Compositions and kits

Abstract

The present application relates to the technical field of in vitro diagnosis, in particular to a composition and a kit, and provides a magnetic microparticle chemiluminescence kit for detecting coxsackievirus A10 IgM, and further provides a recombinant antigen EV71, CA16 and CA6 combination for blocking cross infection and improving the specific detection rate of CA10, which comprises magnetic particles coated with mouse anti-human IgM monoclonal antibody, horseradish peroxidase-labeled coxsackievirus A10 antigen, horseradish peroxidase-labeled coxsackievirus EV71 type, A16 type and A6 type combined blocking antigen. The kit provided by the present application has high sensitivity and specificity, and can distinguish EV71 type, CA16 type and CA6 infection cross in hand-foot-mouth disease, specifically detect A10 pathogens, and has high repeatability and simple operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnosis, in particular to a composition and a kit. BACKGROUND

[0002] Hand-foot-mouth disease is a type of class C infectious disease caused by multiple viruses, and Coxsackievirus A10 (CVA10) is one of the pathogens causing hand-foot-mouth disease, mainly infecting children under the age of 5, and often breaking out in places where children are concentrated, such as kindergartens. The pathogen causing hand-foot-mouth disease also includes Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16).

[0003] CVA10 belongs to the family of Enterovirus of the family Picornaviridae, and the virus is in the shape of a spherical or oval symmetric structure of a regular icosahedron, has no envelope, and has a diameter of 28nm-32nm. The genome of CVA10 is a single-stranded RNA virus with a total length of about 7.4kb, and the 5' and 3' non-coding regions (UTRs) are at both ends of the viral RNA coding region.

[0004] The clinical manifestations of CVA10 are mostly similar to those of EV71 and CVA16, but CVA10 can cause herpangina (HA), onychomadesis, and fatal complications, including infantile myelitis with hypercapnia and sepsis. CVA10 virus can cause severe hand-foot-mouth disease, and the main clinical manifestations are fever, maculopapular rash, and vesicular exudate, limb tremor, or central nervous system involvement. A small number of patients show symptoms of the nervous system and even convulsions or complications of heart and lung function.

[0005] Currently, CVA10 virus detection is mainly through virus isolation, serological neutralization experiments, and molecular biology methods. Virus isolation and identification methods are complicated and time-consuming, and serological detection methods have poor specificity. PCR has high sensitivity and good specificity, but the clinical specimen may contain substances that inhibit PCR amplification, resulting in false negative results or low quantitative values; and PCR requires expensive equipment and has high costs. SUMMARY

[0006] Therefore, the present application provides a composition and a kit, and the recombinant antigen developed by the present application can specifically detect CVA10 virus IgM, which has great significance for the diagnosis and prevention and control of diseases.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] The present application provides a composition comprising a recombinant antigen of coxsackievirus A10, a recombinant antigen of enterovirus 71, a recombinant antigen of coxsackievirus A16 and a recombinant antigen of coxsackievirus A6;

[0009] The recombinant antigen of coxsackievirus A10 comprises an enzyme-labeled recombinant antigen of coxsackievirus A10; and / or

[0010] The enzyme-labeled recombinant antigen of coxsackievirus A10 comprises a horseradish peroxidase-labeled recombinant antigen of coxsackievirus A10; and / or

[0011] The recombinant antigen of coxsackievirus A10 comprises a 3CD protein of coxsackievirus A10 and / or a P1 protein of coxsackievirus A10; and / or

[0012] The recombinant antigen of enterovirus 71 comprises a 3CD protein of enterovirus 71 and / or a P1 protein of enterovirus 71; and / or

[0013] The recombinant antigen of coxsackievirus A16 comprises a 3CD protein of coxsackievirus A16 and / or a P1 protein of coxsackievirus A16; and / or

[0014] The recombinant antigen of coxsackievirus A6 comprises a 3CD protein of coxsackievirus A6 and / or a P1 protein of coxsackievirus A6;

[0015] Wherein:

[0016] The 3CD protein of coxsackievirus A10 has:

[0017] (1) an amino acid sequence as shown in SEQ ID NO: 1; or

[0018] (2) an amino acid sequence obtained by substitution, deletion or addition of one or more residues of the amino acid sequence as shown in (1), and the function is the same or similar to that of (1); or

[0019] (3) an amino acid sequence having at least 70% homology with the amino acid sequence as shown in (1) or (2);

[0020] and / or

[0021] The P1 protein of coxsackievirus A10 has:

[0022] (4) an amino acid sequence as shown in SEQ ID NO: 2; or

[0023] (5) the amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence of (4), and having the same or similar function as that of (4); or

[0024] (6) an amino acid sequence having at least 70% homology with the amino acid sequence of (4) or (5);

[0025] and / or

[0026] The enterovirus 71 3CD protein has:

[0027] (7) the amino acid sequence shown in SEQ ID NO: 5; or

[0028] (8) the amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence of (7), and having the same or similar function as that of (7); or

[0029] (9) an amino acid sequence having at least 70% homology with the amino acid sequence of (7) or (8);

[0030] and / or

[0031] The enterovirus 71 P1 protein has:

[0032] (10) the amino acid sequence shown in SEQ ID NO: 6; or

[0033] (11) the amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence of (10), and having the same or similar function as that of (10); or

[0034] (12) an amino acid sequence having at least 70% homology with the amino acid sequence of (10) or (11);

[0035] and / or

[0036] The coxsackievirus A16 3CD protein has:

[0037] (13) the amino acid sequence shown in SEQ ID NO: 3; or

[0038] (14) the amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence of (13), and having the same or similar function as that of (13); or

[0039] (15) an amino acid sequence having at least 70% homology with the amino acid sequence of (13) or (14);

[0040] and / or

[0041] The Coxsackie virus A16 type P1 protein has:

[0042] (16) the amino acid sequence shown in SEQ ID NO: 4; or

[0043] (17) an amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (16), and having the same or similar functions as (16); or

[0044] (18) an amino acid sequence having at least 70% homology with the amino acid sequence shown in (16) or (17);

[0045] The Coxsackie virus A6 type 3CD protein has:

[0046] (19) the amino acid sequence shown in SEQ ID NO: 7; or

[0047] (20) an amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (19), and having the same or similar functions as (19); or

[0048] (21) an amino acid sequence having at least 70% homology with the amino acid sequence shown in (19) or (20);

[0049] and / or

[0050] The Coxsackie virus A6 type P1 protein has:

[0051] (22) the amino acid sequence shown in SEQ ID NO: 8; or

[0052] (23) an amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (22), and having the same or similar functions as (22); or

[0053] (24) an amino acid sequence having at least 70% homology with the amino acid sequence shown in (22) or (23);

[0054] The plurality is 2 to 200.

[0055] In some specific embodiments of the present application, in the above-mentioned composition:

[0056] The Coxsackie virus A10 type includes one or more of Coxsackie virus A10 type 16-2871-1 strain, Coxsackie virus A10 type S0148 strain, Coxsackie virus A10 type S0273b strain, or Coxsackie virus A10 type BA72 / YN / CHN / 2015 strain; and / or

[0057] The enterovirus 71 includes enterovirus 71 Fuyang.Anhui.P.R.C / 17.08 / 3 strain; and / or

[0058] The coxsackievirus A16 includes coxsackievirus A16 R37 / YN / CHN / 2013 strain; and / or

[0059] The coxsackievirus A6 includes coxsackievirus A6 SHAPHC6069 / SH / CHN / 15 strain.

[0060] In some embodiments of the present application, the preparation method of the horseradish peroxidase-labeled coxsackievirus A10 recombinant antigen in the above composition includes: incubating the coxsackievirus A10 recombinant antigen with HRP at a molar ratio of 1:(1-10) for labeling to obtain the horseradish peroxidase-labeled coxsackievirus A10 recombinant antigen.

[0061] In some embodiments of the present application, the preparation method of the horseradish peroxidase-labeled coxsackievirus A10 recombinant antigen in the above composition includes: incubating the coxsackievirus A10 recombinant antigen with HRP at a molar ratio of 1:5 for labeling to obtain the horseradish peroxidase-labeled coxsackievirus A10 recombinant antigen.

[0062] The present application also provides the use of the above composition in excluding false positives caused by enterovirus in coxsackievirus A10 detection;

[0063] The enterovirus includes one or more of coxsackievirus A16, coxsackievirus A6 or enterovirus 71.

[0064] The present application also provides the use of the above composition in preparing a reagent or kit for detecting anti-coxsackievirus A10 IgM.

[0065] The present application also provides the use of the above composition in the following aspects:

[0066] (a) preparing a diagnostic reagent or diagnostic kit for hand-foot-mouth disease; and / or

[0067] (b) preparing a diagnostic reagent or diagnostic kit for coxsackievirus A10 infection.

[0068] The present application also provides a reagent, including the above composition, and acceptable adjuvants or aids.

[0069] In some embodiments of the present application, the above reagent includes:

[0070] The molar ratio of the enterovirus 71 recombinant antigen, the coxsackievirus A16 recombinant antigen and the coxsackievirus A6 recombinant antigen is (10-1):1:(1-10).

[0071] The application further provides a kit comprising the above-mentioned reagent and an acceptable adjuvant or auxiliary agent.

[0072] In some specific embodiments of the application, the above-mentioned kit further comprises one or more of magnetic particles of a mouse anti-human IgM monoclonal antibody, a magnetic particle preservation solution, a sample diluent, an enzyme conjugate diluent, a washing solution, a chromogenic solution, a termination solution, a negative control or a positive control.

[0073] In some specific embodiments of the application, the above-mentioned kit comprises:

[0074] The positive control comprises a human-mouse chimeric anti-coxsackievirus A10 antibody; and / or

[0075] The negative control comprises a PBS buffer containing 20% BSA; and / or

[0076] The sample diluent comprises a 0.05M Tris buffer containing 2% BSA with a pH of 7.4; and / or

[0077] The washing solution comprises a PBS buffer containing 0.1% Tween 20; and / or

[0078] The enzyme conjugate diluent comprises a 0.05M Tris buffer containing 20% BSA with a pH of 7.4; and / or the chromogenic solution comprises a buffer containing luminol.

[0079] The composition or kit of the application has the following effects:

[0080] 1. The addition of EV71, CA16 and CA6 blocking antigens in the kit can achieve specific detection of hand-foot-mouth disease caused by CA10 infection, avoiding cross-infection caused by EV71, CA16 and CA6 infection.

[0081] 2. By preparing a recombinant CA10 virus-like particle antigen, the risk of infection caused by natural virus source materials can be avoided, and batch consistency can be ensured.

[0082] 3. The recombinant antigen is labeled with horseradish peroxidase and assembled into a coxsackievirus A10 IgM antibody capture kit, which can specifically recognize coxsackievirus A10 IgM positive serum. BRIEF DESCRIPTION OF DRAWINGS

[0083] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or the prior art description will be briefly introduced as follows.

[0084] Figure 1 SDS-PAGE polyacrylamide gel electrophoresis was used to detect the purity of the antigen protein. DETAILED DESCRIPTION

[0085] The present application discloses compositions and kits, and those skilled in the art can refer to the content herein to realize appropriate process parameter improvements. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0086] The application discloses a magnetic particle chemiluminescence kit for specifically detecting IgM of coxsackie virus A10 type, and relates to construction and purification preparation of recombinant virus antigens of coxsackie virus A10 type, and relates to combined use of recombinant antigens EV71, CA16 and CA6 for closed cross infection and improvement of CA10 specificity detection rate. The magnetic particle chemiluminescence kit for specifically detecting IgM of coxsackie virus A10 type comprises magnetic particles coated with mouse anti-human-IgM monoclonal antibodies, horseradish peroxidase-labeled coxsackie virus A10 type antigens, and horseradish peroxidase-labeled coxsackie virus EV71 type, A16 type and A6 type combined blocking antigens. The kit provided by the application has high sensitivity and specificity, and can distinguish EV71 type, CA16 type and CA6 infection cross in hand-foot-mouth disease, specifically detect A10 pathogens, has high repeatability and is simple to operate.

[0087] The application aims at the demand for pathogen detection of hand-foot-mouth disease, and develops a reagent kit for specifically detecting IgM antibodies of coxsackie virus A10 type, and the reagent kit can avoid cross immunoreactions of EV71, CA16 and CA6, wherein,

[0088] The first object of the application is to provide a recombinant antigen construction scheme which can be used for specifically detecting IgM antibodies of coxsackie virus A10 type.

[0089] The second object of the application is to provide a recombinant antigen purification scheme which can be used for specifically detecting IgM antibodies of coxsackie virus A10 type.

[0090] The third object of the present application is to provide a method for specifically detecting coxsackie virus A10 IgM antibody, which avoids cross-immune reaction of EV71, CA16 and CA6.

[0091] The fourth object of the present application is to provide a kit for specifically detecting coxsackie virus A10 IgM antibody.

[0092] To achieve the first object, the technical solution of the present application is that the sequence of the recombinant antigen of coxsackie virus A10 is derived from one or more of 16-2871-1 strain, S0148 strain, S0273b strain and BA72 / YN / CHN / 2015 strain of coxsackie virus A10.

[0093] To achieve the second object, the technical solution of the present application is that the high-purity recombinant antigen of coxsackie virus A10 is obtained by isocratic centrifugation, density gradient centrifugation and anion exchange chromatography using an insect baculovirus expression system.

[0094] To achieve the third object, the technical solution of the present application is that the optimal blocking scheme is obtained by adjusting the addition ratio of blocking antigens of EV71, CA16 and CA6.

[0095] To achieve the fourth object, the technical solution of the present application is that the magnetic microparticle chemiluminescence kit for specifically detecting anti-coxsackie virus A10 IgM antibody comprises any one of the molecules described above.

[0096] The sequences involved in the present application are as follows:

[0097] SEQ ID NO. 1:

[0098] MGPSLDFALSLLRRNIRQAQTDQGHFTMLGVRDRLAILPRHAQPGKTIWIEHKLVNILDAVELVDEQGVNLELTLVTLDTNEKFRDITKFIPETISGASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVIGIHIGGNGRQGFCAGLKRSYFASEQGEIQWMKSNKETGRLNINGPTQTKLEPSVFHDVFEGNKEPAVLTSKDPRLEVDFEQALFSKYVGNTLHEPDEYVTQAALHYANQLKQLDINTNKMSMEDACYGTENLEAIDLHTSAGYPYSAIGIRKRDILDPISRDTTKMKFYMDKYGLDLPYSTYVKDELRSLDKIKKGKSRLIEASSLNDSVYLRMTFGHLYEVFHANPGTITGSAVGCNPDVFWSKLPILLPGALFAFDYSGYDASLSPVWFRALEVVLREIGYSEEAVSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRTLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELARTGKEYGLTMTPADKSPCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMKEIHESIRWTKDARNTQDHVRSLCLLAWHNGKEEYEKFVSTIRSVPIGKALAIPNFENLRRNWLELF

[0099] SEQ ID NO. 2:

[0100] MGAQVSTQKSGSHETGNVATGGSTINFTNINYYKDSYAASATRQDFTQDPKKFTQPVLDSIRELSGPLNSPSVEACGYSDRVAQLTVGNSSITTQEAANIVLAYGEWPEYCPDTDATAVDKPTRPDVSVNRFYTLDSKMWQENSTGWYWKFPDVLNKTGVFGQNAQFHYLYRSGFCLHVQCNASKFHQGALLVAVIPEFVIAGRGANTKPNEAPHPGFTTTFPGTTGATFHDPYILDSGVPLSQALIYPHQWINLRTNNCATIIVPYINAVPFDSAINHSNFGLVVVPVSPLKYSSGATTAIPITITIAPLNSEFGGLRQAVSQGIPAELRPGTNQFLTTDDDTAAPILPGFTPTPVIHIPGEVHSLLELCRVETILEVNNTTEATGLSRLLIPVSSQNKADELCAAFMVDPGRIGPWQSTLVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLIAYSPPGSAQPVNRETAMLGTHVIWDFGLQSSVSLVIPWISNTHFRTAKTGGNYDYYTAGVVTLWYQTNYVVPPETPGEAYIIAMGAAQDNFTLKICKDTDEVTQQAVLQGDPVEDIIHDALGSTVRRAINNTSNVESAANTAPSSHRLETGRVPALQAAETGATSNATDENMIETRCVVNRNGVLEASINHFFSRSGLVGVVNMSDGDTDTTGYATWDIDIMGFVQLRRKCEMFTYMRFNAEFTFVTTTKNGEARPYMLQYMYVPPGAPKPTGRDAFQWQTATNPSIFVKLTDPPAQVSVPFMSPASAYQWFYDGYPTFGQHPETSNTAYGLCPNNMMGTFAVRVVSTVASQLKLQTRVYMKLKHVKAWVPRPIRSQPYLLKNFPNYDNSKIANSARDRSSIKQANM

[0101] SEQ ID NO. 3:

[0102] MGPSLNFALSLLRRNIRQVQTDQGHFTMLGVRDRLPILPRHSQPGKTIWVEHKLINVLDAVELVDEQGVNLELTLVTLDTNEKFRDVTKFIPETITGASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRGYFASEQGEIQWMKSNKETGRLNINGPTRTKLEPSVFYDVFEGSKEPAVLTSKDPRLEVDFEQALFSKYVGNTLHEPDEYATQAALHYANQLKQLDININKMSMEEACYGTEYLEAIDLHTSAGYPYNALGVKKRDILDPITRDTTKMKFYMDKYGLDLPYSTYVKDELRSLDKIKKGKSRLIEASSLNDSVYLRMTFGHLYETFHANPGTVTGSAVGCNPDVFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALEVVLREIGYSEEAVSLIEGINHTHHVYRNRTYCVLGGMPSGCSGTSIFNSMINNIIIRTLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCSELARTGKEYGLTMTPADKSPCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKEEYEEFVSTIRSVPIGKALAIPNFENLRRNWLELF

[0103] SEQ ID NO. 4:

[0104] MGSQVSTQRSGSHENSNSASEGSTINYTTINYYKDAYAASAGRQDMSQDPKKFTDPVMDVIHEMAPPLKSPSAEACGYSDRVAQLTIGNSTITTQEAANIVIAYGEWPEYCPDTDATAVDKPTRPDVSVNRFFTLDTKSWAKDSKGWYWKFPDVLTEVGVFGQNAQFHYLYRSGFCVHVQCNASKFHQGALLVAVLPEYVLGTIAGGTGNENSHPPYATTQPGQVGAVLTHPYVLDAGIPLSQLTVCPHQWINLRTNNCATIIVPYMNTVPFDSALNHCNFGLLVVPVVPLDFNAGATSEIPITVTIAPMCAEFAGLRQAVKQGIPTELKPGTNQFLTTDDGVSAPILPGFHPTPPIHIPGEVHNLLEICRVETILEVNNLKTNETTPMQRLCFPVSVQSKTGELCAAFRADPGRDGPWQSTILGQLCRYYTQWSGSLEVTFMFAGSFMATGKMLIAYTPPGGNVPADRITAMLGTHVIWDFGLQSSVTLVVPWISNTHYRAHARAGYFDYYTTGIITIWYQTNYVVPIGAPTTAYIVALAAAQDNFTMKLCKDTEDIEQTANIQGDPIADMIDQTVNNQVNRSLTALQVLPTAADTEASSHRLGTGVVPALQAAETGASSNASDKNLIETRCVLNHHSTQETAIGNFFSRAGLVSIITMPTTGTQNTDGYVNWDIDLMGYAQLRRKCELFTYMRFDAEFTFVVAKPNGELVPQLLQYMYVPPGAPKPTSRDSFAWQTATNPSVFVKMTDPPAQVSVPFMSPASAYQWFYDGYPTFGEHLQANDLDYGQCPNNMMGTFSIRTVGTEKSPHSITLRVYMRIKHVRAWIPRPLRNQPYLFKTNPNYKGNDIKCTSTSRDKITTL

[0105] SEQ ID NO. 5:

[0106] MGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQGEIQWVKPNKETGRLNINGPTRTKLEPSVFHDIFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTLHEPDEYIKEAALHYANQLKQLEINTSQMSMEEACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSRMKFYMDKYGLDLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYEAFHANPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYSEEAISLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRALLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSPCFNEVNWGNATFLKRGFLPDEQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPNYENLRRNWLELF

[0107] SEQ ID NO. 6:

[0108] MGSQVSTQRSGSHENSNSATEGSTINYTTINYYKDSYAATAGKQSLKQDPDKFANPVKDIFTEMAAPLKSPSAEACGYSDRVAQLTIGNSTITTQEAANIIVGYGEWPSYCSDSDATAVDKPTRPDVSVNRFYTLDTKLWEKSSKGWYWKFPDVLTETGVFGQNAQFHYLYRSGFCIHVQCNASKFHQGALLVAVLPEYVIGTVAGGTGTEDTHPPYKQTQPGADGFELQHPYVLDAGIPISQLTVCPHQWINLRTNNCATIIVPYINALPFDSALNHCNFGLLVVPISPLDYDQGATPVIPITITLAPMCSEFAGLRQAVTQGFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKGELCAVFRADPGRNGPWQSTLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGGPLPKDRATAMLGTHVIWDFGLQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGAPNTAYIIALAAAQKNFTMKLCKDASDILQTGTIQGDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLKGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTL

[0109] SEQ ID NO. 7:

[0110] MGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWVEHKLVNILDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISAASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVIGIHIGGNGRQGFCAGLKRSYFASEQGEIQWVKSNKETGRFNINGPTRTKLEPSVFHDIFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTIHEPDEYIKEAALHYANQLKQLNIDTSQMSMEEACYGTDNLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSKMKFYMDKYGLDLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYETFHANPGTVTGSAVGCNPDVFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYGNEAISLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRSLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCSELARTGKEYGLTMTPADKSPCFNEVNWENATFLKRGFLPDEQFPFLIHPTMPMKEIHESIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSAIRSVPIGKALAIPNYENLRRNWLELF

[0111] SEQ ID NO. 8:

[0112] MGAQVSTEKSGSHETKNVATEGSTINFTNINYYKDSYAASASRQDFAQDPAKFTRPVLDTIREVAAPLQSPSVEACGYSDRVAQLTVGNSTITTQEAANIVLSYGEWPEYCPSTDATAVDKPTRPDVSVNRFYTLSTKSWKTESTGWYWKFPDVLNDTGVFGQNAQFHYLYRSGFCMHVQCNASKFHQGALLVAAIPEFVVAASSPATKPNGQGLYPDFAHTNPGKNGQEFRDPYVLDAGVPLSQALVYPHQWINLRTNNCATIIMPYVNALPFDSALNHSNFGLVVIPISPLKYCNGATTEVPITLTIAPLNSEFSGLRQAIKQGFPTELKPGTNQFLTTDDGTSPPILPGFEPTPLIHIPGEFTSLLDLCQIETILEVNNTTGTTGVSRLLIPVRAQNNVDQLCASFQVDPGRNGPWQSTMVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLIAYTPPGSAQPATREAAMLGTHIVWDFGLQSSVTLVIPWISNTHFRAVKTGGVYDYYATGIVTIWYQTNFVVPPDTPTEANIIALGAAQKNFTLKLCKDTDEIQQTAEYQNDPITNAVESAVSALADTTISRVTAANTAASTHSLGTGRVPALQAAETGASSNASDENLIETRCVMNRNGVNEASVEHFYSRAGLVGVVEVKDSGTSLDGYTVWPIDVMGFVQQRRKLELSTYMRFDAEFTFVSNLNDSTTPGMLLQYMYVPPGAPKPDSRKSYQWQTATNPSVFAKLSDPPPQVSVPFMSPATAYQWFYDGYPTFGEHKQATNLQYGQCPNNMMGHFAIRTVSESTTGKNVHVRVYMRIKHVRAWVPRPLRSQAYMVKNYPTYSQTITNTATDRASITTTDYEGGVPANPQRTS

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials, methods, and examples provided herein are illustrative only and in no way limiting. Other

[0114] The application is further described in conjunction with the following examples:

[0115] Preparation Example

[0116] (I) Virus Antigen Preparation

[0117] 1. Synthesis of Coxsackie virus A10 virus 16-2871-1 strain 3CD gene and P1 gene, as SEQ ID NO. 1 and SEQ ID NO. 2. A16 virus R37 / YN / CHN / 2013 strain 3CD gene and P1 gene, as SEQ ID NO. 3 and SEQ ID NO. 4, EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain 3CD gene and P1 gene, as SEQ ID NO. 5 and SEQ ID NO. 6 and A6 virus SHAPHC6069 / SH / CHN / 15 strain 3CD gene and P1 gene, as SEQ ID NO. 7 and SEQ ID NO. 8.

[0118] 2. Clone the synthesized Coxsackie virus A10 virus 16-2871-1 strain 3CD gene and P1 gene into pFastBac TM Dual Expression Vector at the same time, transform into DH10bac strain, obtain recombinants by blue-white spot screening and PCR identification.

[0119] 3. The preparation method of P3 generation recombinant baculovirus containing Coxsackie virus A10 virus 16-2871-1 strain P1 gene refers to the instruction manual of bac-to-bac baculovirus expression system of Thermo-fisher company.

[0120] 4. Coxsackie virus EV71, A16 and A6 type virus can be constructed according to this method.

[0121] (II) Virus Particle Antigen Purification

[0122] There are many methods for purifying virus particles. In the present application, two different pad media are preferably used in sequence, and virus particle antigens are obtained by isocratic and gradient centrifugation in sequence, and ion exchange affinity purification is also used, and high-purity virus antigens are obtained by three kinds of purification methods in series. The specific operation steps are as follows:

[0123] 1. The supernatant of the cultured cells is first replaced with a tangential flow filtration device to complete the storage solution replacement, and the culture medium is completely replaced with PB buffer, and concentrated, so as to facilitate the next step of ultracentrifugation.

[0124] 2. A sucrose gradient of 30-60% can be selected, with 50% sucrose as the preferred layer. Then, an equal volume of virus solution is placed on top. Centrifuge at 100,000g, 4℃, for 3 hours to purify the viral particle antigen. Carefully remove the sucrose layer and resuspend the precipitate at the bottom of the tube with PB buffer.

[0125] 3. A cesium chloride gradient can be selected from 0% to 60%, with 10%, 20%, 30%, and 40% concentrations being optimal. Add 1-2 ml of resuspended viral particle antigen to the top layer of each tube. Centrifuge at 150,000 g, 4°C for 24 hours. Irradiate with a strong light and use a long needle to aspirate the desired viral particle antigen.

[0126] 4. Using Cytiva's Capto butyl packing material, linear dissociation was performed using 10 mM PB + 1 M NaCl solution to obtain ultrapure viral antigens. Protein purity was assessed using SDS-PAGE polyacrylamide gel electrophoresis. Figure 1 As shown, the VP0 protein size is ~38KD, the VP1 protein size is ~35KD, and the VP2 / 3 protein size is ~26KD.

[0127] (III) Reagent Kit Preparation

[0128] The magnetic microparticle chemiluminescence kit for Coxsackievirus A10 IgM proposed in this invention includes magnetic microparticles conjugated with anti-μ secondary antibody, sample diluent, antigen blocking solution, enzyme-labeled antigen stock solution, enzyme-labeled antigen diluent, washing solution, substrate A solution, substrate B solution, negative control, positive control, calibrator, and instructions.

[0129] The enzyme-labeled antigen was the Coxsackievirus A10 recombinant antigen prepared in (I) and (II), which was obtained by labeling with horseradish peroxidase; the antigen blocking solution was an antigen blocking solution composed of a mixture of Coxsackievirus EV71, A16 and A6 recombinant antigens prepared in (I) and (II) (the mixing ratio was the preferred ratio in Example 1); the negative control, positive control, magnetic microparticles conjugated with anti-μ secondary antibody, sample dilution particles, and enzyme-labeled antigen dilution solution were all prepared by Zhengzhou Yimeno Biotechnology Co., Ltd.; substrate A solution and substrate B solution were prepared by Zhengzhou Antu Biotechnology Co., Ltd.; the detection instrument was the A2000 plus chemiluminescence analyzer of Zhengzhou Antu Biotechnology Co., Ltd.

[0130] The method of using the magnetic particle chemiluminescence reagent kit for Coxsackievirus A10 IgM proposed in this invention is as follows:

[0131] The prepared Coxsackievirus A10 IgM magnetic microparticle chemiluminescence kit was equilibrated at room temperature for 15–30 minutes.

[0132] Calibration curve is performed on the instrument software, the calibrator is placed in the instrument test position, and a fitting curve is obtained from the full-automatic chemiluminescence immunoassay analyzer.

[0133] If the instrument prompts that the fitting passes, the quality control sample test application is performed on the instrument software, the quality control sample is placed in the designated position of the instrument, and the quality control sample test luminescence value output by the full-automatic chemiluminescence analyzer and the concentration value of the quality control sample obtained by fitting the standard curve are obtained.

[0134] If the quality control test value is within the predetermined range, the sample test application can be performed; the sample to be tested is placed in the instrument test position, and the sample to be tested test luminescence value output by the full-automatic chemiluminescence analyzer and the concentration value obtained by fitting the standard curve are obtained.

[0135] The magnetic microparticle chemiluminescence kit for coxsackie virus A10 type IgM provided in the application has the following result determination: the sample concentration value < 20 AU / mL is judged as negative; the concentration value ≥ 25 AU / mL is judged as positive; and 20 AU / mL ≤ the concentration value < 25 AU / mL is judged as suspicious, and it is recommended to re-sample detection after 1-2 weeks or to confirm by using other detection methods.

[0136] Effect example 1: exploration of the addition proportion of blocking antigen

[0137] 1. The blocking antigens of coxsackie viruses EV71, A16 and A6 are added to a buffer solution of 10 mM PBS, 0.6% BSA, 0.2% casein, 0.1% ProClin300, 0.1% Triton X-100, pH 7.2, and different addition amounts are adjusted.

[0138] 2. The molar proportion of the added blocking antigens can be EV71:CA16:CA6 = 10:1:1-1:1:10, and a CA10 negative sample and EV71, CA16 and CA6 positive samples are used for detection verification, and the results are shown in Table 1. Preferably, when the molar proportion approaches EV71:CA16:CA6 = 2:1:1, the specificity is better.

[0139] Table 1

[0140]

[0141] Effect example 2: sensitivity determination

[0142] 10 positive serum samples are stored by the company (coxsackie virus A10 type IgM antibody positive serum samples verified by hospitals clinically), and the coxsackie virus A10 type IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Pharmacy Co., Ltd. is used for re-screening and confirmation of positive, and is established as a positive quality control sample.

[0143] 10 positive quality controls were detected respectively using the kit provided by the preparation example, and the results were shown in Table 2 according to the interpretation of Antu magnetic particle coxsackie virus A10 IgM detection program.

[0144] Table 2

[0145]

[0146] The results showed that 10 positive samples could be detected, and the coincidence rate was 10 / 10, indicating that the sensitivity of the kit met the requirements.

[0147] Effect example 3: specificity determination

[0148] 10 negative serum samples were stored by the company (coxsackie virus A10 IgM antibody negative serum verified by hospital clinical), and were rechecked and screened by coxsackie virus A10 IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Technology Co., Ltd. to confirm negative.

[0149] 10 negative quality controls were detected respectively using the kit provided by the preparation example, and the results were shown in Table 3 according to the interpretation of Antu magnetic particle coxsackie virus A10 IgM detection program.

[0150] Table 3

[0151]

[0152] The results showed that 10 negative samples were not detected, and the coincidence rate was 10 / 10, indicating that the kit had good specificity.

[0153] Effect example 4: EV71, A16 and A6 positive sample blocking ability verification

[0154] 5 EV71 positive, CA10 negative serum samples, 5 CA16 positive, CA10 negative serum samples, 5 CA6 positive, CA10 negative serum samples were stored by the company (coxsackie virus A10 IgM antibody negative serum verified by hospital clinical), and were rechecked and screened by coxsackie virus A10 IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Technology Co., Ltd.

[0155] 15 samples were detected respectively using the kit provided by the preparation example, and the results were shown in Table 4 according to the interpretation of Antu magnetic particle coxsackie virus A10 IgM detection program.

[0156] Table 4

[0157]

[0158] The above results show that the kit of the present application can effectively block the interference of EV71, CA16 and CA6 infection on detection.

[0159] Example 5: PCR identification coincidence

[0160] The prepared coxsackievirus A10 IgM antibody detection kit was used to detect 90 normal human blood samples and 10 samples of clinically diagnosed coxsackievirus A10 infection (PCR identification) of hand-foot-mouth disease. The detection procedure was strictly in accordance with the method provided in the preparation example. The results are shown in Table 5:

[0161] Table 5

[0162]

[0163] The above data show that the coincidence of the kit of the present application and PCR identification is 100%. The magnetic microparticle chemiluminescence kit for coxsackievirus A10 IgM antibody provided in the present application can effectively detect coxsackievirus A10 IgM positive samples in hand-foot-mouth disease.

[0164] Example 6: Stability test

[0165] Three positive sera used for sensitivity test in Example 2 were repeatedly detected 5 times in batch and between batches using the kit provided in the preparation example according to the method in the preparation example. The results were interpreted according to the Antu magnetic microparticle coxsackievirus A10 IgM detection procedure, and the results are shown in Table 6:

[0166] Table 6

[0167]

[0168] The above results show that the coefficient of variation of 5 times of batch detection is within 5%, the coefficient of variation of 5 times of batch detection is within 5%, and the coefficient of variation of batch and batch is within 5%.

[0169] Comparative example: no addition of blocking antigen

[0170] Other reagents were referred to the preparation example, but no EV71, CA16 and CA6 recombinant antigens were added in the antigen blocking reagent. The reagent was used for retest in Example 4, and the results are shown in Table 7:

[0171] Table 7

[0172]

[0173]

[0174] The above results indicate that when the antigen blocking reagent does not contain EV71, CA16, and CA6 recombinant antigens, the CA10 detection results will be affected by the infection of other samples.

[0175] 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. A composition, characterized in that, It consists of enzyme-labeled Coxsackievirus A10 recombinant antigen and blocking antigen; The blocking antigens are enterovirus 71 recombinant antigen, coxsackievirus A16 recombinant antigen, and coxsackievirus A6 recombinant antigen; The enzyme-labeled Coxsackievirus A10 recombinant antigen is horseradish peroxidase-labeled Coxsackievirus A10 recombinant antigen. The recombinant antigen of Coxsackievirus A10 is the Coxsackievirus A10 3CD protein and the Coxsackievirus A10 P1 protein; The recombinant antigen of enterovirus 71 is enterovirus 71 3CD protein and enterovirus 71 P1 protein; The recombinant antigen of Coxsackievirus A16 is the Coxsackievirus A16 3CD protein and the Coxsackievirus A16 P1 protein; The recombinant antigen of Coxsackievirus A6 is the Coxsackievirus A6 3CD protein and the Coxsackievirus A6 P1 protein; in: The amino acid sequence of the Coxsackievirus A10 3CD protein is shown in SEQ ID NO: 1; The amino acid sequence of the Coxsackievirus A10 type P1 protein is shown in SEQ ID NO: 2; The amino acid sequence of the enterovirus 71 3CD protein is shown in SEQ ID NO: 5; The amino acid sequence of the enterovirus 71 P1 protein is shown in SEQ ID NO: 6; The amino acid sequence of the Coxsackievirus A16 3CD protein is shown in SEQ ID NO: 3; The amino acid sequence of the Coxsackievirus A16 type P1 protein is shown in SEQ ID NO: 4; The amino acid sequence of the Coxsackievirus A6 type 3CD protein is shown in SEQ ID NO: 7; The amino acid sequence of the Coxsackievirus A6 type P1 protein is shown in SEQ ID NO: 8; The preparation method of the horseradish peroxidase-labeled Coxsackievirus A10 recombinant antigen is as follows: the Coxsackievirus A10 recombinant antigen is incubated with HRP at a molar ratio of 1:(1~10) to obtain the horseradish peroxidase-labeled Coxsackievirus A10 recombinant antigen. The molar ratio of the enterovirus 71 recombinant antigen, the coxsackievirus A16 recombinant antigen, and the coxsackievirus A6 recombinant antigen is 2:1:

1.

2. The use of the composition as described in claim 1 in excluding false positives caused by enteroviruses in the detection of Coxsackievirus A10; The enteroviruses mentioned are one or more of Coxsackievirus A16, Coxsackievirus A6, or Enterovirus 71.

3. The use of the composition of claim 1 in the preparation of reagents or kits for detecting anti-Coxsackievirus A10 IgM.

4. The use of the composition according to claim 1 in the following aspects: (a) Preparation of diagnostic reagents or kits for hand-foot-mouth disease; or (b) Preparation of diagnostic reagents or diagnostic kits for Coxsackievirus A10 infection.

5. A reagent, characterized in that, Includes the composition as described in claim 1, and acceptable excipients or additives.

6. A reagent kit, characterized in that, It includes the reagent as described in claim 5, as well as acceptable excipients or auxiliaries.

7. The kit according to claim 6, characterized in that, It also includes one or more of the following: magnetic particles of mouse anti-human IgM monoclonal antibody, magnetic particle sealing solution, sample diluent, enzyme conjugate diluent, washing solution, chromogenic solution, stop solution, negative control or positive control.

8. The kit according to claim 7, characterized in that, The positive control was a human-mouse chimeric anti-Coxsackievirus A10 antibody; The negative control was a PBS buffer containing 20% ​​BSA; The sample diluent was a 0.05 M Tris buffer solution containing 2% BSA at pH 7.4; The washing solution is a PBS buffer containing 0.1% Tween 20; The enzyme conjugate dilution solution is a 0.05 M Tris buffer containing 20% ​​BSA at pH 7.4; The colorimetric solution is a buffer solution containing luminol.

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