A chemiluminescent test kit for detecting anti-acetylcholine receptor antibodies

Abstract

The present application provides a chemiluminescence detection kit for detecting anti-acetylcholine receptor antibody, which comprises reagent 1, reagent 2, reagent 3 and reagent 4, wherein the reagent 1 is a magnetic bead suspension coated with acetylcholine receptor antigen, the reagent 2 is a solution of agitoxin, the reagent 3 is a solution of anti-human IgG polyclonal antibody coupled with alkaline phosphatase, and the reagent 4 is a solution of chemiluminescence substrate; the acetylcholine receptor antigen in the reagent 1 is a combination of human acetylcholine receptor alpha subunit recombinant protein fragment with sequence as shown in SEQ ID NO:1, human acetylcholine receptor beta subunit recombinant protein fragment with sequence as shown in SEQ ID NO:2, human acetylcholine receptor delta subunit recombinant protein fragment with sequence as shown in SEQ ID NO:3 and human acetylcholine receptor epsilon subunit recombinant protein fragment with sequence as shown in SEQ ID NO:4. The kit has simpler components and lower preparation cost.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic biochemical detection technology, specifically relating to a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies. Background Technology

[0002] The acetylcholine receptor (AChR) is an ion channel receptor located at the neuromuscular junction. AChR is a transmembrane protein and belongs to the ligand-gated ion channel family. The main function of AChR is to mediate nerve signal transmission, converting nerve impulses into muscle contractions or other cellular responses. There are two subtypes of AChR: embryonic and mature. Embryonic AChR is composed of α2, β, δ, and γ subunits, while mature AChR is composed of α2, β, δ, and ε subunits.

[0003] AChR is an important biomarker protein for myasthenia gravis. Approximately 85% of myasthenia gravis patients have detectable autoantibodies against AChR (anti-acetylcholine receptor antibodies, AChR-Ab), and the detection of these antibodies is an important clinical diagnostic tool for myasthenia gravis. Furthermore, the detection of anti-acetylcholine receptor antibodies is also a valuable indicator for diagnosing Lambert-Eaton syndrome.

[0004] The main methods for detecting AChR autoantibodies are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and cell genetic engineering. Radioimmunoassay utilizes the binding of radiolabeled AChR antigens to antibodies in the patient's serum, determining antibody levels by detecting the intensity of the radioactive signal. Its advantages include high sensitivity and specificity, but it requires radioactive materials and is relatively cumbersome, making full automation difficult. ELISA detects the amount of antibodies in the patient's serum that bind to AChR antigens immobilized on an ELISA plate using enzyme-labeled antibodies, quantifying antibody levels through a colorimetric reaction. Its advantages include simplicity and the ability to process samples in batches, but it is susceptible to various factors during manual operation and is time-consuming. Cell genetic engineering uses transgenic cells or cell fragments expressing AChR as antigens, which bind to AChR-Abs in the patient's serum. The binding is then detected using fluorescently labeled secondary antibodies. This method has the advantages of detecting antibodies with specific conformations and high sensitivity, but it is complex, time-consuming, and requires sophisticated equipment.

[0005] Patent CN118294644A provides a chemiluminescence immunoassay kit for detecting anti-acetylcholine receptor antibodies, which offers improved sensitivity, specificity, and stability. However, patent CN118294644A uses a mixture of magnetically labeled α, β, δ, γ, and ε subunit antigens as the AChR antigen, which is difficult to prepare and relatively expensive, limiting its widespread application in clinical diagnostics. Summary of the Invention

[0006] The purpose of this invention is to provide a chemiluminescent detection reagent for detecting anti-acetylcholine receptor antibodies with simpler components and lower preparation cost, as well as a kit containing the chemiluminescent detection reagent.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] This invention provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies, comprising reagent 1, reagent 2, reagent 3, and reagent 4. Reagent 1 is a magnetic bead suspension coated with acetylcholine receptor antigen; reagent 2 is a krait venom solution; reagent 3 is an anti-human IgG polyclonal antibody solution conjugated with alkaline phosphatase; and reagent 4 is a chemiluminescent substrate solution. The acetylcholine receptor antigen in reagent 1 is a combination of recombinant protein fragments of the human acetylcholine receptor α subunit (as shown in SEQ ID NO:1), the human acetylcholine receptor β subunit (as shown in SEQ ID NO:2), the human acetylcholine receptor δ subunit (as shown in SEQ ID NO:3), and the human acetylcholine receptor ε subunit (as shown in SEQ ID NO:4).

[0009] Preferably, the mass ratio of the recombinant protein fragment of the human acetylcholine receptor α subunit, the recombinant protein fragment of the human acetylcholine receptor β subunit, the recombinant protein fragment of the human acetylcholine receptor δ subunit, and the recombinant protein fragment of the human acetylcholine receptor ε subunit is (1.8~2.2):(0.8~1.2):(0.8~1.2):1.

[0010] Preferably, the solvent of reagent 1 is a Tris buffer solution with a pH of 7.2 to 7.6 containing 100 to 250 mM sodium chloride.

[0011] Preferably, the magnetic beads in reagent 1 are EDC-activated carboxyl-modified magnetic beads.

[0012] More preferably, the average particle size of the carboxyl-modified magnetic beads is 0.5~5μm, and in some embodiments, the average particle size of the carboxyl-modified magnetic beads is 1μm.

[0013] Preferably, the concentration of the cladocarpus venom solution is 0.8~1.2mM.

[0014] Preferably, the solvent for the clams venom solution is a phosphate buffer solution with a pH of 7.2 to 7.6.

[0015] Preferably, the anti-human IgG polyclonal antibody in reagent 3 is a goat anti-human IgG polyclonal antibody, a rabbit anti-human IgG polyclonal antibody, or a chicken anti-human IgG polyclonal antibody.

[0016] Preferably, the solvent of reagent 3 is a Tris buffer solution with a pH of 7.2 to 7.6 containing 100 to 250 mM sodium chloride.

[0017] Preferably, the chemiluminescent substrate in reagent 4 is adenosine 5'-phosphate sulfate.

[0018] Preferably, the solvent of reagent 4 is a Tris buffer solution with a pH of 7.2 to 7.6 containing 100 to 200 mM sodium chloride and 0.5 to 2 mM magnesium chloride.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention uses a mixture of specific sequences of recombinant protein fragments from the α-subunit, β-subunit, δ-subunit, and ε-subunit of the human acetylcholine receptor as an acetylcholine receptor antigen composition. A chemiluminescent detection kit is prepared using this antigen composition. Verification has shown that the anti-acetylcholine receptor antibody exhibits high sensitivity, good specificity, and accurate detection results. Compared to methods using mixtures of full-length recombinant α-subunit, β-subunit, δ-subunit, γ-subunit, and / or ε-subunit recombinant proteins as acetylcholine receptor antigens, the acetylcholine receptor antigen composition of this invention is easier and less expensive to prepare, thereby reducing the cost of chemiluminescent detection kits and promoting the widespread application of anti-acetylcholine receptor antibody chemiluminescent detection kits in clinical diagnosis. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0022] The information on raw materials and reagents in the following examples and comparative examples is as follows:

[0023] Carboxyl-modified magnetic beads (Sera-Mag™, Cytiva); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride, Thermo Fisher); goat anti-human IgG polyclonal antibody conjugated with alkaline phosphatase (Merck, Sigma); positive calibrator for anti-acetylcholine receptor antibody (Merck, Sigma). Both the recombinant full-length protein and the recombinant protein fragment were prepared in-house and verified by amino acid sequence analysis.

[0024] Example 1: This example provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies (kit specification: 100 kits / kit), including reagent 1, reagent 2, reagent 3, reagent 4, and washing solution. The preparation method is as follows:

[0025] Reagent 1:

[0026] 5 mg of carboxyl-modified magnetic beads (1 μm in diameter) were added to 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride. Then, 0.5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) was added for activation for 1 hour. After activation, the magnetic beads were collected using a magnet, the supernatant was discarded, and the beads were washed with 10 mL of 20 mM phosphate buffer (pH 7.4). The beads were collected again using a magnet, the supernatant phosphate buffer was discarded, and the beads were resuspended in 10 mL of 20 mM phosphate buffer (pH 7.4) to obtain the activated magnetic bead suspension.

[0027] Mix 2 mg of recombinant human acetylcholine receptor α subunit protein fragment, 1 mg of recombinant human acetylcholine receptor β subunit protein fragment, 1 mg of recombinant human acetylcholine receptor δ subunit protein fragment, and 1 mg of recombinant human acetylcholine receptor ε subunit protein fragment, add the mixture to a magnetic bead suspension, incubate for 1 hour, collect the magnetic beads using a magnet, discard the supernatant, wash the magnetic beads with 10 mL of 20 mM phosphate buffer (pH 7.4), collect the magnetic beads again using a magnet, discard the supernatant phosphate buffer, and resuspend the magnetic beads coated with the four recombinant protein fragments in 10 mL of 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride, to obtain Reagent 1.

[0028] The amino acid sequence of the recombinant protein fragment of the human acetylcholine receptor α subunit is as follows:

[0029] SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKV LLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL (SEQ IDNO:1).

[0030] The amino acid sequence of the recombinant protein fragment of the human acetylcholine receptor β subunit is as follows:

[0031] SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVSS DGSVRWQPPGIYRSSCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRK (SEQ ID NO:2).

[0032] The amino acid sequence of the recombinant protein fragment of the human acetylcholine receptor δ subunit is as follows:

[0033] LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLVYH YGFVYWLPPAIFRSSCPISVTYFPFDWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSSPRQDITFYLIIRRK (SEQ ID NO:3).

[0034] The amino acid sequence of the recombinant protein fragment of the human acetylcholine receptor ε subunit is as follows:

[0035] KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVY EGGSVTWLPPAIYRSVCAVEVTYFPFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRK (SEQ ID NO:4).

[0036] Reagent 2: Prepare a 1mM solution of krait venom using 5mL of 20mM phosphate buffer (pH 7.4).

[0037] Reagent 3: Mix 0.2 µL of goat anti-human IgG polyclonal antibody conjugated with alkaline phosphatase with 10 mL of 20 mM Tris buffer containing 150 mM sodium chloride at pH 7.4 to obtain Reagent 3.

[0038] Reagent 4: Dissolve 0.085 g of adenosine 5'-phosphate sulfate (APS) in 10 mL of 20 mM Tris buffer at pH 7.4 containing 150 mM sodium chloride and 1 mM magnesium chloride to obtain Reagent 4.

[0039] Cleaning solution: phosphate buffer solution with pH 7.4 containing 0.05% triazolam.

[0040] Comparative Example 1: This comparative example provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies (kit specification: 100 kits / kit), including reagent 1, reagent 2, reagent 3, reagent 4, and washing solution. The only difference between this comparative example's chemiluminescent detection kit and Example 1 is reagent 1. In this comparative example, the preparation of reagent 1 is identical to that of Example 1, except that the 2 mg recombinant protein fragment of the human acetylcholine receptor α subunit is replaced with 4 mg of the full-length recombinant protein of the human acetylcholine receptor α subunit, the 1 mg recombinant protein fragment of the human acetylcholine receptor β subunit is replaced with 2 mg of the full-length recombinant protein of the human acetylcholine receptor β subunit, the 1 mg recombinant protein fragment of the human acetylcholine receptor δ subunit is replaced with 2 mg of the full-length recombinant protein of the human acetylcholine receptor δ subunit, and the 1 mg recombinant protein fragment of the human acetylcholine receptor ε subunit is replaced with 2 mg of the full-length recombinant protein of the human acetylcholine receptor ε subunit.

[0041] In this comparative example, the amino acid sequence of the recombinant full-length protein of the human acetylcholine receptor α subunit is as follows:

[0042] MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNN ADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRL PLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHVMPNWVRKVFIDTIPNIMF FSTMKRPSREKQDKKIFTEDIDISDISGKPGPPPMGFHSPLIKHPEVKSAIEGIKYIAETMKSDQESNNAAAEWKYVAMVMDHILLGVFMLVCIIGTLAVFAGRLIELNQQG (SEQ ID NO:5).

[0043] The amino acid sequence of the recombinant full-length protein of the human acetylcholine receptor β subunit is as follows:

[0044] MTPGALLMLLGALGAPLAPGVRGSEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVA LDISVVVSSDGSVRWQPPGIYRSSCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKPLFYLVNV IAPCILITLLAIFVFYLPPDAGEKMGLSIFALLTLTVFLLLLADKVPETSLSVPIIIKYLMFTMVLVTFSVILSVVVLNLHHRSPHTHQMPLWVRQIFIHKLPLYLRLKRPKPERDLMPEPHCSS PGSGWGRGTDEYFIRKPPSDFLFPKPNRFQPELSAPDLRRFIDGPNRAVALLPELREVVSSISYIARQLQEQEDHDALKEDWQFVAMVVDRLFLWTFIIFTSVGTLVIFLDATYHLPPPDPFP (SEQ ID NO:6).

[0045] The amino acid sequence of the recombinant full-length protein of the human acetylcholine receptor δ subunit is as follows:

[0046] MEGPVLTLGLLAALAVCGSWGLNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCN VLVYHYGFVYWLPPAIFRSSCPISVTYFPFDWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSSPRQDITFYLIIRRKPLFYIINILVPCVLI SFMVNLVFYLPADSGEKTSVAISVLLAQSVFLLLISKRLPATSMAIPLIGKFLLFGMVLVTMVVVICVIVLNIHFRTPSTHVLSEGVKKLFLETLPELLHMSRPAEDGPSPGALVRRSSSLGYISKAEEY FLLKSRSDLMFEKQSERHGLARRLTTARRPPASSEQAQQELFNELKPAVDGANFIVNHMRDQNNYNEEKDSWNRVARTVDRLCLFVVTPVMVVGTAWIFLQGVYNQPPPQPFPGDPYSYNVQDKRFI (SEQ IDNO:7).

[0047] The amino acid sequence of the recombinant full-length protein of the human acetylcholine receptor ε subunit is as follows:

[0048] MARAPLGVLLLLGLLGRGVGKNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAY DANVLVYEGGSVTWLPPAIYRSVCAVEVTYFPFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKPLFYVINII VPCVLISGLVLLAYFLPAQAGGQKCTVSINVLLAQTVFLFLIAQKIPETSLSVPLLGRFLIFVMVVATLIVMNCVIVLNVSQRTPTTHAMSPRLRHVLLELLPRLLGSPPPPEAPRAASPPRRA SSVGLLLRAEELILKKPRSELVFEGQRHRQGTWTAAFCQSLGAAAPEVRCCVDAVNFVAESTRDQEATGEEVSDWVRMGNALDNICFWAALVLFSVGSSLIFLGAYFNRVPDLPYAPCIQP (SEQ ID NO:8).

[0049] Comparative Example 2: This comparative example provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies (kit specification: 100 kits / kit), including reagent 1, reagent 2, reagent 3, reagent 4, and washing solution. The difference between this comparative example's chemiluminescent detection kit and Example 1 is the amino acid sequence and amount of the recombinant protein fragments of the human acetylcholine receptor α subunit, β subunit, δ subunit, and ε subunit used in the preparation of reagent 1.

[0050] In this comparative example, 4 mg of recombinant human acetylcholine receptor α subunit protein fragment was used in the preparation of reagent 1. Its amino acid sequence is as follows:

[0051] MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEI IVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGK (SEQ ID NO:9).

[0052] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor β subunit is as follows:

[0053] MTPGALLMLLGALGAPLAPGVRGSEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVSSDGSVRWQPPGIYRS SCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKPLFYLVNVIAPCILITLLAIFVFYLPPDAGEKMGLSIFALLTLTVFLLLLA (SEQ ID NO:10).

[0054] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor δ subunit is as follows:

[0055] MEGPVLTLGLLAALAVCGSWGLNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLVYHYGFVYWLPPAIFRSSC PISVTYFPFDWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSPSRQDITFYLIIRRKPLFYIINILVPCVLISFMVNLVFYLPADSGEKTSVAISVLLAQSVFLLLISKRL (SEQ ID NO:11).

[0056] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor ε subunit is as follows:

[0057] MARAPLGVLLLLGLLGRGVGKNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLVYEGGSVTWLPPAIYRSVCAVEVTYF PFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRKPLFYVINIIVPCVLISGLVLLAYFLPAQAGGQKCTVSINVLLAQTVFLFLIAQKIPETSLSVPLLGR (SEQ ID NO:12).

[0058] Comparative Example 3: This comparative example provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies (kit specification: 100 kits / kit), including reagent 1, reagent 2, reagent 3, reagent 4, and washing solution. The only difference between this comparative example's chemiluminescent detection kit and Example 1 is the amino acid sequence and amount of the recombinant protein fragments of the human acetylcholine receptor α subunit, β subunit, δ subunit, and ε subunit used in the preparation of reagent 1.

[0059] In this comparative example, 4 mg of recombinant human acetylcholine receptor α subunit protein fragment was used in the preparation of reagent 1. Its amino acid sequence is as follows:

[0060] THHRSPTHVMPNWVRKVFIDTIPNIMFFSTMKRPSREKQDKKIFTEDIDISDISGKPGPPPMGFHSPLIKHPEVKSAIEGIKYIAETMKSDQESNNAAAEWKYVAMVMD (SEQ ID NO: 13).

[0061] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor β subunit is as follows:

[0062] HHRSPHTHQMPLWVRQIFIHKLPLYLRLKRPKPERDLMPEPPHCSSPGSGWGRGTDEYFIRKPPSDFLFPKPNRFQPELSAPDLRRFIDGPNRAVALLPELREVVSSISYIARQLQEQEDHDALKEDWQFVAMVVDR (SEQ ID NO: 14).

[0063] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor δ subunit is as follows:

[0064] HFRTPSTHVLSEGVKKLFLETLPELLHMSRPAEDGPSPGALVRRSSSLGYISKAEEYFLLKSRSDLMFEKQSERHGLARRLTTARRPPASSEQAQQELFNELKPAVDGANFIVNHMRDQNNYNEEKDSWNRVARTVDR (SEQ ID NO: 15).

[0065] The amino acid sequence of the 2 mg recombinant protein fragment of the human acetylcholine receptor ε subunit is as follows:

[0066] SQRTPTTHAMSPRLRHVLLELLPRLLGSPPPPEAPRAASPPRRASSVGLLLRAEELILKKPRSELVFEGQRHRQGTWTAAFCQSLGAAAPEVRCCVDAVNFVAESTRDQEATGEEVSDWVRMGNALDN (SEQ ID NO: 16).

[0067] Comparative Example 4: This comparative example provides a chemiluminescent detection kit for detecting anti-acetylcholine receptor antibodies (kit specification: 100 kits / kit), including reagent 1, reagent 2, reagent 3, reagent 4, and washing solution. The only difference between this comparative example's chemiluminescent detection kit and Example 1 is the amount of recombinant protein fragments of the human acetylcholine receptor α subunit, β subunit, δ subunit, and ε subunit during the preparation of reagent 1, and the addition of a recombinant protein fragment of the human acetylcholine receptor γ subunit.

[0068] In this comparative example, the amount of recombinant protein fragment of the human acetylcholine receptor α subunit was 4 mg, the amount of recombinant protein fragment of the human acetylcholine receptor β subunit was 2 mg, the amount of recombinant protein fragment of the human acetylcholine receptor δ subunit was 1 mg, the amount of recombinant protein fragment of the human acetylcholine receptor ε subunit was 1 mg, and the amount of recombinant protein fragment of the human acetylcholine receptor γ subunit was 1 mg. The amino acid sequence of the recombinant protein fragment of the human acetylcholine receptor γ subunit is as follows:

[0069] RNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNVLVS PDGCIYWLPPAIFRSACSISVTYFPFDWQNCSLIFQSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRK (SEQ ID NO:17).

[0070] The method of using the chemiluminescent detection kits for anti-acetylcholine receptor antibodies in Example 1 and each comparative example is as follows:

[0071] (1) Place 50µL of reagent 2 and 100µL of reagent 1 in a reaction vessel and incubate at 37°C for 10 minutes;

[0072] (2) Use a magnet to attract the magnetic beads in the reaction cup, and clean the magnetic beads with 5 mL of cleaning solution;

[0073] (3) Dilute 2µL of the sample to be tested in 18µL of PBS buffer with a pH of 7.4 and add it to the reaction vessel. Incubate at 37°C for 10 minutes.

[0074] (4) Use a magnet to attract the magnetic beads in the reaction cup, and clean the magnetic beads with 5 mL of cleaning solution;

[0075] (5) Add 100µL of reagent 3 and incubate at 37℃ for 5 minutes;

[0076] (6) Use a magnet to attract the magnetic beads in the reaction cup, and clean the magnetic beads with 5 mL of cleaning solution;

[0077] (7) Add 50µL of reagent 4 and incubate at 37℃ for 5 minutes;

[0078] (8) The total number of internal photons generated in 10 seconds of reaction was detected and counted by the Yingkai I2900 chemiluminescence detector as the reaction signal.

[0079] 1. Detect linear range

[0080] The high-value anti-acetylcholine receptor antibody positive calibrator was diluted in PBS buffer (pH 7.4) to six concentrations: 10 nmol / L, 5 nmol / L, 2 nmol / L, 1 nmol / L, 0.5 nmol / L, and 0.3 nmol / L. Each diluted sample was measured three times using the kits from Example 1 and Comparative Examples 1-4, and the mean response signal y was calculated. The linear regression equation and correlation coefficient |r| of the standard curve were obtained with each diluted sample as the independent variable x and y as the dependent variable. The results are shown in Table 1.

[0081]

[0082] 2. Limit of detection

[0083] Using PBS buffer with a pH of 7.4 as the test sample, the chemiluminescence detection kits of Example 1 and Comparative Examples 1-4 were used to repeatedly measure the test sample 20 times. The detected reaction signal values ​​were substituted into the standard curve of the corresponding kit in Table 1 to obtain the measured concentration. The mean (M) and standard deviation (SD) of the measured concentration were calculated, and M+2SD was obtained as the limit of detection. The results are shown in Table 2.

[0084]

[0085] Table 2 shows that the limit of detection of the chemiluminescence detection kit in Example 1 is 0.2 nmol / L, which is significantly lower than that of Comparative Examples 1-4.

[0086] 3. Repeatability

[0087] The chemiluminescence detection kits of Example 1 and Comparative Examples 1-4 were used to repeatedly test quality control 1 (high-value anti-acetylcholine receptor antibody positive calibrator diluted to 0.5 nmol / L in PBS buffer at pH 7.4) and quality control 2 (high-value anti-acetylcholine receptor antibody positive calibrator diluted to 5 nmol / L in PBS buffer at pH 7.4) 10 times each. The test results are shown in Table 3. The test results are in nmol / L. The coefficient of variation (CV) was calculated based on the test results. If the CV is not greater than 10%, the performance of the kit meets the requirements.

[0088]

[0089] 4. Positive and Negative Reference Values

[0090] Sixty samples with positive values ​​were tested using the chemiluminescence detection kits of Example 1 and Comparative Examples 1-4, including 32 positive samples and 28 negative samples. The results are shown in Table 4.

[0091]

[0092] Table 4 shows that the sample detection sensitivity of the chemiluminescence detection kit in Example 1 was 93.75% (30 / 32), and the sample detection specificity was 92.86% (26 / 28). The sample detection sensitivity of the chemiluminescence detection kit in Comparative Example 1 was 46.87% (15 / 32), and the sample detection specificity was 64.29% (18 / 28). The sample detection sensitivity of the chemiluminescence detection kit in Comparative Example 2 was 62.50% (20 / 32), and the sample detection specificity was 71.4% (20 / 28). The sample detection sensitivity of the chemiluminescence detection kit in Comparative Example 3 was 15.63% (5 / 32), and the sample detection specificity was 57.14% (16 / 28). The sample detection sensitivity of the chemiluminescence detection kit in Comparative Example 4 was 93.75% (30 / 32), and the sample detection specificity was 96.43% (27 / 28).

[0093] Twenty clinically positive serum samples were tested using the chemiluminescence detection kit described in Example 1 and the Euromon anti-acetylcholine receptor antibody (AChR antibody) enzyme-linked immunosorbent assay kit (Anti-Acetylcholine Receptor ELISA (IgG)). The detection method of the EU enzyme-linked immunosorbent assay kit was performed according to its instructions. The detection results are shown in Table 5.

[0094]

[0095] Table 5 shows that the correlation between the detection results of the chemiluminescence detection kit of Example 1 and the detection results of the widely used mature ELISA kit for clinical positive serum samples is 0.98, indicating that the chemiluminescence detection method of Example 1 has high consistency with the results of the widely used mature ELISA detection method.

[0096] The results above show that the chemiluminescence detection kit of Example 1 has high sensitivity and affinity for anti-acetylcholine receptor antibodies, better linear range, low detection limit, and good repeatability. Compared with Comparative Examples 1-4, it not only has better overall performance, but its antigen composition is also easier to prepare than the full-length antigen protein composition, resulting in lower cost and thus reducing the cost of the chemiluminescence detection kit.

Claims

1. A chemiluminescent test kit for detecting anti-acetylcholine receptor antibodies, characterized by, The kit comprises reagent 1, reagent 2, reagent 3 and reagent 4, the reagent 1 is a magnetic bead suspension coated with acetylcholine receptor antigen, the reagent 2 is a solution of Bungarotoxin, the reagent 3 is a solution of anti-human IgG polyclonal antibody coupled with alkaline phosphatase, and the reagent 4 is a solution of chemiluminescent substrate, the acetylcholine receptor antigen in the reagent 1 is a composition of human acetylcholine receptor alpha subunit recombinant protein fragment with sequence as shown in SEQ ID NO:1, human acetylcholine receptor beta subunit recombinant protein fragment with sequence as shown in SEQ ID NO:2, human acetylcholine receptor delta subunit recombinant protein fragment with sequence as shown in SEQ ID NO:3 and human acetylcholine receptor epsilon subunit recombinant protein fragment with sequence as shown in SEQ ID NO:

4.

2. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, characterized by, The mass ratio of the human acetylcholine receptor alpha subunit recombinant protein fragment, the human acetylcholine receptor beta subunit recombinant protein fragment, the human acetylcholine receptor delta subunit recombinant protein fragment and the human acetylcholine receptor epsilon subunit recombinant protein fragment is (1.8~2.2):(0.8~1.2):(0.8~1.2):

1.

3. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, characterized by, The solvent of the reagent 1 is Tris buffer with pH value of 7.2~7.6 containing 100~250mM sodium chloride.

4. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, wherein, The concentration of the Bungarotoxin solution is 0.8~1.2mM.

5. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 4, characterized by, The solvent of the Bungarotoxin solution is phosphate buffer with pH value of 7.2~7.

6.

6. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, wherein, The anti-human IgG polyclonal antibody in the reagent 3 is goat anti-human IgG polyclonal antibody, rabbit anti-human IgG polyclonal antibody or chicken anti-human IgG polyclonal antibody.

7. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 6, characterized by, The solvent of the reagent 3 is Tris buffer with pH value of 7.2~7.6 containing 100~250mM sodium chloride.

8. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, wherein, The chemiluminescent substrate in the reagent 4 is adenosine 5'-phosphosulfate.

9. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, wherein, The solvent of the reagent 4 is Tris buffer with pH value of 7.2~7.6 containing 100~200mM sodium chloride and 0.5~2mM magnesium chloride.

10. The chemiluminescent test kit for detecting anti-acetylcholine receptor antibody according to claim 1, wherein, The chemiluminescent detection kit further comprises a washing solution, which is phosphate buffer with pH value of 7.2~7.6 containing 0.01wt%~0.1wt% Triton X-100.

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