CAM or Cell-CAM120/80, is a single-chain glycoprotein, the calcium-dependent Cell adhesion family (Ca 2+ dependent Cell adhesion molecule), an adhesion molecule that mediates intercellular mutual aggregation in the presence of Ca ²⁺ When present, can resist hydrolysis by proteases; macrophage inflammatory protein-1 delta (MIP-1 delta) belongs to the C-C chemokine family.

Wherein FC1 is E of neuromyelitis pedigree disease-fold difference in E-Cadherin expression levels of Cadherin and multiple sclerosis; FC2 is the difference multiple of IGFBP-3 expression of neuromyelitis pedigree disease and IGFBP-3 expression of multiple sclerosis; FC3 is the difference multiple of GCP-2 expression of neuromyelitis pedigree diseases and GCP-2 expression of multiple sclerosis; FC4 is the fold difference between INF gamma expression of neuromyelitis pedigree disease and INF gamma expression of multiple sclerosis; FC5 is the multiple difference of IL-2 expression of neuromyelitis pedigree diseases and IL-2 expression of multiple sclerosis; FC6 is the multiple, log, of the difference between the expression levels of MCP-1 of neuromyelitis spectrum diseases and MCP-1 of multiple sclerosis ₂ |FC1|>0.263；log ₂ |FC2|>0.263；log ₂ |FC3|>0.263；log ₂ |FC4|>0.263；log ₂ |FC5|>0.263；log ₂ |FC6|>0.263。

FC7 is the fold difference between SDF-1a of neuromyelitis spectrum disease and SDF-1a expression of multiple sclerosis; FC8 is the multiple, log, of the difference between MIP-1delta of neuromyelitis spectrum diseases and MIP-1delta expression of multiple sclerosis ₂ |FC7|>0.263；log ₂ |FC8|>0.263。

Specifically, the E-Cadherin blood content of the patient with neuromyelitis spectrum disease is 0-5056pg/ml; IGFBP-3 blood content of neuromyelitis pedigree disease patient is 0-136736pg/ml; the GCP-2 blood content of the patient with neuromyelitis pedigree disease is 0-81pg/ml; the blood content of INF gamma of patients with neuromyelitis pedigree disease is 0-131pg/ml; IL-2 blood levels of 0-154pg/ml in patients with neuromyelitis spectrum disease; MCP-1 blood content of patients with neuromyelitis spectrum disease is 0-49pg/ml.

Specifically, the E-Cadherin blood content of the multiple sclerosis patient is more than 5056pg/ml; IGFBP-3 blood in multiple sclerosis patients is greater than 136736pg/ml; the GCP-2 blood of the multiple sclerosis patients is more than 81pg/ml; the INF gamma blood content of the patients with multiple sclerosis is more than 131pg/ml; IL-2 blood in multiple sclerosis patients is greater than 154pg/ml; MCP-1 blood of multiple sclerosis patients is greater than 49pg/ml.

Specifically, the SDF-1a blood content of the patient with neuromyelitis neurona lineage disease is more than 1.65pg/ml; the MIP-1delta blood content of the patient with the neuromyelitis pedigree disease is 0-2175pg/ml.

Specifically, the blood level of SDF-1a in patients with multiple sclerosis is 0-6.9pg/ml; the delta blood of MIP-1 in patients with multiple sclerosis is more than 2175pg/ml.

More preferably, the E-Cadherin blood content of the patient with neuromyelitis spectrum disease is 2368-5056pg/ml; the IGFBP-3 blood content of the patient with the neuromyelitis pedigree disease is 91772-136736pg/ml; the GCP-2 blood content of the patient with neuromyelitis pedigree disease is 7-81pg/ml; the INF gamma blood content of the patient with neuromyelitis pedigree disease is 47-131pg/ml; IL-2 blood content of the patient with neuromyelitis spectrum disease is 50-154pg/ml; MCP-1 blood content of patients with neuromyelitis spectrum disease is 23-49pg/ml.

More preferably, the blood content of E-Cadherin in a patient with multiple sclerosis is greater than 5752pg/ml; IGFBP-3 blood in multiple sclerosis patients is greater than 137219pg/ml; the GCP-2 blood of the multiple sclerosis patients is more than 118pg/ml; the INF gamma blood content of the patients with multiple sclerosis is more than 137pg/ml; IL-2 blood in patients with multiple sclerosis is greater than 217pg/ml; MCP-1 blood of multiple sclerosis patients is greater than 67pg/ml.

More preferably, the blood of SDF-1a of a patient with multiple sclerosis is 0-6.9pg/ml; MIP-1delta blood of a patient with multiple sclerosis is more than 3219pg/ml; preferably, the blood level of SDF-1a in a patient with neuromyelitis spectrum disease is 1.65 to 21.19pg/ml.

Preferably, the test sample of neuromyelitis spectrum disease is serum or plasma, and the test sample of multiple sclerosis is serum or plasma.

In a third aspect, the present application provides a protein chip for diagnosing cns demyelinating diseases, which comprises a first antibody for a biomarker, a protein chip for diagnosing cns demyelinating diseases, comprising the first antibody for the biomarker set, or the first antibody for the biomarker set used and a carrier, the first antibody for each biomarker being sequentially immobilized on the carrier, and the first antibody for each biomarker specifically binding to the corresponding biomarker.

Wherein, the carrier can be a glass slide or other substances used for protein chips.

It should be noted that, in order to quantitatively detect multiple markers of demyelinating diseases of the central nervous system, the protein chip and the protein chip kit which can simultaneously quantitatively detect multiple markers provided by the invention can be the conventional protein chip and protein chip kit. The protein chip comprises a glass slide on which a first antibody of a biomarker is fixed and the first antibody of a biomarker group, wherein the biomarker comprises stromal cell derived factor-1 (SDF-1 a), granulocyte chemotactic protein 2 (GCP-2), monocyte chemotactic protein-1 (MCP-1), interleukin-2 (IL-2), gamma-interferon (INF gamma), insulin-like growth factor binding protein-3 (IGFBP-3), E-Cadherin (E-Cadherin) and macrophage inflammatory protein-1 delta (MIP-1 delta).

When the kit is used, the first antibodies of the biomarker groups are independently fixed on the glass slide to form a plurality of independent recognition sites, and the first antibodies of the biomarker groups can be monoclonal antibodies or polyclonal antibodies. The tested sample is used as antigen, namely, matrix cell derived factor-1 (SDF-1 a), granulocyte chemotactic protein 2 (GCP-2), monocyte chemotactic protein-1 (MCP-1), interleukin-2 (IL-2), gamma-interferon (INF gamma), insulin-like growth factor binding protein-3 (IGFBP-3), E-Cadherin (E-Cadherin) and macrophage inflammatory protein-1 delta (MIP-1 delta) are used as antigens, the biomarker of the tested sample is used as antigen to have antibody-antigen reaction with a first antibody of each biomarker of the protein chip, and then whether a substance capable of having antibody-antigen reaction with the specific biomarker group exists in the tested sample can be detected through a multiple sandwich ELISA method.

Specifically, a single concentration of the first antibody for each biomarker may be immobilized on the independent recognition site of the slide, and one or more concentrations of the first antibody for each biomarker may be immobilized on each independent recognition site of the slide.

Specifically, the fixed content of the first antibody of the biomarker is 0.02 ng-2 ng.

Specifically, the protein chip technology is adopted, 10 markers of the demyelinating disease of the central nervous system can be detected, the defects of complex operation, single detection index, need of expensive instruments, low sensitivity and the like in the prior art are overcome, and the method has the advantages of low price, convenience, sensitivity, accuracy, high flux, small sample consumption, capability of being popularized and scaled in a common laboratory and the like.

In a third aspect, the present application provides a protein chip kit for diagnosing a demyelinating disease of the central nervous system, comprising said protein chip and a plurality of detection reagents, each of said detection reagents specifically binding to a corresponding one of each of said biomarkers.

Preferably, the detection reagent comprises a second antibody corresponding to each of the biomarkers and a detectable label component, the second antibody corresponding to each of the biomarkers being conjugated to the detectable label component. The biomarker of the detected sample is specifically combined with the first antibody of the biomarker, and the biomarker of the detected sample is specifically combined with the second antibody of the biomarker, so that a sandwich structure is formed, and the biomarker of the detected sample is qualitatively and quantitatively detected through the detectable labeling component on the detection reagent.

Preferably, the detectable label element comprises one of an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance or a radioactive substance.

In a third aspect, the present application provides an ELISA kit for diagnosing a central nervous system demyelinating disease, comprising a first antibody for each of the biomarkers and reagents for an enzyme-linked immunosorbent assay.

In a preferred embodiment of the present invention, the detection reagent is labeled with biotin. Preferably, the detection reagent further comprises streptavidin labeled with recognition biotin, and the streptavidin is labeled with fluorescent dye, and the fluorescent dye is Cy3 or fluorescent dye with similar absorption wavelength.

In a preferred embodiment of the present invention, the detection reagent further comprises a protein standard, which is a mixture having step concentrations of the several biomarker panels. The different biomarker groups are mixed together according to a certain quantitative ratio, and are dried by a freeze-drying method after being subpackaged. Each protein standard is separately reacted with a first antibody reaction antigen-antibody reaction of the corresponding biomarker of the protein chip, and a standard curve of protein fluorescence is constructed so as to quantitatively and qualitatively detect the biomarker group in the detected sample.

The present invention also provides a method for preparing a protein chip kit for diagnosing a demyelinating disease of the central nervous system, comprising the step of immobilizing a first antibody to a biomarker on a glass slide, the step comprising:

(1) 100-1000 picoliters of PBS buffer (containing 0.01-10g/100ml bovine albumin) containing 0.02-2ng of the primary antibody of the biomarker was spotted on the slide;

(2) And standing the spotted slide glass at room temperature overnight, and storing at 2-8 ℃ for later use.

Wherein, the sample application operation is completed by adopting a full-automatic sample application instrument in the step (1), and the first antibody of each biomarker is arranged on the glass slide in a chip lattice manner.

Because the method of the invention adopts the excellent characteristics of the sample solution, and the difference between the use method of the protein chip kit of the invention in detecting the biomarker group and the prior art is combined, the step of fixing the first antibody of the biomarker on the glass slide in the method of the invention is greatly simplified, and the operation step of sealing the effective components on the glass slide after sample application commonly adopted in the prior art is not needed.

In one embodiment of the present invention, the spotting process of step (1) is performed by using a fully automated spotting apparatus manufactured by Perkin Elmer (Perkin Elmer) of the United states of America. The first antibody dot matrix of each biomarker is arranged on the glass slide, and in the specific operation process, the arrangement of the first antibodies of each biomarker can be adjusted according to the experimental design requirements, and the full-automatic sample spotting instrument is controlled according to the arrangement arrays of different protein chips to prepare the required intermediate product.

The protein chip kit of the invention realizes the joint detection of multiple samples and multiple indexes, overcomes the defects of complex operation, single detection index, need of expensive instruments, low sensitivity and the like in the prior art, and has the advantages of low price, convenience, sensitivity, accuracy, high flux, small sample consumption, capability of being popularized and scaled in a common laboratory and the like. The protein chip kit can be used for population census and is beneficial to establishing baseline data. Monitoring changes in the biomarker panels of the invention facilitates early detection of lesions and corresponding measures. Timely discovery and early treatment can greatly improve the cure rate and reduce the medical expense.

The application adopts a high-flux protein chip to detect the protein of a biomarker group in the serum of the central nervous system demyelinating disease patient with AQP4 antibody negativity, and the difference of the biomarker group is used for assisting diagnosis and identification of a multiple sclerosis patient or a neuromyelitis pedigree disease patient, and has important significance for researching pathogenesis and treatment of the two central nervous system demyelinating diseases.

According to the technical scheme, the method has the following advantages:

the application provides an application of a biomarker group in preparing a product for diagnosing demyelinating diseases of the central nervous system, under the condition that a water channel protein 4 antibody in serum is negative, the expression quantity of 6 proteins of GCP-2, MCP-1, IL-2, INF gamma, IGFBP-3 and E-Cadherin in the serum of a patient with neuromyelitis spectrum disease and a patient with multiple sclerosis can be reliably, accurately and quickly diagnosed and identified, and the expression quantity of the 6 proteins of GCP-2, MCP-1, IL-2, INF gamma, IGFBP-3 and E-Cadherin can be obviously different.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Figure 1 is a volcanic plot of the biomarkers histones of the present application;

FIG. 2 is a graph of a cluster analysis of the biomarker panel of the present application;

FIG. 3 is a GO functional analysis graph of a biomarker panel of the present application;

figure 4 is a graph of a KEGG signal pathway analysis for a biomarker panel of the present application.

Detailed Description

The invention provides application of a biomarker group and a protein chip, a protein chip kit and an ELISA kit thereof, wherein the biomarker group is applied to preparation of products for diagnosing central nervous system demyelinating diseases, and is used for solving the technical defect that no reliable products for diagnosing Multiple Sclerosis (MS) and neuromyelitis spectrum diseases (NMOSD) under the negative of aquaporin 4 antibodies exist in the current clinic.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Wherein, the reagents and solvents used in the following examples are all commercially available or self-made;

the invention discovers that E-Cadherin (E-Cadherin), insulin-like growth factor binding protein-3 (IGFBP-3), granulocyte chemotactic protein 2 (GCP-2), gamma-interferon (INF gamma), interleukin-2 (IL-2) and monocyte chemotactic protein-1 (MCP-1) have specific proportion relation in neuromyelitis pedigree diseases negative to aquaporin 4 antibody and multiple sclerosis negative to aquaporin 4 antibody, and particularly, FC1 is the multiple difference of the E-Cadherin expression quantity of the neuromyelitis pedigree diseases and the E-Cadherin expression quantity of the multiple sclerosis; FC2 is the difference multiple of IGFBP-3 expression of neuromyelitis pedigree disease and IGFBP-3 expression of multiple sclerosis; FC3 is neuromyelitisFold difference in GCP-2 expression of lineage disease and multiple sclerosis; FC4 is the fold difference between INF gamma expression of neuromyelitis spectrum diseases and INF gamma expression of multiple sclerosis; FC5 is the multiple difference of IL-2 expression of neuromyelitis pedigree diseases and IL-2 expression of multiple sclerosis; FC6 is the multiple, log, of the difference between the expression levels of MCP-1 of neuromyelitis spectrum diseases and MCP-1 of multiple sclerosis ₂ |FC1|>0.263；log ₂ |FC2|>0.263；log ₂ |FC3|>0.263；log ₂ |FC4|>0.263；log ₂ |FC5|>0.263；log ₂ |FC6|>0.263。

The biomarker panel of the present application further includes that stromal cell derived factor-1 (SDF-1 a), macrophage inflammatory protein-1 delta (MIP-1 delta) can form a new biomarker panel with the above six biomarkers, wherein, FC7 is the difference multiple of the expression quantity of SDF-1a of neuromyelitis neuronic lineage diseases and SDF-1a of multiple sclerosis; FC8 is the difference multiple, log, of MIP-1delta expression of neuromyelitis pedigree diseases and MIP-1delta expression of multiple sclerosis ₂ |FC7|>0.263；log ₂ |FC8|>0.263。

Example 1

The embodiment of the invention relates to a serum sample preparation process, which comprises the following specific steps:

1. 12 sera of multiple sclerosis patients and 9 sera of aquaporin 4 antibody (AQP 4 Ab-systemic NMOSD) patients were collected, and the sera were referred to as MS group and nmo.neg group. Differential diagnosis of Multiple Sclerosis (MS) patients versus neuromyelitis spectrum disease (NMO) patients all serum samples were processed according to standard procedures as shown in table 1: the whole blood was collected in a vacutainer serum tube, allowed to stand at room temperature for 30 minutes, and then centrifuged at 2000rpm in a 4 ℃ centrifuge for 15 minutes. The supernatant was collected and frozen at-80 ℃ until use. All clinical serum samples used in this example were approved by the ethical review committee of human subjects at the second hospital affiliated with Guangzhou medical university (China, guangzhou). Hereinafter, the AQP4 antibody-negative multiple sclerosis group was referred to as the MS group, and the AQP4 antibody-negative neuromyelitis spectrum disease group was referred to as the nmo.

TABLE 1 differential diagnosis of Multiple Sclerosis (MS) and neuromyelitis lineages disease (NMO)

Example 2

The embodiment of the invention is to detect the protein expression level of the serum of the AQP4 antibody negative multiple sclerosis patient and the AQP4 antibody negative neuromyelitis pedigree disease patient in example 1, and comprises the following specific steps:

1. protein chip technology assay detection of the expression levels of 200 proteins in serum of AQP4 antibody negative multiple sclerosis patients and AQP4 antibody negative neuromyelitis spectrum disease patients of example 1: protein levels in serum were measured using sandwich-based antibody microarrays. Protein screening was performed using a RayBiotech Human Cytokine Antibody Array Q4000 (QAH-CAA-4000, rayBiotech, inc., norcross, GA), which contains a combination of 5 non-overlapping arrays to quantitatively measure 200 Human cytokines.

The preparation steps of the protein chip of the application are as follows:

1. complete drying of the slide chip: taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1-2 hours.

2. Gradient dilution of protein standards

2.1 Add 500. Mu.l of PBS sample dilution to a small tube of standard mix (standard of 200 human cytokines) and re-dissolve the standard. Before opening the vial, the vial is rapidly centrifuged and gently pipetted up and down to dissolve the powder, and the vial is labeled Std1.

2.2, label 6 clean centrifuge tubes as Std2, std3 to Std7, respectively, and add 200. Mu.l of sample diluent to each vial.

2.3, 100. Mu.l of Std1 was added to Std2 and mixed gently, and then 100. Mu.l was extracted from Std2 and added to Std3, and thus diluted to Std7 with gradient.

2.4, draw 100. Mu.l of the sample dilution into another new centrifuge tube, labeled CNTRL, as a negative control.

3. Chip operation process

3.1, 100. Mu.l of sample diluent is added to each well, incubated on a shaker at room temperature for 30 minutes, and the quantitative protein chip is sealed.

3.2 the sample dilution from each well was aspirated, 100. Mu.l of standard (200 human cytokine standards) and sample added to the wells and incubated on a shaker overnight at 4 ℃. The samples were sera naturally precipitated after venous blood collection (sera of AQP4 antibody-negative multiple sclerosis patients and AQP4 antibody-negative neuromyelitis spectrum disease patients), and before use, the samples were diluted with a diluent 1:1.

3.3, cleaning: and (3) drawing out the standard solution or the sample from each well, washing the IX lotion I for 5 times, shaking the IX lotion I in a shaking table at room temperature for 5min each time, drawing out 150 mu l of IX lotion I from each well, cleaning the lotion I for each time, and diluting the lotion I by 20 times with deionized water. And (3) pumping out the IX lotion I in each hole, adding the IX lotion II, washing for 2 times, shaking by a shaking table at room temperature for 5min every time, pumping out 150 mu l of IX lotion II in each hole, and diluting 20 multiplied by lotion II by deionized water. IX Wash I and IX Wash II are currently conventional protein chip washes.

3.4, incubation of the detection antibody mixed solution: and centrifuging the detection antibody mixed solution small tube, adding 1.4ml of sample diluent, uniformly mixing, and quickly centrifuging again. Add 80. Mu.l of detection antibody to each well and incubate for 2 hours on a shaker at room temperature.

3.5, cleaning: and (3) extracting the detection antibody in each hole, washing the IX lotion I for 5 times, shaking the IX lotion I for 5min at room temperature for each time, extracting 150 mu l of I X lotion I for each hole, cleaning the lotion for each time, then adding I X lotion II for cleaning for 2 times, shaking the IX lotion II for 5min at room temperature for each hole, and extracting the lotion for each time.

3.6 incubation of Cy 3-streptavidin: the Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and centrifuged quickly again. Add 80ul Cy 3-streptavidin to each well, wrap the slide with aluminum foil paper and incubate in the dark for 1 hour on a shaker at room temperature. Cy 3-labeled streptavidin is a conventional protein chip detection reagent.

3.7, cleaning: and (3) extracting Cy 3-streptavidin from each hole, washing the IX lotion I for 5 times, shaking the IX lotion I for 5min at room temperature every time, extracting the lotion I for 150 mu l of IX lotion I for each hole, washing the IX lotion I for 2 times, adding the IX lotion II for washing the IX lotion I for 5min at room temperature every time, shaking the IX lotion II for 150 mu l of IX lotion I for each hole, and extracting the lotion for each washing.

3.8 fluorescence detection

1) The slide frame was removed, taking care not to touch the antibody-printed side of the slide by hand.

2) Placing the slide in a slide cleaning tube, adding about 30ml of IX washing solution I to completely cover the slide, shaking for 15min on a room temperature shaking table, discarding the IX washing solution I, adding about 30ml of IX washing solution II, and shaking for 5min on the room temperature shaking table.

3) The residual wash solution of the slide was removed. The slides were placed in slide wash/dry tubes without lid and centrifuged at 1000rpm for 3min.

4) The signal is scanned using a laser scanner, such as Innopsys, using either Cy3 or green channels (excitation frequency of 532 nm).

3.9, data extraction of the chip and data analysis with analysis software.

1) The fluorescence values of the biochip were read with Mapix software. The microarray parameters of the chip were 14 (rows) × 12 (columns), and the dot diameters were 140 μm.

2) The value selected after reading is the Median reading (F532 media-LocalBack) with the surrounding background removed. The standard curve for each recombinant protein was made using the specific quantitative chip computing software QAH-CAA-Q4000.

The normalization of the data was performed using the same two positive control spots on each chip (noted as POSs and POS 2) as a reference frame before calculating the protein (8 biomarkers of the present application) concentration for different targets for different samples (12 sera from multiple sclerosis patients, 9 sera from AQP4 antibody negative neuromyelitis spectrum disease patients). The signal values of the two positive controls differ by a factor of approximately 4. The positive control value POS = (POSl +4 × POS 2)/2 was calculated for each chip before normalization. This value is then used to normalize all data: correction = original value x (sample mean POS)/POS.

Example 3

The embodiment of the application provides the protein data conversion and graph generation of the embodiment 2, and the specific steps are as follows:

after the concentrations of the 8 biomarkers of the application are calculated according to specific software of the protein chip, data are converted before data analysis, and the conversion formula is as follows: log (log) ₂ (X + 1). And X is the concentration of different proteins corresponding to each sample. The transformed data is used for subsequent data analysis, including volcano graph analysis and heat map cluster analysis. All data were generated directly in RStudio and the pictures were then integrated using illuminator CC5 (Adobe, san Diego, CA).

Example 4

The present application provides the differential protein expression profiling of example 2, comprising the following steps:

to identify significantly varying proteins (based on Log) in different serum samples ₂ Fold difference) which is the ratio of the amount of protein of a patient with neuromyelitis spectrum disease to the amount of protein of a patient with multiple sclerosis. This example calculates False-Discovery Rates (FDR) for each protein by non-parametric p-value analysis using the p.adjust function in R studio. Differentially Expressed Proteins (DEPs) between different serum samples were defined as having FDRs<0.2 and absolute log ₂ Multiple of difference>0.263 protein, all proteins FDR and log using ggplot function in R studio ₂ The fold difference was plotted in the volcano, the proteins with differences in expression are shown as blue dots, and the proteins with no differences in expression are shown as red dots.

As a result, FC1 is a fold difference between the expression levels of E-Cadherin of neuromyelitis spectrum diseases and E-Cadherin of multiple sclerosis; FC2 is IGFBP-3 for neuromyelitis spectrum diseases and IGFBP-3 for multiple sclerosisFold difference in expression level; FC3 is the difference multiple of GCP-2 expression of neuromyelitis pedigree diseases and GCP-2 expression of multiple sclerosis; FC4 is the fold difference between INF gamma expression of neuromyelitis pedigree disease and INF gamma expression of multiple sclerosis; FC5 is the multiple difference of IL-2 expression of neuromyelitis pedigree diseases and IL-2 expression of multiple sclerosis; FC6 is the difference multiple of MCP-1 expression of neuromyelitis pedigree diseases and MCP-1 expression of multiple sclerosis; log (log) ₂ |FC1|>0.263；log ₂ |FC2|>0.263；log ₂ |FC3|>0.263；log ₂ |FC4|>0.263；log ₂ |FC5|>0.263；log ₂ |FC6|>0.263; FC7 is the fold difference between SDF-1a of neuromyelitis spectrum disease and SDF-1a expression of multiple sclerosis; FC8 is the difference multiple, log, of MIP-1delta expression of neuromyelitis pedigree diseases and MIP-1delta expression of multiple sclerosis ₂ |FC7|>0.263；log ₂ |FC8|>0.263. Wherein, the multiple sclerosis is MS group, and the neuromyelitis pedigree disease is NMO.

Example 5

The embodiment of the application provides a functional annotation and a protein interaction network for the differential protein of the embodiment 2, and the specific steps are as follows:

protein functional annotation analysis, including GO function and KEGG signal pathway analysis, was performed by the "clusterProfiler" and "DOSE" analysis packages in R speech.

It was found in the examples of the present application that 8 proteins of the present application, namely, stromal cell derived factor-1 (SDF-1 a), granulocyte chemotactic protein 2 (GCP-2), monocyte chemotactic protein-1 (MCP-1), interleukin-2 (IL-2), gamma-interferon (INF gamma), insulin-like growth factor binding protein-3 (IGFBP-3), E-Cadherin (E-Cadherin), and macrophage inflammatory protein-1 delta (MIP-1 delta), were significantly different between the neuromyelitis lineage disease group negative to aquaporin 4 antibody and the multiple sclerosis group negative to aquaporin 4 antibody, and FIG. 1 is a volcano graph of the biomarker histones of the present application showing the neuromyelitis disease lineage group negative to aquaporin 4 antibody and the multiple sclerosis group negative to aquaporin 4 antibody in the volcano graphLog of significance and fold change in proteins of the scleroderma group ₂ Distribution after transformation. Of the 200 proteins analyzed, 8 protein levels of the present application were significantly different between MS group and nmo.neg group sera (FDR)<0.2 and fold change>1.2 ); fig. 2 is a graph of a cluster analysis of the biomarker panel of the present application, showing that, based on the expression of the 10 proteins of the present application between the two groups, the 8 differential proteins of the present application were subjected to a thermographic cluster analysis, and then almost all MS samples were clustered together, and all nmo.neg group samples were clustered together, and the clustering heatmap clearly shows the expression of the 8 proteins of the present application in the two groups of serum samples; fig. 3 is a GO functional analysis diagram of the biomarker panel of the present application, and 8 proteins of the present application were found after GO functional analysis: the 5 proteins E-cadherin (CDH 1), SDF-1 (CXCL 12), IFNG, IL2 and MCP-1 (CCL 2) are involved in regulating cell-cell adhesion; 4 proteins, GCP-2, SDF-1a, MCP-1 and MIP-1, are associated with bone marrow leukocyte dissociation (myeloid leukocyte migration), leukocyte chemotaxis (leukocytic chemotaxis) and cell chemotaxis (cell chemotaxis); 4 proteins, GCP-2, SDF-1a, IL-2 and MIP-1, are involved in the positive regulation of response to external stimuli (positive regulation of response to external stimuli); SDF-1a, IFN g, IL-2, MCP-1, 4 proteins are involved in regulating the cell-cell adhesion between white blood cells (regulation of leukocyte cell-cell attachment); 3 proteins SDF-1a, MCP-1 and MIP1-d are related to monocyte chemotaxis (monocyte chemotaxis) and monocyte migration (monocyte cell migration); four proteins, GCP-2, SDF-1a, MCP-1 and MIP-1d, are involved in chemokine-mediated signaling pathways (chemokine-mediated signaling pathways); the two proteins SDF-1 and MCP-1 are involved in positive regulation of calcium ion influx. Fig. 4 is a KEGG signal pathway analysis graph of the biomarker panel of the present application, which shows that after performing the KEGG signal pathway analysis on the biomarker panel of the present application: 6 proteins of GCP-2, SDF-1, IFN g, IL-2, MCP-1 and MIP-1 participate in the interaction of receptors among cytokines (Cytokine-Cytokine receptor interaction); four proteins, GCP-2, SDF-1, IFN g and MCP-1, are involved in Rheumatoid arthritis (Rheumatoid arthritis)) A signal path; the IFNg and IL-2 proteins participate in a JAK-STAT signal pathway; four proteins, GCP-2, SDF-1, MCP-1 and MIP-1, are involved in chemokine signaling pathway (chemokine signaling pathway); three proteins, namely GCP-2, IFN g and MCP-1, participate in an IL-17 signal path; three proteins, IFG g, IL-2 and MCP-1, are involved in the ancylostomiasis signaling pathway (Chagas disease); IFN g and MCP-1 are involved in Fluid shear stress and the atherosclerotic signaling pathway; IFN g and IL-2 are involved in signal pathways such as Allograft rejection (Allograft rejection), graft-versus-host disease (Graft-cover-host disease), type I diabetes (Type I diabetes mellitis), and the like; SDF-1 and IL-2 are involved in the intestinal immune network for IgA production; both IFN g and MCP-1 proteins are involved in the signaling pathway for malarial diseases.

When the biomarker panel of the present application is used to diagnose neuromyelitis spectrum diseases negative to aquaporin 4 antibody and multiple sclerosis negative to aquaporin 4 antibody, the steps are as follows:

the biomarker panel is used for indicating whether patients suffer from neuromyelitis pedigree diseases or multiple sclerosis in a mixed mode, the patients have common clinical characteristics of the neuromyelitis pedigree diseases and the multiple sclerosis, aquaporin 4 antibodies are negative patients, the expression levels of the biomarker panels of the patients are obtained, whether the expression levels of the biomarker panels are in a preset biomarker panel range of the neuromyelitis pedigree diseases negative to the aquaporin 4 antibodies or not are in a preset biomarker panel range of the multiple sclerosis negative to the aquaporin 4 antibodies is judged, and a preliminary diagnosis conclusion is obtained according to the judgment result.

Example 6

The embodiments of the present application provide a test for verifying the feasibility of a biomarker panel, comprising the steps of:

1. serum from 6 patients with established multiple sclerosis (Nos. 1 to 6 of Table 2) and serum from 6 patients with neuromyelitis spectrum disease (AQP 4 Ab-serogenic NMOSD) negative to the established aquaporin 4 antibody (AQP 4 antibody) (Nos. 7 to 12 of Table 2) were collected, and blood was drawn from the above 12 patients, and the protein content of E-Cadherin (E-Cadherin), insulin-like growth factor binding protein-3 (IGFBP-3), granulocyte chemotactic protein 2 (GCP-2), gamma-interferon (INF gamma), interleukin-2 (IL-2) and monocyte chemotactic protein-1 (MCP-1), stromal cell-derived factor-1 (SDF-1 a) and macrophage inflammatory protein-1 delta (MIP-1 delta) in the blood was examined, and the results are shown in Table 2.

2. Judging whether the protein content in the serum of 12 patients meets the following requirements: the E-Cadherin blood content of the patients with the multiple sclerosis is more than 5056pg/ml; IGFBP-3 blood from multiple sclerosis patients is greater than 136736pg/ml; the GCP-2 blood of the multiple sclerosis patients is more than 81pg/ml; the INF gamma blood content of the patients with multiple sclerosis is more than 131pg/ml; IL-2 blood in patients with multiple sclerosis is greater than 154pg/ml; MCP-1 blood of a patient with multiple sclerosis is greater than 49pg/ml; SDF-1a blood of multiple sclerosis patients is 0-6.9pg/ml; MIP-1delta blood of greater than 2175pg/ml for multiple sclerosis patients; preferably, MIP-1delta blood in a patient with multiple sclerosis is greater than 3219pg/ml.

The E-Cadherin blood content of the patient with the neuromyelitis spectrum disease is 0-5056pg/ml; IGFBP-3 blood content of neuromyelitis pedigree disease patient is 0-136736pg/ml; the GCP-2 blood content of the patient with neuromyelitis spectrum diseases is 0-81pg/ml; the blood content of INF gamma of patients with neuromyelitis pedigree disease is 0-131pg/ml; IL-2 blood levels of 0-154pg/ml in patients with neuromyelitis spectrum disease; MCP-1 blood content of patients with neuromyelitis pedigree diseases is 0-49pg/ml; the SDF-1a blood content of the patient with the neuromyelitis spectrum disease is more than 1.65pg/ml; the delta blood content of MIP-1 of a patient with neuromyelitis spectrum disease is 0-2175pg/ml.

2. As can be seen from Table 2, samples Nos. 6 and 12 had protein concentrations that did not match the protein range of the biomarker panel (including E-Cadherin, IGFBP-3, GCP-2, SDF-1a, INF gamma, IL-2, MCP-1 and MIP-1 delta). The biomarker panel of the present application example has high accuracy in diagnosing multiple sclerosis and neuromyelitis spectrum diseases negative to aquaporin 4 antibody (AQP 4 antibody), and the accuracy in diagnosing multiple sclerosis patients is 5/6 × 100% =83%; the accuracy of diagnosing the patients with neuromyelitis pedigree diseases negative to the aquaporin 4 antibody (AQP 4 antibody) reaches 5/6 × 100% =83%.

TABLE 2

The terms "first," "second," "third," "fourth," and the like in the description of the application and the above-described figures, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the application described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. Use of a biomarker panel consisting of several biomarkers including stromal cell derived factor-1, granulocyte chemotactic protein 2, monocyte chemotactic protein-1, interleukin-2, gamma-interferon, insulin-like growth factor binding protein-3, E-cadherin, and macrophage inflammatory protein-1 δ for the preparation of a product for the diagnosis of a demyelinating disease of the central nervous system; the central nervous system demyelinating diseases include neuromyelitis spectrum diseases negative to aquaporin 4 antibody, and multiple sclerosis negative to aquaporin 4 antibody;

the tested sample of the neuromyelitis spectrum disease is serum or plasma, and the tested sample of the multiple sclerosis is serum or plasma.