Chronic inflammatory demyelinating multiple nerve root neuropathy antibody detection material and preparation method thereof

Abstract

The invention relates to an antibody detection material for chronic inflammatory demyelinating multiple nerve root neuropathy and a preparation method of the antibody detection material. The antibody detection material for the chronic inflammatory demyelinating multiple nerve root neuropathy comprises modified silicon dioxide, a positive control, a negative control, a working solution and an auxiliary agent, the nano silicon dioxide is modified by using the Chinese yam polysaccharide, and a large amount of hydroxyl groups on the surface of the Chinese yam polysaccharide and hydroxyl groups on the surface of silicon dioxide can form hydrogen bond connection and coat the surface of silicon dioxide, so that the dispersity of the modified silicon dioxide in components is improved; and the specific surface area of the modified silicon dioxide is increased, so that the adsorption capacity to cells is remarkably improved, the binding efficiency of an antibody and an antigen is enhanced, and the sensitivity of antibody detection is improved. Secondly, the antibody detection material can realize simultaneous detection of multiple antigens, so that the problem of insufficient detection coverage of a single antigen in a traditional method is solved.

Description

Technical Field

[0001] The invention relates to the technical field of cell detection, in particular to an antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy and a preparation method thereof. Background Art

[0002] Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare chronic neuropathy caused by the autoimmune system attacking the myelin sheath of peripheral nerves. It usually manifests as gradually worsening or recurrent weakness and paresthesia in the limbs, and even leads to severe limb dysfunction. Its pathological features mainly include demyelinating changes in nerve roots and peripheral nerves, accompanied by axonal degeneration. Although the disease is relatively rare, due to its similarities in clinical manifestations with other neurological diseases, the diagnosis process is complicated, which brings certain challenges to the early diagnosis and treatment of patients.

[0003] The clinical diagnosis of CIDP currently relies on traditional methods such as medical history, physical signs, neuroelectrophysiological examination, cerebrospinal fluid analysis, and nerve biopsy. However, each of these detection methods has its limitations. Electrophysiological examination can indicate demyelination characteristics, but its sensitivity is not high, and it requires experienced operators to correctly interpret the results. Although protein cell separation in cerebrospinal fluid examination is common in CIDP, it is not a specific indicator, and similar phenomena may also occur in other inflammatory neuropathies. Nerve biopsy is an invasive examination. Although it can provide intuitive pathological evidence, its clinical application is relatively limited due to its complexity and risk. Therefore, finding reliable and non-invasive biomarkers for CIDP has become a research hotspot, especially the detection of autoantibodies against specific antigens of the nervous system.

[0004] In the prior art, the detection of CIDP-related antibodies usually adopts immunological methods, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), etc. These methods rely on the specific binding of antigens and antibodies and have certain sensitivity and specificity. However, these traditional methods have several limitations, which restrict their wide clinical application. First, ELISA is a common antibody detection method. Although the operation is relatively simple and the sensitivity is high, the preparation of antigens is relatively complicated, and the structural stability and activity of antigens are easily affected during the detection process, thereby affecting the accuracy of the test results. Secondly, although immunoblotting (Western Blot) can detect specific proteins, the operation process is cumbersome, time-consuming, and the quantitative accuracy is low, which is easily affected by fluctuations in experimental conditions. In addition, these methods can usually only detect single target antibodies, and it is difficult to screen multiple related antigens in one experiment, resulting in low detection efficiency.

[0005] Another problem is that most of the antigens in the existing antibody detection system are artificially synthesized or recombinant proteins, and their structures may be somewhat different from natural antigens, which affects the recognition of antibodies. In addition, the stability and activity of cells are difficult to maintain for a long time during the detection process, resulting in a decrease in the reliability of the test results. These problems are particularly prominent in clinical applications, which makes it difficult for doctors to determine the cause of the disease and increases the risk of misdiagnosis.

[0006] Therefore, in view of the problems in the existing technology such as complex antigen preparation, insufficient detection sensitivity and specificity, and limited coverage of multi-antigen detection, an improved antibody detection technology is urgently needed to improve the efficiency, accuracy and stability of detection. Summary of the invention

[0007] Based on this, the present invention provides a more efficient, sensitive and stable chronic inflammatory demyelinating polyradiculoneuropathy antibody detection material, which can provide strong technical support for the early diagnosis, typing and precise treatment of the disease.

[0008] One object of the present invention is to provide an antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy, wherein the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy comprises: modified silica, a positive control, a negative control, a working solution, and an adjuvant;

[0009] Wherein, the modified silica is adsorbed with Hela cells containing antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG;

[0010] The modified silicon dioxide is nano silicon dioxide modified with yam polysaccharide.

[0011] Furthermore, the particle size of the nano-silicon dioxide is 50-80nm.

[0012] Furthermore, the working fluid is goat serum or bovine serum.

[0013] Furthermore, the auxiliary agent is selected from one or more of a sealing agent, a fluorescent secondary antibody, and a PBS buffer.

[0014] Furthermore, the fluorescent secondary antibody is selected from one or more of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and tetraethylrhodamine.

[0015] Furthermore, the sealing medium is glycerol.

[0016] Furthermore, the negative control is 8-12 wt % goat serum.

[0017] Furthermore, the positive control is a recombinant antigen containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins.

[0018] Specifically, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a type of immune-mediated motor and sensory peripheral neuropathy with a chronic progressive (>2 months) or remission-relapse course, often accompanied by cerebrospinal fluid protein-cell separation, electrophysiological manifestations of slowed peripheral nerve conduction velocity, conduction block and abnormal waveform dispersion, and pathological features of multifocal demyelination of myelinated fibers, endoneurial edema, inflammatory cell infiltration, etc. CIDP includes classic and variant types, the latter including pure motor type, pure sensory type, distal acquired demyelinating symmetrical neuropathy, multifocal acquired demyelinating sensorimotor neuropathy, focal type, etc. Classic CIDP is relatively easy to diagnose, but variant CIDP often has a unique phenotype, which brings certain challenges to clinical practice. In recent years, the discovery of IgG4 antibodies in the nodal and paranodal regions of Ranvier nodes has brought new progress to the study of the clinical phenotype of CIDP. The nodes of Ranvier are important structures of myelinated fibers, including the node region, paranode region, juxtanode region and internode region, a total of four structural regions. Each region is orderly arranged by different ion channels, cell adhesion molecules, signal transduction proteins, cytoskeletal proteins, etc., which together maintain the normal structure and function of nerves. The node region is densely distributed with sodium ion channels and potassium ion channels, which are mainly involved in the conduction of nerve impulses. Neurofascin (NF) 186, neuronal cell adhesion molecule (NrCAM) and gliomedin participate in maintaining the stability of the nodes of Ranvier and the aggregation of sodium channels. The important structure of the paranode region is a complex composed of contactin-1 (CNTN1) and contactin associated protein 1 (CASPR1) located at the axonal end and neurofascin (NF) 155 located at the myelin end. The function of this complex is to block the sodium channels in the node region and the potassium channels in the juxtanode region, and anchor the myelin sheath to the axonal end. Myelin-associated glycoprotein (MAG) is also distributed in the paranodal region, which participates in the formation of myelin and the maintenance of its integrity by mediating the interaction between glial cells and axons. Potassium channels, contactin-2 (CNTN2) and contactin-associated protein 2 (CASPR2) are distributed in the near paranodal region. CIDP is a group of highly heterogeneous diseases, and its diagnosis and differential diagnosis are mainly based on clinical manifestations, autoantibody detection, imaging examinations and electromyography, among which autoantibody detection is particularly important.In recent years, with the continuous deepening of disease research and the development of antibody detection technology, more and more antibodies have been reported, especially the discovery of Ranvier nodes / paranode antibodies, which provide a new perspective for revealing the disease mechanism.

[0019] In recent years, nanomaterials have gradually attracted the attention of scientists due to their unique physical and chemical properties, especially their advantages in biomolecule adsorption and sensing. Nanosilica, as a material with a large specific surface area and good biocompatibility, has great potential in the application of antigen carriers. By chemically modifying silica, its adsorption efficiency and stability on cells can be further improved, thereby improving the sensitivity and specificity of antibody detection.

[0020] In addition, HeLa cells, as a commonly used cell model, can efficiently express a variety of cell targets. By fixing the specific neural antigens expressed by HeLa cells on modified nano-silica, efficient adsorption and stable display of a variety of cell targets can be achieved. This method not only simplifies the antigen preparation process, but also greatly improves the efficiency of antibody-antigen binding, which facilitates the simultaneous detection of multiple targets, thereby significantly improving the diagnostic accuracy of complex autoimmune diseases such as CIDP.

[0021] In summary, the existing CIDP antibody detection technology has many deficiencies in terms of sensitivity, specificity, multi-antigen screening capability, and cell stability, which restricts its wide application in clinical diagnosis. By introducing a new antibody detection system of modified nano-silica and HeLa cells co-expressing multiple neural antigens, it is expected to overcome these technical bottlenecks and provide an efficient, accurate, and sensitive CIDP antibody detection method, providing strong support for early clinical diagnosis and precision treatment.

[0022] The present invention also provides a method for preparing the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy, comprising the following steps:

[0023] S1. Soaking nano-silica in a yam polysaccharide solution and ultrasonically heating the solution to obtain an intermediate product;

[0024] S2. Add PBS buffer of Hela cells containing antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG to the intermediate product for soaking and adsorption to obtain modified silica;

[0025] S3. Spotting the modified silica onto a glass slide, cutting it to form an antibody detection strip, and obtaining the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy.

[0026] The present invention also provides the use of the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy in a kit.

[0027] The present invention has the following beneficial effects:

[0028] The present invention first uses yam polysaccharide to modify nano-silicon dioxide. A large number of hydroxyl groups on the surface of yam polysaccharide can form hydrogen bonds with hydroxyl groups on the surface of silica, and are coated on the surface of silica, thereby improving the dispersibility of modified silica in components; and the specific surface area of ​​modified silica increases, which significantly improves the adsorption capacity of cells, enhances the efficiency of antibody-antigen binding, and improves the sensitivity of antibody detection. Secondly, the antibody detection material can realize the simultaneous detection of multiple antigens, solves the problem of insufficient coverage of single antigen detection in traditional methods, can screen multiple autoantibodies at one time, expands the coverage of detection, ensures comprehensive diagnosis and accurate classification of CIDP, and helps to formulate personalized treatment plans for patients. In addition, modified silica enhances the stability and biological activity of cells during the detection process, avoiding easy inactivation of cells. The high stability of cells ensures the reliability and consistency of antibody detection results, thereby reducing experimental errors and improving the accuracy of experiments. The present invention simplifies the detection process and significantly improves the detection efficiency by adsorbing Hela cells on modified silica. Compared with traditional methods, this technology makes experimental operations more convenient, shortens the diagnosis cycle, and is particularly suitable for clinical rapid testing needs. DETAILED DESCRIPTION

[0029] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0030] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0031] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified in all instances by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary depending on the desired properties to be obtained by the present invention.

[0032] HeLa cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0033] Example 1

[0034] A chronic inflammatory demyelinating polyradiculoneuropathy antibody detection material, comprising the following components: 1 part of modified silica, 1 part of working solution (10g / 100ml goat serum), 15 parts of PBS (0.01mol / L), 1 part of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 2 parts of mounting medium (glycerol), 1 part of positive control (containing recombinant antigens of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, MAG proteins) and 1 part of negative control (8wt% goat serum);

[0035] The method for preparing the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0036] S1. Soak 6 parts of nano-silica in a sufficient amount of yam polysaccharide aqueous solution (ensuring that the solute is 30wt%), and ultrasonically heat at 65°C for 10h to obtain an intermediate product;

[0037] S2. Add 5 mg / ml of PBS buffer containing HeLa cells of antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG to the intermediate product for soaking and adsorption to obtain modified silica;

[0038] S3. Spot the modified silica onto a glass slide, and then spot it onto a glass slide at a temperature of 25°C and a humidity of 35%. After cutting, antibody detection strips are formed, and then packaged together with other components according to the above-mentioned mass fractions, and frozen in liquid nitrogen to obtain the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy.

[0039] Example 2

[0040] A chronic inflammatory demyelinating polyradiculoneuropathy antibody detection material, comprising the following components: 2 parts of modified silica, 2 parts of working solution (10g / 100ml goat serum), 20 parts of PBS (0.01mol / L), 2 parts of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 4 parts of mounting medium (glycerol), 2 parts of positive control (containing recombinant antigens of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, MAG protein) and 2 parts of negative control (8wt% goat serum);

[0041] The method for preparing the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0042] S1. Soak 6 parts of nano-silica in a sufficient amount of yam polysaccharide aqueous solution (ensuring that the solute is 30wt%), and ultrasonically heat at 65°C for 10h to obtain an intermediate product;

[0043] S2. Add 5 mg / ml of PBS buffer containing HeLa cells of antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG to the intermediate product for soaking and adsorption to obtain modified silica;

[0044] S3. Spot the modified silica onto a glass slide, and then spot it onto a glass slide at a temperature of 25°C and a humidity of 35%. After cutting, antibody detection strips are formed, and then packaged together with other components according to the above-mentioned mass fractions, and frozen in liquid nitrogen to obtain the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy.

[0045] Example 3

[0046] A chronic inflammatory demyelinating polyradiculoneuropathy antibody detection material, comprising the following components: 1 part of modified silica, 1 part of working solution (10g / 100ml goat serum), 15 parts of PBS (0.01mol / L), 1 part of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 2 parts of mounting medium (glycerol), 1 part of positive control (containing recombinant antigens of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, MAG proteins) and 1 part of negative control (8wt% goat serum);

[0047] The method for preparing the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0048] S1. Soak 10 parts of nano-silica in a sufficient amount of yam polysaccharide aqueous solution (ensuring that the solute is 30wt%), and ultrasonically heat at 65°C for 10h to obtain an intermediate product;

[0049] S2. Add 5 mg / ml of PBS buffer containing HeLa cells of antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG to the intermediate product for soaking and adsorption to obtain modified silica;

[0050] S3. Spot the modified silica onto a glass slide, and then spot the modified silica onto a glass slide at a temperature of 30°C and a humidity of 35%. After cutting, antibody detection strips are formed, and then packaged together with other components according to the above-mentioned mass fractions, and frozen in liquid nitrogen to obtain the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy.

[0051] Comparative Example 1

[0052] A material for detecting antibodies of chronic inflammatory demyelinating polyradiculoneuropathy. In this comparative example, unmodified nano-silica is used to replace the modified silica, and other components and preparation methods are the same.

[0053] Test Case

[0054] The antibody detection materials for chronic inflammatory demyelinating polyradiculoneuropathy prepared in Example 1 and Comparative Example 1 were used as a kit for performance testing.

[0055] The antibody detection materials for chronic inflammatory demyelinating polyradiculoneuropathy prepared in Example 1 and Comparative Example 1 were used as kits to detect 30 NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, MAG negative sera and 30 NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, MAG positive sera;

[0056] Test method:

[0057] A1. Equilibrate room temperature: Take out the cell matrix slide and place it at room temperature for 30 minutes;

[0058] A2. Sample incubation: Add 100 μl of the diluted serum sample to be tested to each well of the reaction plate in a fume hood, place the cell matrix slide upside down on the reaction plate, and incubate at room temperature for 1 hour;

[0059] A3. Washing: Immerse the cell matrix slide in a washing tank filled with PBS for 5 minutes, take out the cell matrix slide, and wipe off the excess liquid on the edge and back with absorbent paper;

[0060] A4. Secondary antibody incubation: Add 100 μl of fluorescent secondary antibody to each well of a dry and clean reaction plate, quickly invert the cell matrix slide onto the reaction plate, and incubate at room temperature for 1 hour;

[0061] A5. Washing: Immerse the cell matrix slide in a washing tank filled with PBS for 5 minutes in a dark environment. Take out the cell matrix slide and wipe off the excess liquid on the edge and back with absorbent paper.

[0062] A6. Sealing: Add 100 μl of sealing medium to each reaction area and cover with a coverslip;

[0063] A7. Observe the fluorescence signal: read the green fluorescence result under the microscope, observe with a 20x objective lens, and analyze the result. Table 1 Performance test results of antibody detection materials for chronic inflammatory demyelinating polyradiculoneuropathy in Example 1 and Comparative Example 1

[0064]

[0065] It can be concluded from Table 1 that the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy of Example 1 of the present invention effectively improves the stability and biological activity of cells during the detection process, simplifies the detection process, and significantly improves the detection efficiency.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0067] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy, characterized in that: The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy comprises: modified silica, a positive control, a negative control, a working solution, and an adjuvant; Wherein, the modified silica is adsorbed with Hela cells containing antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG; The modified silicon dioxide is nano silicon dioxide modified with yam polysaccharide.

2. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The particle size of the nano silicon dioxide is 50-80nm.

3. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The working solution is goat serum or bovine serum.

4. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The auxiliary agent is selected from one or more of a sealing agent, a fluorescent secondary antibody, and a PBS buffer.

5. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 4, characterized in that: The fluorescent secondary antibody is selected from one or more of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and tetraethylrhodamine.

6. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 4, characterized in that: The mounting medium is glycerol.

7. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The negative control is 8-12 wt % goat serum.

8. The antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The positive control is a recombinant antigen containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins.

9. The method for preparing the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to any one of claims 1 to 8, characterized in that: The steps include: S1. Soaking nano-silica in a yam polysaccharide solution and ultrasonically heating the solution to obtain an intermediate product; S2. Add PBS buffer of Hela cells containing antigenic proteins of NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG to the intermediate product for soaking and adsorption to obtain modified silica; S3. Spotting the modified silica onto a glass slide, cutting it to form an antibody detection strip, and obtaining the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy.

10. Use of the antibody detection material for chronic inflammatory demyelinating polyradiculoneuropathy according to any one of claims 1 to 9 in a kit.

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