MuSK antibody detection method and corresponding product

Abstract

The invention relates to a MuSK antibody detection method and a corresponding product. The polynucleotide adopted by the invention is the polynucleotide for coding the recombinant human Flag-MuSK-ECD protein, and the recombinant human Flag-MuSK-ECD protein has the advantages that the recombinant human Flag- The sequence of the recombinant human Flag-MuSK-ECD protein is as shown in SEQ ID NO. 2; the sequence of the polynucleotide is as shown in SEQ ID NO. 1. According to the MuSK antibody detection method, eukaryotic cells are transfected through artificially constructed target gene expression plasmids, secreting type Flag-MuSK-ECD protein in supernatant is collected, the secreting type Flag-MuSK-ECD protein is used as a detection antigen, and the MuSK antibody is detected through the detection antigen. Factors which influence antigen activity detection due to reasons such as detection of antigen species difference, expression of immature MuSK full-length protein by prokaryotic cells, low protein purification purity caused by partial anti-MuSK transmembrane segment and / or intracellular segment antibodies and a poor-specificity protein tag purification system are avoided, so that the antigen activity is relatively good; therefore, the specificity and the sensitivity of pathogenic antibody detection are improved, and a more valuable biomarker detection method is provided for clinical diagnosis and treatment schemes.

Description

Technical Field

[0001] The present application relates to the field of antibody detection, and in particular to a MuSK antibody detection method and corresponding products, such as polynucleotides, recombinant protein expression plasmids, transformants, expression systems, proteins, compositions, product combinations, reagents, kits, systems or systems. Background Art

[0002] Myasthenia grais (MG) is a common clinical autoimmune disease characterized by fluctuating muscle weakness caused by autoimmune antibodies attacking the neuromuscular junction (NMJ). AChR and MuSK antibodies are currently recognized as two antibodies with independent pathogenicity for MG. The detection of these two antibodies is very important for the diagnosis and efficacy observation of MG. The diagnosis of myasthenia gravis requires the main clinical symptoms plus one of the three laboratory diagnostic markers. In addition, there are many types of myasthenia gravis, which need to be grouped. The significance of MuSK antibody detection is to assist in the diagnosis of MuSKMG patients.

[0003] The detection method for AChR antibody positivity is already very mature. However, the detection method for MuSK antibody is still immature. Currently, there are three main methods for detecting autoimmune antibodies: radioimmunoassay, ELISA, and CBA. Among them, radioimmunoassay is the standard antibody detection method recommended by the current guidelines, but because it involves radioactive detection qualification requirements, it is difficult to be promoted and used. The implementation of the CBA detection method requires expensive equipment such as professional cell culture and fluorescence microscopes, which limits its promotion and use in clinical practice. The advantage of the ELISA detection method is that the detection is convenient and rapid, and it is easy to construct a commercial rapid detection kit. It does not require high configuration of the detection site and is suitable for clinical promotion and use. In addition, most of the mature antibody detection technologies for other autoimmune diseases in clinical practice use ELISA detection technology, so it is worth further developing ELISA detection methods for MG autoimmune antibodies including MuSK. Summary of the invention

[0004] The first aspect of the present application is to propose a polynucleotide to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0005] A polynucleotide encoding a recombinant human Flag-MuSK-ECD protein;

[0006] Preferably, the sequence of the recombinant human Flag-MuSK-ECD protein is as shown in SEQ ID NO.2;

[0007] Preferably, the sequence of the polynucleotide is as shown in SEQ ID NO.1.

[0008] The second aspect of the present application is to propose a recombinant protein expression plasmid to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0009] A recombinant protein expression plasmid comprises a plasmid vector and a target gene, wherein the target gene is the polynucleotide as described in the first aspect, and the plasmid vector is pCMV.

[0010] Preferably, the recombinant protein expression plasmid is pCMV-Flag-human-MuSK-ECD.

[0011] The third aspect of the present application is to propose a recombinant protein transformant or expression system to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0012] A recombinant protein transformant or expression system, comprising a receptor cell and a recombinant protein expression plasmid, wherein the recombinant protein expression plasmid is as described in the second aspect above; the receptor cell is a eukaryotic cell;

[0013] Preferably, the eukaryotic cell is a human cell;

[0014] More preferably, the human-derived cells are human-derived epithelial cells;

[0015] Still more preferably, the human epithelial cells are human kidney epithelial cells;

[0016] Even more preferably, the human kidney epithelial cells are 293T.

[0017] The fourth aspect of the present application is to propose a protein to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0018] A protein, wherein the protein is a recombinant human Flag-MuSK-ECD protein, and the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; preferably, the protein purity of the recombinant human Flag-MuSK-ECD protein is above 80-90%, and the concentration is above 0.4-0.5 ug / uL.

[0019] The fifth aspect of the present application is to propose a use of a recombinant protein to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0020] A use of a recombinant protein, the protein being as described in the fourth aspect above; the use of the recombinant protein is to be used as a detection antigen for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis, or to be used for preparing a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis;

[0021] Preferably, the sequence of the recombinant human Flag-MuSK-ECD protein is as shown in SEQ ID NO.2;

[0022] Preferably, the detection is an enzyme-labeled antibody detection;

[0023] More preferably, the enzyme-labeled antibody detection is an indirect method.

[0024] The sixth aspect of the present application is to propose a method for preparing a protein to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0025] A method for preparing a protein, wherein the protein is as described in the fourth aspect above; the method for preparing the protein comprises product expression and product purification steps, wherein the product expression step comprises obtaining a culture supernatant containing a recombinant human Flag-MuSK-ECD protein using the recombinant protein transformant or expression system described in the third aspect above, and the product purification step comprises purification using Flag magnetic beads;

[0026] Preferably, the product purification step comprises the following steps:

[0027] a. contacting the culture supernatant containing the recombinant human Flag-MuSK-ECD protein with Flag magnetic beads, preferably, the Flag magnetic beads include anti-Flag tag monoclonal antibodies, the anti-Flag tag monoclonal antibody is M1, and the amount is 10-20uFlag magnetic beads per 1mL of culture supernatant containing recombinant human Flag-MuSK-ECD protein, 1ug / uL;

[0028] b. removing non-specifically bound proteins by washing, leaving specifically bound recombinant human Flag-MuSK-ECD protein; preferably, the washing buffer used for washing is 1% (v / v) Tween20 in PBS, with an amount of 200 uL / well;

[0029] c. Elution to obtain recombinant human Flag-MuSK-ECD protein; preferably, the elution adopts 3*Flag polypeptide competitive inhibition elution, and the dosage is 200ug, 1ug / uL.

[0030] The seventh aspect of the present application is to propose a composition, product combination, reagent, kit, system or system to solve the current technical problems in detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis.

[0031] A composition, product combination, reagent, kit, system or system, wherein the composition, product combination, reagent, kit, system or system comprises a detection antigen, wherein the detection antigen is a protein as described in the fourth aspect above, or a protein prepared according to the method for preparing the protein as described in the sixth aspect above.

[0032] Furthermore, the composition, product combination, reagent, kit, system or system also includes: a secondary antibody, a chromogenic substrate;

[0033] Furthermore, the composition, product combination, reagent, kit, system or system also includes: positive standard control serum and control protein; the positive standard control serum is a serum sample of a patient diagnosed with MuSK MG, and the control protein is a protein purified by flag magnetic beads from the supernatant of cells transfected with an empty vector.

[0034] Still further, the composition, product combination, reagent, kit, system or system also includes: coating buffer, blocking buffer, washing buffer, terminator; the chromogenic substrate is a chromogenic substrate diluted with chromogenic substrate buffer.

[0035] Based on any of the above technical solutions, preferably, the secondary antibody is an alkaline phosphatase-labeled goat anti-human IgG (H+L) secondary antibody; and the color developing substrate is p-nitrophenyl phosphate.

[0036] The eighth aspect of the present application is to propose a composition, product combination, reagent, kit, system or system to solve the above-mentioned technical problems existing in the current MuSK antibody detection.

[0037] This application adopts the following technical solutions to solve the above technical problems.

[0038] A use of a composition, product combination, reagent, kit, system or system, wherein the composition, product combination, reagent, kit, system or system is as described in any technical solution of the seventh aspect above, and the use of the composition, product combination, reagent, kit, system or system is for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis, or for preparing a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis;

[0039] Preferably, the detection is an enzyme-labeled antibody detection;

[0040] More preferably, the enzyme-labeled antibody detection is an indirect method.

[0041] The ninth aspect of the present application is to propose a method for detecting MuSK antibodies for non-disease diagnosis purposes or to assist in the group diagnosis of myasthenia gravis, so as to solve the above-mentioned technical problems existing in the current MuSK antibody detection.

[0042] A method for detecting MuSK antibodies for non-disease diagnosis purposes or assisting in group diagnosis of myasthenia gravis, comprising the following steps:

[0043] 1) contacting the serum or plasma sample to be tested with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect above;

[0044] Contacting the serum or plasma sample to be tested with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect above;

[0045] contacting the positive standard control serum with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect above;

[0046] contacting the positive standard control serum with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect above;

[0047] Set up a blank control;

[0048] 2) Measure the OD value;

[0049] 3) Calculation results and judgment: Preferably, the detection OD value is first converted as follows: (Flag-MuSK-ECD protein OD value-blank control OD value) / (control protein OD value-blank control OD value) to obtain the calibration expression value, and the calibration expression value of the positive standard control serum is calibrated as 1, and the relative expression value of the serum antibody to be tested is calculated, and compared with the boundary value drawn from the detection data of healthy people to determine whether it is a positive result of the antibody test.

[0050] The beneficial effects of this application are as follows:

[0051] 1. The existing technology uses non-human proteins as detection antigens, which leads to obvious species differences and greatly affects the sensitivity and specificity of the detection results. This application uses recombinant human MuSK protein as the detection antigen, thereby avoiding the test result errors caused by species differences and significantly improving the sensitivity and specificity of the detection.

[0052] 2. Since MuSK is a single transmembrane protein, the existing MuSK detection technology uses a recombinant MuSK full-length protein as the detection antigen, so that antibody-positive patients include antibodies against the transmembrane segment and / or intracellular segment of MuSK, and these antibodies are considered to have no direct pathogenicity, resulting in false positive results and affecting the specificity of the test. The antigen MuSK extracellular segment protein used in this application does not include the MuSK transmembrane segment and / or intracellular segment, thereby greatly reducing the possibility of non-pathogenic false positive antibodies being detected and significantly improving the specificity of the test.

[0053] 3. The existing methods for obtaining antigens for MuSK antibody ELISA detection all come from recombinant MuSK proteins extracted from prokaryotic cell lysates. Since it is a non-eukaryotic cell expression system, the protein modification is incomplete, and the purified protein is intracellular protein, resulting in most of the obtained immature protein. These factors significantly reduce its antigenic activity, greatly affecting the sensitivity and specificity of ELISA detection. The present application constructs a Flag-MuSK-ECD stable eukaryotic expression cell line, purifies the secretory Flag-MuSK-ECD protein in its supernatant, and obtains mature Flag-MuSK-ECD proteins, which greatly improves its antigenic activity, thereby significantly improving the sensitivity and specificity of ELISA detection.

[0054] 4. The immunoprecipitation tags used in the existing MuSK antibody ELISA detection antigen protein purification are not specific (such as His tags), resulting in low purity and affecting the antigenic activity of the MuSK detection protein, further reducing the specificity of the ELISA detection. The present application constructs a more specific flag-tagged recombinant Flag-MuSK-ECD protein, uses the Flag magnetic bead immunoprecipitation method to purify the secreted recombinant human Flag-MuSK-ECD protein, improves the purity of protein purification, further maintains the antigenic activity of the MuSK detection protein, and significantly improves the specificity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the pCMV-Flag-human-MuSK-ECD expression plasmid map;

[0056] Figure 2 This is a purity analysis diagram of Flag-MuSK-ECD protein purification;

[0057] Figure 3 This is a graph showing the results of a control experiment of a MuSK antibody detection method according to an embodiment.

[0058] The ELISA detection method of the present application was used to test the experimental samples, including 118 healthy controls (HC), 50 disease controls (PC, patient control), and 291 MG patients (MG), and the antibody positive cutoff value was calculated based on the test results of HC patients. The results showed that 10 patients in the MG group were positive for MuSK antibodies, and no positive patients were detected in the disease control group. DETAILED DESCRIPTION

[0059] The sources of reagents are as follows:

[0060] Thermo: Thermo Fisher Scientific Inc.

[0061] Gibco:Thermo Fisher Scientific Inc.

[0062] sigma: Merck KGaA, Darmstadt, Germany

[0063] MCE: MedChemExpress LLC

[0064] Abcam: Abcamlc

[0065] The term "system" should be understood in a broad sense, and may refer to a composition, a product combination, a reagent, a kit, or an instrument containing the aforementioned composition, product combination, reagent, kit, or a mixture (system) formed when the composition, product combination, reagent, kit is used for detection, as well as an instrument containing the aforementioned mixture.

[0066] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0067] The present application uses Flag magnetic beads with better specificity to purify the secreted mature recombinant Flag-MuSK-ECD protein expressed in the supernatant of eukaryotic cells as the detection antigen, thereby avoiding factors that affect the activity of the detection antigen due to species differences in the detection antigen, prokaryotic cells expressing immature MuSK full-length protein, antibodies against some anti-MuSK transmembrane segments and / or intracellular segments, and low protein purification purity caused by a protein tag purification system with poor specificity. Therefore, the antigen activity is better, thereby significantly improving the specificity and sensitivity of pathogenic antibody detection, providing a more valuable biomarker detection method for clinical diagnosis and treatment plans.

[0068] In the first aspect of this specific embodiment, a polynucleotide is proposed, which is a polynucleotide encoding a recombinant human Flag-MuSK-ECD protein; the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; the sequence of the polynucleotide is shown in SEQ ID NO.1. The Flag tag is also called the DYKDDDDK tag, which is essentially a hydrophilic polypeptide protein tag, a fusion polypeptide composed of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), and its corresponding nucleic acid sequences can be used in this application.

[0069] SEQ ID NO.1MuSK-ECD nucleic acid sequence:

[0070]

[0071] LPKAPVITTPLETVDALVEEVATFMCAVESYPQPEISWTRNKILIKLFDTRYSIRENGQLLTILSVEDSDDGIYCCTANNGVGGAVESCGALQVKMKPKITRPPINVKIIEGLKAVLP CTTMGNPKPSVSWIKGDSPLRENSRIAVLESGSLRIHNVQKEDAGQYRCVAKNSLGTAYSKVVKLEVEVFARILRAPESHNVTFGSFVTLHCTATGIPVPTITWIENGNAVSSGSIQE SVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATISIAEWSKPQKDNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNH IFQECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPTACARLPHLDYNKENLKTFPPMTSSKPSVDIPNLPSSSSSSFSVSPTYSMT

[0072] The second aspect of this specific embodiment proposes a recombinant protein expression plasmid, which includes a plasmid vector and a target gene, including the polynucleotide as described in the first aspect above, that is, the polynucleotide is a polynucleotide encoding a recombinant human Flag-MuSK-ECD protein; the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; the sequence of the polynucleotide is shown in SEQ ID NO.1; the plasmid vector is pCMV. Preferably, the recombinant protein expression plasmid is pCMV-Flag-human-MuSK-ECD

[0073] The third aspect of this specific embodiment proposes a recombinant protein transformant or expression system, including a recipient cell and a recombinant protein expression plasmid, the recombinant protein expression plasmid is as described in the second aspect of this specific embodiment, that is, the recombinant protein expression plasmid includes the polynucleotide as described in the first aspect above, that is, the polynucleotide is a polynucleotide encoding a recombinant human Flag-MuSK-ECD protein; the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; the sequence of the polynucleotide is shown in SEQ ID NO.1. The recipient cell of the recombinant protein expression plasmid is a eukaryotic cell; preferably, the eukaryotic cell is a human cell, more preferably, the human cell is a human epithelial cell, more preferably, the human epithelial cell is a human kidney epithelial cell, and even more preferably, the human kidney epithelial cell is 293T.

[0074] The fourth aspect of this specific embodiment proposes a protein, which is a recombinant human Flag-MuSK-ECD protein, and the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; preferably, the protein purity of the recombinant human Flag-MuSK-ECD protein is above 80-90%, and the concentration is above 0.4-0.5ug / uL; preferably, the recombinant human Flag-MuSK-ECD protein is encoded by the polynucleotide shown in SEQ ID NO.1. Preferably, the recombinant human Flag-MuSK-ECD protein is obtained by expressing the transformant or expression system proposed in the third aspect of this specific embodiment.

[0075] In the fifth aspect of this specific embodiment, a use of a protein is proposed, and the protein is as described in the fourth aspect of this specific embodiment; the use of the protein is to be used as a detection antigen for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis, or to prepare a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis; preferably, the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; preferably, the detection is an enzyme-labeled antibody method detection; more preferably, the enzyme-labeled antibody method detection is an indirect method. There are currently four methods for ELISA, including direct method, indirect method, sandwich method, and competitive method. The indirect method is to combine a known antibody with a solid phase carrier (such as an ELISA plate), and then react with the antigen in the sample to be tested to form an antigen-antibody complex. After washing, an enzyme-labeled secondary antibody is added to react with the antigen-antibody complex to form an enzyme-labeled antigen-antibody complex. Finally, a color-developing substrate is added to develop color, and the result is judged based on the color reaction.

[0076] In a sixth aspect of this specific embodiment, a method for preparing a protein is provided, wherein the protein is as described in the fourth aspect of this specific embodiment; the method for preparing the protein comprises product expression and product purification steps, wherein the product expression step comprises obtaining a culture supernatant containing a recombinant human Flag-MuSK-ECD protein using the transformant or expression system as described in the third aspect of this specific embodiment, and the product purification step comprises purification using Flag magnetic beads;

[0077] Preferably, the product purification step comprises the following steps:

[0078] a. Adding Flag magnetic beads to the culture supernatant containing the recombinant human Flag-MuSK-ECD protein, preferably, the Flag magnetic beads include anti-Flag tag monoclonal antibodies, the anti-Flag tag monoclonal antibodies are M1, and the amount is 10-20uL Flag magnetic beads per 1mL of culture supernatant containing the recombinant human Flag-MuSK-ECD protein, 1ug / uL;

[0079] b. removing non-specifically bound proteins by washing, leaving specifically bound recombinant human Flag-MuSK-ECD protein; preferably, the washing buffer used for washing is 1% (v / v) Tween20 in PBS, with an amount of 200 uL / well;

[0080] c. Elution to obtain recombinant human Flag-MuSK-ECD protein; preferably, the elution adopts 3*Flag polypeptide competitive inhibition elution, and the dosage is 200ug, 1ug / uL.

[0081] In the seventh aspect of this specific embodiment, a composition, product combination, reagent, kit, system or system is provided, wherein the composition, product combination, reagent, kit, system or system includes a detection antigen, wherein the detection antigen is a protein as described in the fourth aspect of this specific embodiment, or a protein prepared according to the method for preparing a protein as described in the sixth aspect of this specific embodiment. The composition, product combination, reagent, kit, system or system also includes: a secondary antibody and a chromogenic substrate.

[0082] The composition, product combination, reagent, kit, system or system also includes: positive standard control serum and control protein; the positive standard control serum is a serum sample of a patient diagnosed with MuSK MG, and the control protein is a protein purified by flag magnetic beads from the supernatant of cells transfected with an empty vector.

[0083] The composition, product combination, reagent, kit, system or system further includes: a coating buffer, a blocking buffer, a washing buffer, and a terminator; the chromogenic substrate is a chromogenic substrate diluted with a chromogenic substrate buffer.

[0084] Preferably, the secondary antibody is an anti-human antibody. Exemplarily, the secondary antibody includes but is not limited to goat anti-human antibody, rat anti-human antibody, mouse anti-human antibody, pig anti-human antibody, donkey anti-human antibody, sheep anti-human antibody, chicken anti-human antibody, horse anti-human antibody, rabbit anti-human antibody, hamster anti-human antibody, dog anti-human antibody, bovine anti-human antibody, etc., preferably goat anti-human antibody. More preferably, the goat anti-human antibody is an alkaline phosphatase-labeled goat anti-human IgG (H+L) secondary antibody.

[0085] The chromogenic substrate is p-nitrophenyl phosphate.

[0086] In an eighth aspect of this specific embodiment, a use of a composition, product combination, reagent, kit, system or system is proposed. The composition, product combination, reagent, kit, system or system is as described in any technical scheme of the seventh aspect of this specific embodiment, and the use of the composition, product combination, reagent, kit, system or system is for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis, or for preparing a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis; preferably, the detection is enzyme-labeled antibody detection; more preferably, the enzyme-labeled antibody detection is an indirect method.

[0087] In a ninth aspect of this specific embodiment, a method for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis for non-disease diagnosis purposes is proposed, comprising the following steps:

[0088] 1) contacting the serum or plasma sample to be tested with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect of this specific embodiment;

[0089] Contacting the serum or plasma sample to be tested with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the further technical solution of the seventh aspect of this specific embodiment;

[0090] contacting the positive standard control serum with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system described in the seventh aspect of this specific embodiment;

[0091] Contacting the positive standard control serum with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system, or system described in the further technical solution of the seventh aspect of this specific embodiment;

[0092] Set up a blank control;

[0093] 2) Measure the OD value;

[0094] 3) Calculation results and judgment: Preferably, the detection OD value is first converted as follows: (Flag-MuSK-ECD protein OD value-blank control OD value) / (control protein OD value-blank control OD value) to obtain the calibration expression value, and the calibration expression value of the positive standard control serum is calibrated as 1, and the relative expression value of the serum antibody to be tested is calculated, and compared with the boundary value drawn from the detection data of healthy people to determine whether it is a positive result of the antibody test.

[0095] Example 1

[0096] Since MuSK is a single transmembrane protein, the existing MuSK detection technology uses a recombinant MuSK full-length protein as the coated antigen, resulting in the detected antibodies including some anti-MuSK transmembrane and / or intracellular segment antibodies, which reduces the specificity of pathogenic antibody detection. The present application improves its antigen activity by constructing and purifying the secretory MuSK-ECD protein in the supernatant of a stably expressed cell line, thereby improving the sensitivity and specificity of the ELISA detection kit as an antigen detection kit. The MuSK antibody detection method of one embodiment comprises the following steps:

[0097] Step 1: Construct recombinant human MuSK-ECD protein expression plasmid. See the plasmid map for details. Figure 1 ;

[0098] Step 2: constructing a 293T eukaryotic cell transformant or expression system of recombinant human MuSK-ECD protein;

[0099] Step 3: Use flag gel to purify the recombinant human MuSK-ECD protein from the supernatant of the recombinant human MuSK-ECD protein 293T eukaryotic cell transformants and the supernatant of the empty plasmid vector 293T eukaryotic cell transformants (see Figure 2 ) and control protein;

[0100] Step 4: Dissolve the purified recombinant human MuSK-ECD protein and control protein in sodium carbonate buffer and coat a 96-well ELISA plate (NUNC maxisorpApogent Cat. No. 80040LE 0903), 10 ng per well, and place at 4°C overnight;

[0101] Step 5: Wash 6 times with washing buffer (0.5% PBST), 5 minutes each time;

[0102] Step 6: Add 100ul of blocking buffer (2% BSA in 0.5% PBST) to each well and incubate at room temperature for 2 hours;

[0103] Step 7. According to the experimental design, add patient serum and positive control serum diluted with blocking buffer (1:100) to different wells and incubate at 4°C overnight;

[0104] Step 8: Wash 6 times with washing buffer, 5 minutes each time;

[0105] Step 9. Add the secondary antibody diluted in blocking buffer: Goat anti-Mouse / Human IgG (H+L) AP 1:1000 to all wells and incubate at room temperature for 2 hours;

[0106] Step 10: Wash 6 times with washing buffer, 5 minutes each time;

[0107] Step 11: Add the reaction substrate pnpp (15 mg in 100 μl) diluted in the substrate reaction solution to all wells and incubate at room temperature for 15 minutes;

[0108] Step 12, adding a stop reaction solution (3M NaOH);

[0109] Step 13: Immediately measure the optical density (OD value) of each well at a wavelength of 405 nm using an ELISA reader.

[0110] Step 14: Analyze each group of data, calculate the relative expression of antibodies, compare with the cutoff value drawn from the test data of healthy people, and obtain the antibody test results.

[0111] The MuSK antibody detection method and kit will be described in more detail below.

[0112] Step 1: Plasmid construction

[0113] A recombinant protein expression plasmid pCMV-Flag-human-MuSK-ECD was constructed by gene editing technology, such as subcloning technology; the plasmid map is shown in Figure 1 .

[0114] Step 2: Transfection: Use Lippo3000 (Thermo, L3000075) to transfect pCMV-Flag-human-MuSK-ECD plasmid or empty plasmid vector into HEK293T cells.

[0115] Step 3: Product expression

[0116] After culturing for 48 hours with 10% (v / v) FBS (Gibco, 10099141C) in DMEM medium (Gibco, 11995-65), the medium was replaced with DMEM (Gibco, 11995-65) to promote protein secretion. After 24-48 hours, the culture supernatant containing recombinant human Flag-MuSK-ECD protein and control protein was collected.

[0117] Step 4: Purification

[0118] The secreted mature recombinant human Flag-MuSK-ECD protein and the control protein were purified from the supernatant of the aforementioned protein 293T eukaryotic cell transformants using Flag magnetic beads (Cat. No.: Sigma M8823). The control protein was obtained by purifying the supernatant of cells transfected with an empty plasmid containing a Flag tag using Flag magnetic beads. The process was the same as the Flag-MuSK-ECD protein purification process and was performed simultaneously. The concentration of Flag-MuSK-ECD protein was 0.4-0.5ug / uL, and the concentration of the control protein was 0.4-0.5ug / ul (BSA protein quantification method).

[0119] The step 4 comprises the following steps:

[0120] a. Add Flag magnetic beads to the culture supernatant containing the recombinant human Flag-MuSK-ECD protein and the control protein. The Flag magnetic beads include anti-Flag tag monoclonal antibodies. The anti-Flag tag monoclonal antibody is M1. The amount is 20uFlag magnetic beads per 1mL supernatant, 1ug / uL.

[0121] b. The non-specifically bound proteins were removed by washing, leaving the specifically bound recombinant human Flag-MuSK-ECD protein and the control protein; the washing buffer used for the washing was 1% (v / v) Tween20 in PBS, and the amount was 200uL / well;

[0122] c. Elution to obtain recombinant human Flag-MuSK-ECD protein and control protein; the elution was carried out using 3*Flag polypeptide (MCE, catalog number: HY-P0319) competitive inhibition elution, with a dosage of 200ug, 1ug / uL.

[0123] Purity analysis of recombinant human Flag-MuSK-ECD protein is shown in Figure 2 ImageJ software was used to analyze Figure 2 Grayscale analysis showed a purity of 80-90% and a Flag-MuSK-ECD protein concentration of 0.4-0.5ug / uL (Coomassie blue staining). The recombinant human Flag-MuSK-ECD protein of the present application, after adopting the purification method of the present application, can be used as a protein for MuSK antibody detection without further concentration and / or purification.

[0124] Step 5: Wrap

[0125] The Flag-MuSK-ECD protein and the control protein were dissolved in sodium carbonate buffer (pH 9.6), coated on ELISA plates, 10 ng per well, and placed at 4° C. for 12 hours;

[0126] Step 6: Washing

[0127] The ELISA plate was washed several times with a washing buffer (1% (v / v) Tween 20 in PBS (PBST)), and 100 ul of a blocking buffer (2% (v / v) BSA in PBST) was added to each well, followed by incubation at 25°C for 2 hours.

[0128] Step 7: Closing

[0129] The test serum and positive standard control serum (MusK antibody concentration 8 nmol / L) diluted with the blocking buffer were added to the target wells of the ELISA plate at a dilution ratio of 1:100 (volume ratio). In one plate, each sample had 3 replicate wells, columns 1-3 and 7-9 were incubated with control proteins, columns 4-6 and 10-12 were incubated with detection antigen recombinant human Flag-MuSK-ECD proteins, and placed at 4°C for 12 hours.

[0130] Step 8: Add secondary antibody after washing

[0131] The ELISA plate was rinsed six times with the washing buffer, and the secondary antibody diluted with the blocking buffer was added to the target well: alkaline phosphatase-labeled goat anti-human IgG (H+L) secondary antibody (Goat anti-Human IgG (H+L) AP, abcam, ab6859), with a dilution ratio of 1:1000, and incubated at 25°C for 2 hours.

[0132] Step 9: Add colorimetric substrate after washing

[0133] The ELISA plate was washed six times with the washing buffer, and the reaction substrate (i.e., chromogenic substrate) pNPP (p-nitrophenyl phosphate, Bio-Tech, ST1042-5g) diluted with the chromogenic substrate reaction solution, i.e., the chromogenic substrate developer described below (0.015 g p-NITROPHENYL Phosphate (sigma ca#N-3254) dissolved in chromogenic substrate buffer (10 mM Diethanolamme (sigma, ca#D8885), 0.5 mM Magnesium chloride, pH 9.8 dissolved in PBS) was added to the target well and incubated at 25°C for 15 minutes.

[0134] Step 10: Termination, adding terminator (3M NaOH).

[0135] Step 11: Measure the OD value

[0136] The optical density (OD value) of each well was measured at a wavelength of 405 nm using an enzyme-labeled instrument, and the optical density of the target well was measured.

[0137] Step 12: Calculate the results

[0138] First, the detection OD value was converted as follows: (Flag-MuSK-ECD protein OD value - blank control OD value) / (control protein OD value - blank control OD value) to obtain the calibration expression value, and the positive standard control serum calibration expression value was calibrated as 1, and the relative expression value of the serum antibody to be tested was calculated and compared with the cutoff value drawn from the healthy person test data (see Figure 3 ) to determine whether the antibody test result is positive.

[0139] Example:

[0140] The same 96-well plate assay:

[0141] Positive standard control serum calibration expression value = (Flag-MuSK-ECD protein OD value 1.044333333-blank control OD value 0.075) / (control protein OD value 0.124333333-blank control OD value 0.076333333) = 20.32867133,

[0142] The calibrated expression value of the serum antibody to be tested (Flag-MuSK-ECD protein OD value 1.731666667-blank control OD value 0.075) / (control protein OD value 0.16-blank control OD value 0.076333333) = 19.80079681,

[0143] The relative expression value of the serum antibody to be tested = 19.80079681 / 20.32867133 = 0.974033005.

[0144] A total of 118 healthy subjects were included in this experiment. The relative expression value was obtained according to the above formula, and the positive cut-off value was calculated as cut line = mean + 4 * SD = 0.070916712 + 4 * 0.030071085 = 0.191201051

[0145] The tested serum was judged to be MuSK antibody positive.

[0146] The positive cut-off value is to assess whether the patient's antibodies are positive and provide information for clinical diagnosis, but it is not a direct basis for diagnosis.

[0147] The kit of one embodiment includes the Flag-MuSK-ECD protein in the above detection method, and also includes positive standard control serum, coating buffer, blocking buffer (2% <v v>BSA in PBST), washing buffer (1% <v v>Tween20 in PBS (PBST)), colorimetric substrate developer (0.015 g p-NITROPHENYL Phosphate (sigma ca#N-3254) dissolved in colorimetric substrate buffer (10 mM Diethanolamme (sigma, ca#D8885), 0.5 mM Magnesium chloride, PH9.8 dissolved in PBS), and stop buffer (3 M NaOH).

[0148] In collaboration with Xuanwu Hospital of Capital Medical University, a single-blind retrospective clinical study was conducted. The kit was used to detect MuSK antibodies in 118 healthy subjects, 291 confirmed MG patients and 50 other disease control groups. The results of MuSK radioimmunoassay (Beijing North Institute of Biotechnology) were used as positive controls (the results of MuSK radioimmunoassay were used as positive controls in the prior art, see Oeztuerk M, Henes A, Schroeter CB, Nelke C, Quint P, Theissen L, Meuth SG, Ruck T. Current biomarker strategies in autoimmuneneuromuscular diseases. Cells. 2023; 12). Figure 3 .

[0149] The results showed that 10 patients with positive MuSK antibodies were detected among 291 MG patients tested using this application, and no MuSK antibodies were detected in healthy controls and other disease control groups. This result is consistent with the results of the MuSK radioimmunoassay test. After unblinding, it was found that 19 patients were previously diagnosed as MuSK positive, of which 9 positive patients were consistent with the positive results of this application and radioimmunoassay, but the other 10 positive patients were negative in this application and radioimmunoassay. And there was 1 patient who was positive in both this application and radioimmunoassay, but was previously diagnosed as MuSK negative. After an in-depth analysis of the clinical manifestations and signs of 20 patients with positive results in the three methods, it was found that the clinical manifestations of patients with positive MuSK antibodies detected by the ELISA test of this application and the radioimmunoassay were more consistent with the characteristic manifestations of MuSKMG, while more patients with atypical disease characteristics of MuSK MG in the past were judged to be positive, and 1 patient with typical disease characteristics of MuSK MG was judged to be negative. This shows that the specificity and sensitivity of the kit under this specific embodiment are more ideal, especially the specificity has been significantly improved, which can avoid misdiagnosis of MG patients. The above results suggest that the present application significantly improves the consistency of the results of MuSK antibody ELISA detection and MuSK radioimmunoassay detection, thereby improving the sensitivity and specificity of MuSK antibody ELISA detection.

[0150] The present application uses the more specific Flag gel to purify the secreted mature Flag-MuSK-ECD protein expressed in the supernatant of eukaryotic cells as the detection antigen, thereby avoiding factors that affect the activity of the detection antigen due to species differences in the detection antigen, prokaryotic cells expressing immature MuSK full-length protein, antibodies against some anti-MuSK transmembrane segments and / or intracellular segments, and low protein purification purity caused by a protein tag purification system with poor specificity, thereby significantly improving the specificity and sensitivity of pathogenic antibody detection.

[0151] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.< / v> < / v>

Claims

1. A polynucleotide, characterized in that The polynucleotide is a polynucleotide encoding a recombinant human Flag-MuSK-ECD protein; Preferably, the sequence of the recombinant human Flag-MuSK-ECD protein is as shown in SEQ ID NO.2; Preferably, the sequence of the polynucleotide is as shown in SEQ ID NO.

1.

2. A recombinant protein expression plasmid, comprising a plasmid vector and a target gene, characterized in that: The target gene is the polynucleotide as claimed in claim 1, and the plasmid vector is pCMV.

3. A recombinant protein transformant or expression system, comprising a recipient cell and a recombinant protein expression plasmid, characterized in that: The recombinant protein expression plasmid is as described in claim 2; the recipient cell is a eukaryotic cell; Preferably, the eukaryotic cell is a human cell, more preferably, the human cell is a human epithelial cell, even more preferably, the human epithelial cell is a human kidney epithelial cell, and even more preferably, the human kidney epithelial cell is 293T.

4. A protein, characterized in that The protein is a recombinant human Flag-MuSK-ECD protein, and the sequence of the recombinant human Flag-MuSK-ECD protein is shown in SEQ ID NO.2; Preferably, the recombinant human Flag-MuSK-ECD protein has a protein purity of 80-90% or more and a concentration of 0.4-0.5 ug / uL or more.

5. A use of a recombinant protein, characterized in that: The recombinant protein is as described in claim 4; the use of the recombinant protein is to be used as a detection antigen for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis, or to be used to prepare a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in the group diagnosis of myasthenia gravis; Preferably, the sequence of the recombinant human Flag-MuSK-ECD protein is as shown in SEQ ID NO.2; Preferably, the detection is an enzyme-labeled antibody method detection; more preferably, the enzyme-labeled antibody method detection is an indirect method.

6. A method for preparing a protein, characterized in that: The protein is as described in claim 4; the method for preparing the protein comprises product expression and product purification steps, wherein the product expression step comprises obtaining a culture supernatant containing recombinant human Flag-MuSK-ECD protein using the transformant or expression system as described in claim 3, and the product purification step comprises purification using Flag magnetic beads; Preferably, the product purification step comprises the following steps: a. contacting the culture supernatant containing the recombinant human MuSK-ECD-Flag protein with Flag magnetic beads, preferably, the Flag magnetic beads include anti-Flag tag monoclonal antibodies, the anti-Flag tag monoclonal antibodies are M1, and the amount is 10-20uFlag magnetic beads per 1mL of culture supernatant containing recombinant human MuSK-ECD-Flag protein, and the concentration of Flag magnetic beads is 1ug / uL; b. removing non-specifically bound proteins by washing, leaving specifically bound recombinant human Flag-MuSK-ECD protein; preferably, the washing buffer used for washing is 1% (v / v) Tween20 in PBS, with an amount of 200 uL / well; c. Elution to obtain recombinant human Flag-MuSK-ECD protein; preferably, the elution adopts 3*Flag polypeptide competitive inhibition elution, and the dosage is 200ug / 200uL.

7. A composition, product combination, reagent, kit, system or system, characterized in that: The composition, product combination, reagent, kit, system or system includes a detection antigen, and the detection antigen is the protein according to claim 4, or a protein prepared according to the method for preparing the protein according to claim 6.

8. The composition, product combination, reagent, kit, system or system according to claim 7, characterized in that: The composition, product combination, reagent, kit, system or system also includes: a secondary antibody and a color developing substrate.

9. The composition, product combination, reagent, kit, system or system according to claim 8, characterized in that: The composition, product combination, reagent, kit, system or system also includes: positive standard control serum and control protein; the positive standard control serum is a serum sample of a patient diagnosed with MuSKMG, and the control protein is a protein purified by flag magnetic beads from the supernatant of cells transfected with an empty vector.

10. The composition, product combination, reagent, kit, system or system according to claim 9, characterized in that: The composition, product combination, reagent, kit, system or system further includes: a coating buffer, a blocking buffer, a washing buffer, and a terminator; the chromogenic substrate is a chromogenic substrate diluted with a chromogenic substrate buffer.

11. The composition, product combination, reagent, kit, system or system according to any one of claims 8 to 10, characterized in that: The secondary antibody is alkaline phosphatase-labeled goat anti-human IgG (H+L) secondary antibody; the color development substrate is p-nitrophenyl phosphate.

12. Use of a composition, product combination, reagent, kit, system or system, wherein the composition, product combination, reagent, kit, system or system is as described in any one of claims 7 to 11, and the composition, product combination, reagent, kit, system or system is used for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis, or for preparing a composition, product combination, reagent, kit, system or system for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis; Preferably, the detection is an enzyme-labeled antibody method detection; more preferably, the enzyme-labeled antibody method detection is an indirect method.

13. A method for detecting MuSK antibodies or assisting in group diagnosis of myasthenia gravis for non-disease diagnosis purposes, characterized in that: The following steps are involved: contacting the serum or plasma sample to be tested with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system according to any one of claims 9 to 11; Contacting the serum or plasma sample to be tested with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system or system according to any one of claims 9 to 11; contacting the positive standard control serum with the recombinant human Flag-MuSK-ECD protein, the secondary antibody, and the chromogenic substrate in the composition, product combination, reagent, kit, system or system of any one of claims 9 to 11; Contacting the positive standard control serum with the control protein, secondary antibody, and chromogenic substrate in the composition, product combination, reagent, kit, system or system of any one of claims 9 to 11; The positive standard control serum was a serum sample from a patient diagnosed with MuSKMG, and the control protein was a protein purified by flag magnetic beads from the supernatant of cells transfected with an empty vector; Set up a blank control; Measure the OD value; Calculation results and judgments; Preferably, the detection OD value is first converted as follows: (Flag-MuSK-ECD protein OD value-blank control OD value) / (control protein OD value-blank control OD value) to obtain the calibration expression value, and the positive standard control serum calibration expression value is calibrated as 1, and the relative expression value of the serum antibody to be tested is calculated and compared with the boundary value drawn from the healthy person detection data to determine whether the antibody test result is positive.

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