Antibody detection kit for detecting chronic inflammatory demyelinating multiple nerve root neuropathy and preparation method thereof

Abstract

The invention relates to an antibody detection kit for detecting chronic inflammatory demyelinating multiple nerve root neuropathy and a preparation method of the antibody detection kit. The antibody detection kit for detecting the chronic inflammatory demyelinating multiple nerve root neuropathy comprises an IgG antibody detection strip, an IgM antibody detection strip, a working solution and an auxiliary agent. The antibody detection kit disclosed by the invention can be used for simultaneously detecting nine autoantibodies related to the chronic inflammatory demyelinating multiple nerve root neuropathy, wherein the nine autoantibodies comprise NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM and MAG. The design of simultaneous detection of multiple antigens greatly improves the comprehensiveness of detection, so that multiple possible pathogenic factors can be covered in single detection, and the problem of missed diagnosis caused by single antibody detection is avoided.

Description

Technical Field

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

[0002] 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, and inflammatory cell infiltration. CIDP includes classic and variant types, the latter of which includes 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.

[0003] 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 four structural regions: the nodal region, the paranodal region, the proximal paranodal region, and the internodal region. 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 nodal 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 are involved in maintaining the stability of Ranvier nodes and the aggregation of sodium channels. The important structure of the paranodal 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 nodal region and the potassium channels in the proximal paranodal region, and anchor the myelin sheath to the axonal membrane. Myelin-associated glycoprotein (MAG) is also distributed in the paranodal region, which participates in the formation of myelin sheath 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 proximal paranodal region.

[0004] CIDP is a group of highly heterogeneous diseases. Its diagnosis and differential diagnosis are mainly based on clinical manifestations, autoantibody detection, imaging examinations and electromyography examinations, 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 provides a new perspective for revealing the disease mechanism.

[0005] In the existing technology, CIDP-related antibody detection can often only detect antibodies against a single antigen, such as MAG antibodies or NF155 antibodies. This single-target detection method has obvious limitations. First, the immune response of CIDP is complex, and multiple antibodies may be involved at the same time. Only detecting a specific antibody may lead to missed diagnosis. In addition, there are problems such as complicated operating procedures and long detection time. Since each test can only target a single antigen, the laboratory needs to conduct multiple tests repeatedly to cover different antibodies, which not only increases the detection time and cost, but may also cause patients to delay treatment.

[0006] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0007] Based on this, the kit of the present invention provides a basis for the diagnosis and differential diagnosis of clinical autoimmune Ranvier's node disease by detecting 9 autoantibodies including NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM and MAG.

[0008] One object of the present invention is to provide an antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, wherein the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprises: an IgG antibody detection strip, an IgM antibody detection strip, a working solution, a control group, and an auxiliary agent;

[0009] Wherein, the IgG antibody test strip is spotted with Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG antigen proteins;

[0010] The IgM antibody detection strip is spotted with Hela cells containing MAG antigen protein.

[0011] Furthermore, the working solution is selected from one or more of goat serum and bovine serum.

[0012] Furthermore, the auxiliary agent is selected from one or more of a sealing agent, an IgG fluorescent secondary antibody, an IgM fluorescent secondary antibody, and a PBS buffer.

[0013] Further, the IgG fluorescent secondary antibody is selected from goat anti-human IgG combined with fluorescein isothiocyanate;

[0014] The IgM fluorescent secondary antibody is selected from goat anti-human IgM combined with fluorescein isothiocyanate.

[0015] Furthermore, the sealing medium is glycerol containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane, and thymol.

[0016] Furthermore, a control group is included.

[0017] Furthermore, the control group includes a positive control and a negative control.

[0018] Furthermore, the negative control is 8-12 wt % goat serum.

[0019] Furthermore, the positive control is positive goat serum containing the recombinant antigen of CASPR1 protein.

[0020] The present invention also provides a method for preparing the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, comprising the following steps:

[0021] Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins were spotted onto slides and cut to form IgG antibody detection strips;

[0022] Hela cells containing MAG protein were spotted onto a glass slide and cut to form an IgM antibody detection strip;

[0023] The IgG antibody detection strip, the IgM antibody detection strip and other components are packaged to obtain the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy.

[0024] The present invention has the following beneficial effects:

[0025] The antibody detection kit of the present invention can simultaneously detect 9 autoantibodies related to chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), including NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM and MAG. This design of simultaneous detection of multiple antibodies greatly improves the comprehensiveness of the detection, so that multiple possible pathogenic factors can be covered in a single detection, avoiding the problem of missed diagnosis caused by single antibody detection.

[0026] In addition, the simultaneous detection of multiple antibodies helps to more accurately evaluate the patient's own antibody spectrum and effectively supports personalized disease diagnosis. Different patients may have different antibody combinations. Through this kit, multiple antibody results can be obtained at one time, which is helpful for early detection, accurate typing and targeted treatment of the disease. Compared with the traditional method that requires separate detection of single antigen antibodies, the present invention simplifies the detection process and reduces the complexity of operation. By integrating multiple antigens into one detection kit, the detection of all antibodies can be completed in a short time, saving detection time and cost and improving work efficiency. The kit also has a wide range of applicability, which is suitable for the initial diagnosis of CIDP, as well as for subsequent disease course monitoring and treatment effect evaluation, and can provide continuous diagnostic support and guidance for clinicians. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] HeLa cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0031] HeLa cells containing CASPR1 protein were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0032] Goat anti-human IgM conjugated with fluorescein isothiocyanate and goat anti-human IgG conjugated with fluorescein isothiocyanate were purchased from Omen Medical Diagnostics (China) Co., Ltd.

[0033] Example 1

[0034] An antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprising the following components: 1 IgG antibody detection strip, 1 IgM antibody detection strip, 1 working solution (10 g / 100 ml goat serum), 15 PBS buffers (0.01 mol / L), 2 sealing agents (containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, 1,4-diazobicyclo [2.2.2] octane, thymol glycerol, mass ratio of 8:0.2:1.5:0.25:0.1:30), 1 IgG fluorescent secondary antibody (goat anti-human IgG combined with fluorescein isothiocyanate), 1 IgM fluorescent secondary antibody (goat anti-human IgM combined with fluorescein isothiocyanate), 1 positive control (positive goat serum containing recombinant antigen of CASPR1 protein), 1 negative control (8wt% goat serum);

[0035] Wherein, the IgG antibody test strip is spotted with Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG antigen proteins;

[0036] The IgM antibody detection strip is spotted with Hela cells containing MAG antigen protein.

[0037] The preparation method of the above-mentioned antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0038] A PBS solution of HeLa cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins at 5 mg / ml was spotted onto a glass slide at 25°C and 40% humidity, and then cut to form an IgG antibody detection strip;

[0039] A PBS solution of Hela cells containing MAG protein at 5 mg / ml was spotted onto a glass slide at 25°C and 40% humidity, and then cut to form an IgM antibody detection strip;

[0040] According to the above mass fractions, the IgG antibody test strip, the IgM antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy.

[0041] Example 2

[0042] An antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprising the following components: 2 IgG antibody detection strips, 2 IgM antibody detection strips, 2 working solutions (10 g / 100 ml goat serum), 25 PBS buffers (0.01 mol / L), 4 sealing agents (containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, 1,4-diazobicyclo [2.2.2] octane, thymol glycerol, with a mass ratio of 8:0.2:1.5:0.25:0.1:30), 2 IgG fluorescent secondary antibodies (goat anti-human IgG combined with fluorescein isothiocyanate), 2 IgM fluorescent secondary antibodies (goat anti-human IgM combined with fluorescein isothiocyanate), 2 positive controls (positive goat serum containing recombinant antigen of CASPR1 protein), and 2 negative controls (8 wt% goat serum);

[0043] Wherein, the IgG antibody test strip is spotted with Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG antigen proteins;

[0044] The IgM antibody detection strip is spotted with Hela cells containing MAG antigen protein.

[0045] The preparation method of the above-mentioned antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0046] A PBS solution of HeLa cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins at 5 mg / ml was spotted onto a glass slide at 25°C and 40% humidity, and then cut to form an IgG antibody detection strip;

[0047] A PBS solution of Hela cells containing MAG protein at 5 mg / ml was spotted onto a glass slide at 25°C and 40% humidity, and then cut to form an IgM antibody detection strip;

[0048] According to the above mass fractions, the IgG antibody test strip, the IgM antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy.

[0049] Example 3

[0050] An antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprising the following components: 1 IgG antibody detection strip, 1 IgM antibody detection strip, 1 working solution (10 g / 100 ml goat serum), 15 PBS buffers (0.01 mol / L), 2 sealing agents (containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, 1,4-diazobicyclo [2.2.2] octane, thymol glycerol, mass ratio of 8:0.2:1.5:0.25:0.1:30), 1 IgG fluorescent secondary antibody (goat anti-human IgG combined with fluorescein isothiocyanate), 1 IgM fluorescent secondary antibody (goat anti-human IgM combined with fluorescein isothiocyanate), 1 positive control (positive goat serum containing recombinant antigen of CASPR1 protein), 1 negative control (8wt% goat serum);

[0051] Wherein, the IgG antibody test strip is spotted with Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG antigen proteins;

[0052] The IgM antibody detection strip is spotted with Hela cells containing MAG antigen protein.

[0053] The preparation method of the above-mentioned antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprises the following steps:

[0054] 8 mg / ml of HeLa cell PBS solution containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins was spotted onto a glass slide at 25°C and 40% humidity, and then cut to form an IgG antibody test strip;

[0055] Spot 8 mg / ml of PBS solution of Hela cells containing MAG protein on a glass slide at 25°C and 40% humidity, and cut it to form an IgM antibody detection strip;

[0056] According to the above mass fractions, the IgG antibody test strip, the IgM antibody test strip and other components are packaged and frozen in liquid nitrogen to obtain the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy.

[0057] Comparative Example 1

[0058] An antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy. In this comparative example, trehalose of equal mass is used to replace modified trehalose, and other components and preparation methods are the same.

[0059] Test Case

[0060] The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy prepared in Example 1 and Comparative Example 1 was used as a kit for performance testing.

[0061] The antibody detection kits for detecting chronic inflammatory demyelinating polyradiculoneuropathy prepared in Example 1 and Comparative Examples 1-2 were used as the 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;

[0062] Test method:

[0063] A1. Balance room temperature: Take out the IgG antibody test strip and IgM antibody test strip respectively and place them at room temperature for 30 minutes.

[0064] A2. Sample incubation: Add 100 μL of the diluted sample to be tested to each well of the reaction plate, place the IgG antibody test strip and IgM antibody test strip upside down on the reaction plate, and incubate at room temperature for 1 hour. Be careful to avoid generating bubbles.

[0065] A3. Cleaning: Immerse the IgG antibody test strip and the IgM antibody test strip in a washing tank filled with PBS washing solution for 5 minutes; take out the IgG antibody test strip and the IgM antibody test strip, and wipe off the excess liquid on the edge and the back with absorbent paper.

[0066] A4. Secondary antibody incubation: Add 100 μL of IgG fluorescent secondary antibody to each well of a dry and clean reaction plate under light-proof conditions, quickly invert the IgG antibody test strip onto the reaction plate, and incubate at room temperature for 1 hour, taking care to avoid bubbles.

[0067] In a dark environment, add 100 μL of IgM fluorescent secondary antibody to each well of a dry and clean reaction plate, quickly turn the IgM antibody test strip upside down onto the reaction plate, and incubate at room temperature for 1 hour, taking care to avoid bubbles.

[0068] A5. Cleaning: Immerse the IgG antibody test strip and the IgM antibody test strip in a washing tank filled with PBS washing solution for 5 minutes; take out the IgG antibody test strip and the IgM antibody test strip, and wipe off the excess liquid on the edge and the back with absorbent paper.

[0069] A6. Sealing: Add 40 μL of sealing medium to each reaction area and cover with a coverslip, taking care to avoid creating bubbles.

[0070] A7. Observe the fluorescence signal: Read the green fluorescence result under the microscope, observe with a 20x objective lens, and analyze the result.

[0071] Table 1 Kit performance test results of Example 1 and Comparative Example 1

[0072]

[0073] According to Table 1, it can be concluded that the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy of Example 1 of the present invention can effectively detect 9 different antibodies including NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM and MAG.

[0074] 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.

[0075] 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 kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy, characterized in that: The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy comprises: an IgG antibody detection strip, an IgM antibody detection strip, a working solution, and an auxiliary agent; Wherein, the IgG antibody test strip is spotted with Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG antigen proteins; The IgM antibody detection strip is spotted with Hela cells containing MAG antigen protein.

2. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The working solution is selected from one or more of goat serum and bovine serum.

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

4. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: The IgG fluorescent secondary antibody is selected from goat anti-human IgG combined with fluorescein isothiocyanate; The IgM fluorescent secondary antibody is selected from goat anti-human IgM combined with fluorescein isothiocyanate.

5. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 3, characterized in that: The sealing medium is glycerol containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane and thymol.

6. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 1, characterized in that: A control group was also included.

7. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 6, characterized in that: The control group includes a positive control and a negative control.

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

9. The antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to claim 7, characterized in that: The positive control is positive goat serum containing the recombinant antigen of CASPR1 protein.

10. The method for preparing the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy according to any one of claims 1 to 9, characterized in that: The steps include: Hela cells containing NF155, NF186, CNTN1, CNTN2, CASPR1, CASPR2, GLIOMEDIN, NRCAM, and MAG proteins were spotted onto slides and cut to form IgG antibody detection strips; Hela cells containing MAG protein were spotted onto a glass slide and cut to form an IgM antibody detection strip; The IgG antibody detection strip, the IgM antibody detection strip and other components are packaged to obtain the antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy.

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