A fusion protein for detecting aquaporin 4 antibody and a preparation method and application thereof

Abstract

The application provides a fusion protein for detecting aquaporin 4 antibody and a preparation method and application thereof, and belongs to the technical field of aquaporin 4 antibody detection. The nucleotide sequence of the fusion protein is shown as SEQ ID NO. 5, the fusion protein is used as a coating antigen, and M1 type and M23 type aquaporin 4 can be simultaneously detected. The sensitivity and accuracy of the aquaporin 4 antibody detection are high, and the repeatability is good. When the kit of the application is used for aquaporin 4 antibody, the detection method is simple and rapid, and is suitable for large-scale screening of aquaporin 4 antibody in clinical and scientific research.

Description

Technical Field

[0001] This invention belongs to the field of aquaporin 4 antibody detection technology, and particularly relates to a fusion protein for detecting aquaporin 4 antibodies, its preparation method and application. Background Technology

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease of the central nervous system, primarily affecting the optic nerve and spinal cord. It commonly affects young adults, predominantly women, and is characterized by high recurrence and disability rates. It is easily misdiagnosed as primary open-angle glaucoma, macular degeneration, or other eye diseases. Without timely treatment, most patients suffer from severe visual impairment, limb dysfunction, and urinary and bowel dysfunction.

[0003] The vast majority of NMOSD patients have detectable aquaporin 4 antibody (AQP4-IgG) in their blood. This antibody mistakenly attacks a specific protein in the central nervous system, namely aquaporin-4 (AQP4). AQP4 was isolated using homologous clones of the aquaporin family. It is widely distributed in the central nervous system and is the most abundant aquaporin in the mammalian brain. Currently, 13 subtypes of aquaporins have been identified in humans, namely AQP0 to AQP12. AQP4 is one of them. Its gene is located at the junction of human chromosome 18q11.2 and q12.1, containing four exons responsible for encoding the amino acid sequences at positions 127, 55, 27, and 92, with three introns located in between. Structurally, AQP4, located on the cell membrane, consists of six transmembrane helical structures forming five ring structures. Rings A, C, and E are located extracellularly, while rings B, D, and the carboxyl and amino terms are located intracellularly, and it is mainly expressed in astrocytes. AQP4 has two main isoforms: M1 (32 kDa) and M23 (30 kDa). Both have the same extracellular domain, but M1 has an extra 22 amino acid sequence at its intracellular N-terminus. AQP4 usually exists in tetramer form, with the ratio of M23 to M1 being 3:1.

[0004] The discovery of AQP4-IgG, a specific biomarker for diagnosing NMOSD, laid the foundation for the immunopathogenesis of neuromyelitis optica. Currently, serum AQP4-IgG positivity has been included as one of the diagnostic criteria for NMOSD. Therefore, obtaining high-quality target proteins as raw materials for detection reagents to detect AQP4-IgG is of great significance and helps improve the accuracy of early diagnosis.

[0005] Furthermore, using only M1 or M23 proteins in detection reagents can easily lead to false negatives. Therefore, improving the accuracy of AQP4-IgG detection is an urgent problem to be solved. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a fusion protein for detecting aquaporin 4 antibodies, its preparation method and application, which can simultaneously detect M1 and M23 aquaporin 4, and has high sensitivity, accuracy and good repeatability in detecting aquaporin 4 antibodies.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a fusion protein for detecting aquaporin 4 antibodies, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0009] This invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps:

[0010] The coding gene sequences of M1 type AQP4 and M23 type AQP4 were double-digested and then ligated into a backbone vector to obtain a recombinant vector.

[0011] The recombinant vector was transformed into E. coli, and the recombinant vector was amplified to obtain the amplified recombinant vector.

[0012] The amplified recombinant vector was transfected into human embryonic kidney cells HEK293 and cultured to obtain transfected cells;

[0013] Finally, the fusion protein was extracted from the transfected cells.

[0014] Preferably, the coding gene sequence of the M1 type AQP4 is shown in SEQ ID NO.1, and the coding gene sequence of the M23 type AQP4 is shown in SEQ ID NO.3; the dual enzymes are EcoRI enzyme and XhoI enzyme; and the backbone vector is pET-28a(+) vector.

[0015] Preferably, the Escherichia coli includes Escherichia coli DH5α.

[0016] Preferably, the transfection method includes: adding the amplified recombinant vector and PEI transfection reagent to human embryonic kidney cells HEK293, mixing, allowing to stand, culturing, and obtaining transfected cells.

[0017] Preferably, the extraction method includes: mixing transfected cells with cell lysis buffer, repeatedly freezing and thawing, centrifuging at 2-6°C and 800-1200g for 5-15 min to collect the supernatant, centrifuging the supernatant at 2-6°C and 12000-16000g for 25-35 min to collect the precipitate, dissolving the precipitate with membrane protein extraction buffer to obtain a cell membrane protein solution; mixing His purification resin with the cell membrane protein solution to obtain a mixture, centrifuging the mixture at 2-6°C and 800-1200g for 5-15 min, collecting the first precipitating resin, washing the first precipitating resin to obtain the second precipitating resin, then eluting with an imidazole-containing elution buffer, centrifuging to collect the supernatant to obtain the fusion protein.

[0018] The present invention also provides the application of the above-described fusion protein or the fusion protein obtained by the preparation method in the preparation of an antibody product for detecting aquaporin 4.

[0019] The present invention also provides a kit for detecting aquaporin 4 antibodies, the kit comprising the fusion protein described above or a fusion protein obtained by the preparation method described above.

[0020] Preferably, the kit includes an ELISA kit.

[0021] Preferably, the kit further includes horseradish peroxidase-labeled secondary antibody, standards, quality control products, enzyme substrate, washing solution, and stop solution.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a fusion protein for detecting aquaporin 4 antibodies, its preparation method, and its applications. Using this fusion protein as a coating antigen, it can simultaneously detect both M1 and M23 types of aquaporin 4, exhibiting high sensitivity, accuracy, and reproducibility in antibody detection. Furthermore, this invention utilizes this fusion protein as a coating antigen to prepare an enzyme-linked immunosorbent assay (ELISA) kit. The detection method is simple, rapid, and suitable for large-scale screening of aquaporin 4 antibodies in clinical and research settings. Attached Figure Description

[0024] Figure 1 This is an electrophoresis image of SDS-PAGE for detecting the expression of the fusion protein. The left lane represents the fusion protein, and the right lane represents the marker. Detailed Implementation

[0025] This invention provides a fusion protein for detecting aquaporin 4 antibodies, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0026] This invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps:

[0027] The coding gene sequences of M1 type AQP4 and M23 type AQP4 were double-digested and then ligated into a backbone vector to obtain a recombinant vector.

[0028] The recombinant vector was transformed into E. coli, and the recombinant vector was amplified to obtain the amplified recombinant vector.

[0029] The amplified recombinant vector was transfected into human embryonic kidney cells HEK293 and cultured to obtain transfected cells;

[0030] Finally, the fusion protein was extracted from the transfected cells.

[0031] In the above-mentioned method for preparing the fusion protein, the coding gene sequences of M1 type AQP4 and M23 type AQP4 are respectively double-digested with enzymes and then ligated into a backbone vector to obtain a recombinant vector. The coding gene sequence of M1 type AQP4 is shown in SEQ ID NO.1, and the coding gene sequence of M23 type AQP4 is shown in SEQ ID NO.3; the double enzymes are EcoRI enzyme and XhoI enzyme; and the backbone vector is a pET-28a(+) vector.

[0032] After obtaining the recombinant vector, it is transformed into *E. coli*, and the recombinant vector is amplified to obtain the amplified recombinant vector. The *E. coli* includes *E. coli* DH5α. This invention does not specifically limit the transformation method; conventional transformation methods in the art can be used to achieve the purpose of amplifying the recombinant vector.

[0033] After obtaining the amplified recombinant vector, it was transfected into human embryonic kidney cells (HEK293) and cultured to obtain transfected cells. The transfection method included: adding the amplified recombinant vector and PEI transfection reagent to HEK29 human embryonic kidney cells, mixing well, allowing to stand, and culturing to obtain transfected cells. The mass-to-volume ratio of the recombinant vector to the PEI transfection reagent was 5–15:15–25 μg / μL; the standing time was preferably 10–20 min, more preferably 12–18 min, and even more preferably 13, 14, 15, 16, or 17 min. The complete culture medium refers to a basal culture medium prepared by adding 10% fetal bovine serum (FBS) to it and mixing thoroughly. The basal culture medium included DMEM or MEM. The second standing time was preferably 10–20 min, more preferably 12–18 min, and even more preferably 13, 14, 15, 16, or 17 min. The culture conditions are 37°C and 5% CO2 for 40–50 h, more preferably 37°C and 5% CO2 for 42–48 h, and even more preferably 37°C and 5% CO2 for 48 h. This invention utilizes HEK293 cells to express fusion proteins, enabling efficient production of fusion proteins to meet the needs of large-scale production and facilitating the production of more bioactive fusion proteins.

[0034] After obtaining transfected cells, the fusion protein was extracted from the transfected cells. The extraction method included: mixing the transfected cells with cell lysis buffer, repeatedly freezing and thawing, centrifuging at 2–6°C and 800–1200g for 5–15 min to collect the supernatant, centrifuging the supernatant at 2–6°C and 12000–16000g for 25–35 min to collect the precipitate, dissolving the precipitate in membrane protein extraction buffer to obtain a cell membrane protein solution; mixing His purification resin with the cell membrane protein solution to obtain a mixture, centrifuging the mixture at 2–6°C and 800–1200g for 5–15 min, collecting the first precipitated resin, washing the first precipitated resin to obtain a second precipitated resin, eluting with an imidazole-containing elution buffer, centrifuging to collect the supernatant to obtain the fusion protein. The cell lysis buffer was prepared by mixing 4.95 mL of 1×PBS and 50 μL of 100× protease inhibitor (PI) evenly (5 mL per 5 mL). The repeated freeze-thaw cycles were performed three times to achieve cell disruption. The 1 mL cell membrane protein extract was prepared by mixing 50 μL of 1M Tris-HCl (pH 7.4), 150 μL of 1M NaCl, 10 μL of NP-40, 5 μL of Triton-X-100, 10 μL of 100×PI, and 775 μL of H2O evenly. The His purification resin was preferably His purification resin washed with PBS, more preferably His purification resin washed with PBS three times. The washing method involved washing the first precipitated resin 2-4 times with 1×PBS pre-cooled at 2-6°C, more preferably washing the first precipitated resin 3 times with 1×PBS pre-cooled at 4°C; each washing was performed by centrifugation at 1000g for 5 min at 4°C, and the precipitated resin was collected.

[0035] The present invention can successfully obtain a fusion protein with the sequence shown in SEQ ID NO.5 by means of the above preparation method.

[0036] The present invention also provides the application of the above-described fusion protein or the fusion protein obtained by the preparation method in the preparation of an antibody product for detecting aquaporin 4.

[0037] In this invention, the product is preferably a reagent or a kit.

[0038] The present invention also provides a kit for detecting aquaporin 4 antibodies, the kit comprising the fusion protein described above or a fusion protein obtained by the preparation method described above.

[0039] In this invention, the kit includes an ELISA kit. Further, the kit also includes a secondary antibody labeled with HRP, standards, quality controls, enzyme substrate, washing buffer, and stop solution. Taking the ELISA kit as an example, the ELISA kit contains a microplate coated with a fusion protein. The preparation method of the microplate coated with the fusion protein includes: coating a 96-well microplate with the 8-12 μg / mL fusion protein solution, adding 100 μL of the 8-12 μg / mL fusion protein solution to each well, incubating overnight at 4°C, blocking with 200 μL of phosphate buffer containing 1% bovine serum albumin per well, incubating at 37°C for 0.5-1.5 h, and washing 2-4 times with PBS buffer to obtain the microplate coated with the fusion protein. The HRP-labeled secondary antibody is an anti-human IgG secondary antibody labeled with HRP. The method for preparing the HRP-labeled anti-human IgG secondary antibody includes: labeling the anti-human IgG secondary antibody with horseradish peroxidase (HRP), and diluting it with PBS buffer containing 1% BSA to a volume ratio of 1:4500-5500 to obtain the HRP-labeled anti-human IgG secondary antibody. The source of the anti-human IgG secondary antibody is not particularly limited in this invention; commercially available products in the art can be used. The enzyme substrate is tetramethylbenzidine. The washing buffer is preferably phosphate buffer containing 0.1% Tween. The stop solution is preferably a 0.5M sulfuric acid solution. The standard is a PBS solution containing anti-AQP4 antibody at concentrations of 81 RU / mL, 27 RU / mL, 9 RU / mL, 3 RU / mL, and 1 RU / mL. The quality control includes positive and negative controls. The positive control is a sample dilution containing anti-AQP4 antibody at a concentration range of 5-10 RU / mL; the negative control is a sample dilution without anti-AQP4 antibody.

[0040] The present invention utilizes the above-mentioned kit to rapidly detect AQP4-IgG, and the detection of AQP4-IgG has high sensitivity, accuracy and good repeatability.

[0041] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] In the following examples, the 100× protease inhibitor was purchased from Thermo Fisher Scientific, catalog number 87785; the NP-40 was purchased from Thermo Fisher Scientific, catalog number 85125; and the anti-human IgG secondary antibody was purchased from Merck, catalog number AP101P.

[0044] The 250mM imidazole eluent is prepared by weighing 1.702g of imidazole, dissolving it in deionized water, and making up to 100mL.

[0045] The eluent containing 0.5 mM imidazole is prepared by taking 0.2 mL of 250 mM imidazole solution and diluting it to 100 mL with deionized water.

[0046] The sample diluent is PBS.

[0047] Example 1

[0048] The purpose of this embodiment is to prepare a protein for the accurate detection of AQP4-IgG. The specific steps of the method for preparing the protein for detecting AQP4-IgG are as follows:

[0049] 1. Preparation of recombinant vector pET-28a(+)-M1-M23: The coding sequences of M1-type AQP4 and M23-type AQP4 were directly synthesized, digested with EcoRI and XhoI respectively, and ligated into the pET-28a(+) vector to construct the recombinant vector pET-28a(+)-M1-M23. After confirmation by PCR and double enzyme digestion, the recombinant vector pET-28a(+)-M1-M23 was transformed into Escherichia coli DH5α, the plasmid was amplified, and the plasmid was extracted according to the instructions of the plasmid extraction kit.

[0050] The encoding sequence for M1 type AQP4 is as follows:

[0051] aggcggtggggtaagtgtggacctttgtgtaccagagagaacatcatggtggctttcaaaggggtctggactcaag

[0052] ctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccaccatcaactggggtg

[0053] gaacagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaaccatggtgcagtgctttg

[0054] gccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatcagcatcgccaagtct

[0055] gtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacctcccagtgtggtgg

[0056] gaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataatcacatttcaattgg

[0057] tgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattggattttctgttgcaattg

[0058] gacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatcatgggaaattggg

[0059] aaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgtcttctgtccagatgt

[0060] tgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaggaagctacatggaggtggaggaca

[0061] acaggagtcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttgaccggggagagga

[0062] gaagaaggggaaagaccaatctggagaggtattgtcttcagtatga(SEQ ID NO.1).

[0063] The M1 protein sequence is as follows:

[0064] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIF

[0065] VLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTV

[0066] AMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLT

[0067] AGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGAS

[0068] MNPARSFAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQV

[0069] ETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV(SEQ ID NO.2)。

[0070] The coding sequence of M23-type AQP4 is as follows:

[0071] atggtggctttcaaaggggtctggactcaagctttctggaaagcagtcacagcggaatttctggccatgcttatttttg

[0072] ttctcctcagcctgggatccaccatcaactggggtggaacagaaaagcctttaccggtcgacatggttctcatctccctttgc

[0073] tttggactcagcattgcaaccatggtgcagtgctttggccatatcagcggtggccacatcaaccctgcagtgactgtggcca

[0074] tggtgtgcaccaggaagatcagcatcgccaagtctgtcttctacatcgcagcccagtgcctgggggccatcattggagca

[0075] ggaatcctctatctggtcacacctcccagtgtggtgggaggcctgggagtcaccatggttcatggaaatcttaccgctggtc

[0076] atggtctcctggttgagttgataatcacatttcaattggtgtttactatctttgccagctgtgattccaaacggactgatgtcactg

[0077] gctcaatagctttagcaattggattttctgttgcaattggacatttatttgcaatcaattatactggtgccagcatgaatcccgcc

[0078] cgatcctttggacctgcagttatcatgggaaattgggaaaaccattggatatattgggttgggcccatcataggagctgtcct

[0079] cgctggtggcctttatgagtatgtcttctgtccagatgttgaattcaaacgtcgttttaaagaagccttcagcaaagctgccca

[0080] gcaaacaaaaggaagctacatggaggtggaggacaacaggagtcaggtagagacggatgacctgattctaaaacctgg

[0081] agtggtgcatgtgattgacgttgaccggggagaggagaagaaggggaaagaccaatctggagaggtattgtcttcagtat

[0082] ga(SEQ ID NO.3)。

[0083] The amino acid sequence of M23 protein is as follows:

[0084] MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDM

[0085] VLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLG

[0086] AIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDS

[0087] KRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIY

[0088] WVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRS

[0089] QVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV(SEQ ID NO.4)。

[0090] The correct fusion protein sequence identified by PCR and double digestion is as follows:

[0091] aggcggtggggtaagtgtggacctttgtgtaccagagagaacatcatggtggctttcaaaggggtctggactcaag

[0092] ctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccaccatcaactggggtg

[0093] gaacagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaaccatggtgcagtgctttg

[0094] gccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatcagcatcgccaagtct

[0095] gtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacctcccagtgtggtgg

[0096] gaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataatcacatttcaattgg

[0097] tgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattggattttctgttgcaattg

[0098] gacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatcatgggaaattggg

[0099] aaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgtcttctgtccagatgt

[0100] tgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaagaagctacatggaggtggaggaca

[0101] acaggatcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttgaccggggagagga

[0102] gaaaggggaaagaccaatctggagaggtattgtcttcagtatgaggggsggggggggsatggtggctttcaaaggg

[0103] gtctggactcaagctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccacc

[0104] atcaactggggtggaacaagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaacat

[0105] ggtgcagtgctttggccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatca

[0106] gcatcgccaagtctgtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacc

[0107] tcccagtgtggtgggaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataa

[0108] tcacatttcaattggtgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattgga

[0109] ttttctgttgcaattggacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatc

[0110] atgggaaattgggaaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgt

[0111] cttctgtccagatgttgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaggaagctacatg

[0112] gaggtggaggacaacaggagtcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttga

[0113] ccggggagaggagaagaaggggaaagaccaatctggagaggtattgtcttcagtatga (SEQ ID NO. 5).

[0114] 2. Expression of fusion protein

[0115] (1) Cell inoculation

[0116] HEK293 cells were seeded into culture dishes at a rate of approximately 6 million cells per dish, so that the density could reach 70-80% confluence by the next day.

[0117] (2) Cell transfection

[0118] Before transfection, each dish needs to be replaced with 12 mL of complete culture medium. Take two clean, sterile centrifuge tubes and add 500 μL of LMEM high-glucose medium to each. Then, add 10 μg of plasmid to one tube and gently mix with a pipette. Add 20 μL of PEI transfection reagent to the other tube (the mass-to-volume ratio of plasmid to transfection reagent is 1:2) and mix thoroughly. Then, gently add the culture medium containing the plasmid to the culture medium containing the transfection reagent with a pipette and gently mix with a pipette. Let it stand at room temperature for 15 min to obtain the plasmid-PEI mixed solution. Drop the prepared plasmid-PEI mixed solution into the culture dish, mix gently, and incubate in a cell culture incubator for 48 hours.

[0119] 3. Extraction of fusion proteins

[0120] A. If the transfection efficiency is above 90% when observed under an inverted microscope, remove the culture medium directly, removing it as completely as possible, taking care not to perform any digestion of the cells. (If not used immediately, store in a -80℃ freezer).

[0121] B. Add 5 mL of cell lysis buffer to the cell culture plate, and use a pipette to transfer all the cells into a centrifuge tube. The 5 mL cell lysis buffer is prepared by mixing 4.95 mL of 1×PBS and 50 μL of 100×protease inhibitor (PI) thoroughly.

[0122] C. Place the entire tube of cells in liquid nitrogen for 1 minute, and repeat the freeze-thaw cycle three times to destroy the cells.

[0123] D. Removal of cell nuclei and undisturbed cells: Centrifuge the cells at 4℃ and 1000g for 10 min and collect the supernatant.

[0124] E. Precipitate cell membrane debris: Centrifuge the supernatant obtained in step D at 4°C and 14000g for 30 min, and collect the precipitate.

[0125] F. The precipitated cells obtained in step E are resuspended and dissolved in 500 μL of membrane protein extraction solution to obtain a cell membrane protein solution; wherein, the 1 mL cell membrane protein extraction solution is prepared by mixing 50 μL of 1 M Tris-HCl, 150 μL of 1 M NaCl, 10 μL of NP-40, 5 μL of Triton-X-100, 100×PI, and 775 μL of H2O at pH 7.4.

[0126] G. Take 250 μL of His-tagged purification resin, wash it 3 times with PBS, add 500 μL of the obtained cell membrane protein solution, and shake it in a shaker at room temperature for 60 min to obtain a mixture.

[0127] H. Centrifuge the mixture obtained in step G at 4°C and 1000g for 5 min, and collect the first precipitated resin. Wash the collected first precipitated resin three times with 1×PBS pre-cooled at 4°C to obtain the second precipitated resin. Each washing step involves centrifuging at 4°C and 1000g for 5 min, and collecting the precipitated resin.

[0128] I. The second precipitating resin was washed sequentially with 1 mL of elution buffer containing 0.5 mM imidazole and 500 μL of 250 mM imidazole elution buffer, respectively. The mixture was vortexed at 4 °C for 60 min, centrifuged at 1000 g for 5 min, and the supernatant was collected to obtain the fusion protein.

[0129] The expression of the fusion protein was detected by SDS-PAGE, and the results are shown in [Figure number missing]. Figure 1 Meanwhile, the sequence of the fusion protein was obtained by sequencing as shown in SEQ ID NO.5.

[0130] Figure 1 The results show that the present invention successfully obtained the fusion protein as shown in SEQ ID NO.5.

[0131] Example 2

[0132] An ELISA kit for improving the accuracy of AQP4-IgG detection includes: a microplate coated with a fusion protein, a secondary antibody labeled with HRP and containing anti-human IgG, standards, quality controls, phosphate buffer with a volume fraction of 0.1% Tween, tetramethylbenzidine (TMB), and 0.5 M sulfuric acid solution.

[0133] The preparation of the microplate coated with the target protein is as follows: The fusion protein prepared in Example 1 is diluted to 10 μg / mL with phosphate buffer (pH 7.4). The resulting 10 μg / mL fusion protein solution is used to coat a 96-well microplate. 100 μL of the 10 μg / mL fusion protein solution is added to each well, and the plate is incubated overnight at 4°C. The plate is then blocked with 200 μL of phosphate buffer (PBS) containing 1% bovine serum albumin (BSA) per well, incubated at 37°C for 1 h, and washed three times with PBS buffer to obtain the microplate coated with the fusion protein.

[0134] Preparation of the HRP-labeled anti-human IgG secondary antibody: The horseradish peroxidase (HRP)-labeled anti-human IgG secondary antibody was diluted with PBS buffer containing 1% BSA to a volume ratio of 1:5000 to obtain the HRP-labeled anti-human IgG secondary antibody. This HRP-labeled anti-human IgG secondary antibody is used to form a complex with AQP4-IgG in the sample and generate a signal.

[0135] Using TMB as an enzyme substrate, it reacts with horseradish peroxidase to produce a measurable color change, resulting in an ELISA kit for improving the accuracy of AQP4-IgG detection.

[0136] The standard is a sample diluent containing different concentrations of anti-AQP4 antibody, with five concentrations of 81RU / mL, 27RU / mL, 9RU / mL, 3RU / mL and 1RU / mL.

[0137] The quality control materials are positive and negative controls. The positive control is a sample diluent containing anti-AQP4 antibody, with a concentration range of 5–10 RU / mL; the negative control is a sample diluent without anti-AQP4 antibody.

[0138] Comparative Example 1

[0139] The difference between this comparative example and Example 2 is that the target protein in this comparative example is the M1 type target protein with the nucleotide sequence SEQ ID NO.1, which replaces the fusion protein prepared in Example 1. This results in a microplate coated with the M1 type target protein alone, and ultimately an ELISA kit with M1 type as the coating antigen alone. The other kit components are the same as in Example 2, resulting in an ELISA kit with M1 type as the coating antigen alone.

[0140] Example 3

[0141] The purpose of this embodiment is to simultaneously detect AQP4-IgG in the serum of 40 clinical cases using the ELISA kit provided in Embodiment 2 of the present invention and the ELISA kit with M1 type as the coating antigen alone in Comparative Example 1, and to compare it with the cell-based assay (CBA) reagent, which is considered the "gold standard". The specific steps are as follows:

[0142] 1. Detect AQP4-IgG in serum using the ELISA kit of Example 2 or Comparative Example 1.

[0143] (1) Place the kit at room temperature, take out the microplate coated with fusion protein or the microplate coated with M1 type target protein alone, and add 100 μL of the serum sample to be tested diluted with sample diluent (1:100 dilution), standard, negative control and positive control to each well. Incubate at 37°C for 30 min to allow AQP4-IgG in the serum sample to bind to the coated fusion protein or M1 type target protein (antigen);

[0144] (2) Remove the sample from the well of the microplate, add 300 μL of 0.1% Tween phosphate buffer per well, let stand for 10 s, remove the liquid in the well, repeat three times to wash away the unbound sample, and obtain AQP4-IgG complex.

[0145] (3) Add 100 μL of horseradish peroxidase (HRP)-anti-human IgG secondary antibody to each well of the microplate and incubate at 37°C for 30 min to allow the secondary antibody to bind to the AQP4-IgG complex.

[0146] (4) Add 300 μL of 0.1% Tween phosphate buffer per well, let stand for 10 seconds, remove the liquid in the well, repeat three times to wash away the unbound complex.

[0147] (5) Add 100 μL of TMB enzyme substrate to each well and incubate at room temperature for 15 min to allow the enzyme to react with the substrate and produce a color change; add 50 μL of stop solution (0.5 M sulfuric acid solution) to each well to stop the reaction, and the solution color changes from blue to yellow; read the absorbance (OD) of the microplate with an ELISA reader at a wavelength of 450 nm and calculate the concentration of AQP4-IgG. The intensity of the solution color is directly proportional to the concentration of the antibody.

[0148] (6) Plot the standard curve: Plot the concentration value of the calibrator on the x-axis and the absorbance OD value of the corresponding calibrator on the y-axis. Use double logarithmic (LOG10) linear regression or other suitable mathematical models to fit the dose-response curve and plot the standard curve.

[0149] (7) Substitute the absorbance OD value of the sample to be tested into the linear regression equation of the standard curve to obtain the AQP4-IgG concentration value of the sample to be tested.

[0150] When using the ELISA kit provided in Example 2 of the present invention, a positive result can be obtained when the concentration of AQP4-IgG in the serum sample to be tested is greater than 3RU / mL, that is, the serum sample to be tested contains the antibody AQP4-IgG.

[0151] 2. Use the CBA reagent kit for detection.

[0152] (1) Place the kit at room temperature, remove the slide, add 30 μL of the serum sample to be tested diluted with sample diluent (1:10 dilution) to the reaction area of ​​the slide, and incubate at 37°C for 1 h to allow the AQP4-IgG in the sample to bind to the cell antigen on the slide.

[0153] (2) Add washing solution and repeat three times to wash away unbound samples to obtain AQP4-IgG complex;

[0154] (3) Drain the slide thoroughly, add 30 μL of fluorescent secondary antibody working solution to the reaction area of ​​the slide, and incubate at 37°C for 30 min to allow the secondary antibody to bind to the AQP4-IgG complex;

[0155] (4) Add washing solution and repeat three times;

[0156] (5) Add 30 μL of washing buffer (containing 0.1% Tween phosphate buffer) to each reaction zone;

[0157] (6) Observe and interpret the results using a fluorescence microscope. The presence of green fluorescence indicates that the serum sample to be tested contains the antibody AQP4-IgG.

[0158] 3. Results Analysis

[0159] Table 1 Comparison of results between the ELSIA kit of the present invention and the CBA kit.

[0160]

[0161] Note: "+" indicates that the serum sample to be tested contains antibody AQP4-IgG, and "-" indicates that the serum sample to be tested does not contain antibody AQP4-IgG.

[0162] Table 2 Comparison of results between standalone M1 type ELSIA kit and CBA kit

[0163]

[0164] The results in Tables 1 and 2 show that the ELSIA kit of the present invention has a 100% concordance rate with the CBA kit, while the concordance rate between the M1 type ELSIA kit alone and the CBA kit is not completely consistent, with a concordance rate of 95%. Therefore, the ELSIA kit of the present invention has higher accuracy.

[0165] Example 4

[0166] The analytical performance evaluation methods and results of the kit in Example 2 of this invention are as follows, including the evaluation methods and results of the limit of detection, linear range, and repeatability.

[0167] 1. Limit of detection

[0168] 1.1 Detection Method: Sample diluent (PBS) was used as the test sample. The detection method was the same as the method for detecting AQP4-IgG in serum described in Part "1" of Example 3. The measurement was repeated 20 times, and the absorbance values ​​(OD values) of the 20 measurements were obtained. The average value (M) and standard deviation (SD) were calculated to obtain the OD value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the calibration curve equation of the calibrator used in the kit or based on the concentration-OD value results between the zero-concentration calibrator and adjacent calibrators. The OD value corresponding to M+2SD was substituted into the above equation to obtain the corresponding concentration value, which is the limit of detection.

[0169] 1.2 Acceptance criteria: not greater than 0.5 RU / mL.

[0170] Table 3 Calibration Results

[0171] Concentration (RU / mL) OD value 1 OD value 2 Average OD 81 1.3253 1.3776 1.351 27 0.576 0.5974 0.587 9 0.2315 0.2532 0.242 3 0.1013 0.1073 0.104 1 0.0418 0.0466 0.044

[0172] Based on the results in Table 3, a standard curve for calculating sample concentration was plotted, and the detection limit concentration was obtained according to the results of the lowest detection limit in Table 4.

[0173] Table 4 Results of the Limit of Detection

[0174]

[0175]

[0176] The results in Table 4 show that the limit of detection for the kit of the present invention is 0.24 RU / mL.

[0177] 2. Linear range

[0178] 2.1 Test Method

[0179] Linearity testing was performed on five concentrations of calibrators, with each concentration sample tested twice. The average concentration was calculated, and the average result was fitted to a linear relationship with the dilution ratio using the least squares method. The linear correlation coefficient r was calculated. The testing method was performed according to the method for detecting AQP4-IgG in serum described in Part "1" of Example 3.

[0180] 2.2 Acceptance criteria: r is not less than 0.9900.

[0181] Table 5 Linear Range Results

[0182]

[0183] Based on the results in Table 5, the calculated linear correlation coefficient r is 0.9996. Therefore, the kit of the present invention shows good linearity in the detection of AQP4-IgG.

[0184] 3. Repeatability

[0185] 3.1 Test Method

[0186] Using the same batch of kits from Example 2 of this invention, AQP4 antibody samples at two concentration levels (15 RU / mL and 27 RU / mL, respectively) were tested, with each test repeated 10 times. The mean M and standard deviation SD of the 10 test concentrations were calculated, and the coefficient of variation CV was obtained according to the formula CV = SD / M × 100%.

[0187] 3.2 Acceptance criteria: CV not greater than 12%.

[0188] 3.3 Results:

[0189] Table 6 Repeatability Results

[0190]

[0191]

[0192] The results in Table 6 show that the CV of AQP4-IgG detected by the kit of the present invention is no greater than 12%, which means that the kit of the present invention has good repeatability in detecting AQP4-IgG.

[0193] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a fusion protein of an aquaporin 4 antibody for the manufacture of a product for detecting an aquaporin 4 antibody, characterized in that, A nucleotide sequence of the fusion protein is shown as SEQ ID NO.

5.

2. Use according to claim 1, characterized in that, The preparation method of the fusion protein comprises the following steps: coding sequence of M1 type AQP4 and coding sequence of M23 type AQP4 are double enzyme cut respectively, and then are connected to a skeleton vector to obtain a recombinant expression vector; the recombinant expression vector is transformed into E. coli, and the recombinant expression vector is amplified to obtain an amplified recombinant expression vector; the amplified recombinant expression vector is transfected into HEK293 cells, and the cells are cultured to obtain transfected cells; finally, the fusion protein in the transfected cells is extracted.

3. Use according to claim 2, characterized in that, The coding sequence of M1 type AQP4 is shown as SEQ ID NO. 1, and the coding sequence of M23 type AQP4 is shown as SEQ ID NO.

3. The double enzyme is EcoRI enzyme and XhoI enzyme; and the skeleton vector is pET-28a (+) vector.

4. Use according to claim 2, characterized in that, The E. coli includes E. coli DH5α.

5. Use according to claim 2, characterized in that, The transfection method comprises the following steps: adding the amplified recombinant expression vector and PEI transfection reagent into HEK293 cells, mixing, standing, culturing, and obtaining transfected cells.

6. Use according to claim 2, characterized in that, The extraction method comprises the following steps: mixing the transfected cells with a cell lysis solution, repeatedly freezing and thawing, centrifuging at 2-6 DEG C, 800-1200g for 5-15 min to take supernatant, centrifuging the supernatant at 2-6 DEG C, 12000-16000g for 25-35 min to take sediment, dissolving the sediment with a membrane protein extraction solution to obtain a cell membrane protein solution; mixing His purification resin with the cell membrane protein solution to obtain a mixed solution, centrifuging the mixed solution at 2-6 DEG C, 800-1200g for 5-15 min to take first sediment resin, cleaning the first sediment resin to obtain second sediment resin, then eluting with an eluent containing imidazole, centrifuging to take supernatant, and obtaining the fusion protein.

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