Kit and process for its preparation

Abstract

The present application relates to the technical field of in vitro diagnosis, in particular to a kit and a preparation process thereof, the present application is a recombinant virus antigen of coxsackie virus A16 and EV71, and is used for specific detection of coxsackie virus A16 IgM, a magnetic micro-particle chemiluminescence kit, which comprises magnetic particles coated with mouse anti-human IgM monoclonal antibody, coxsackie virus A16 antigen labeled with horseradish peroxidase and coxsackie virus EV71 recombinant virus antigen. The reagent has excellent sensitivity, specificity and precision, and can distinguish the two pathogens of EV71 and coxsackie A16 in hand-foot-mouth disease, has high repeatability between tests, and is easy to control the standard.

Description

TECHNICAL FIELD

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

[0002] Coxsackievirus A16 (CA16) belongs to the order of Picornavirales, the family of Picornaviridae and the species of Enterovirus. The viral genome is a positive-strand RNA of 8.5 kb in length, and the 5' end of the genomic RNA is covalently coupled to the vpg protein to drive viral genome transcription. The virus has a naked 20-sided virus (T=3) morphology, and the viral particle is about 30 nm in diameter.

[0003] Hand-foot-mouth disease is an infectious disease caused by enterovirus. There are more than 20 types of enterovirus that can cause hand-foot-mouth disease, among which coxsackievirus A16 (Cox A16) and enterovirus 71 (EV 71) are the most common.

[0004] Hand-foot-mouth disease mainly occurs in children under the age of 5, and is characterized by oral pain, anorexia, low fever, and small vesicles or small ulcers on the hands, feet, mouth, and other parts. Most patients recover within a week, but a small number of patients can develop complications such as myocarditis, pulmonary edema, and aseptic meningitis. In rare cases, the disease progresses rapidly and leads to death. There is currently no effective treatment, and the main treatment is symptomatic.

[0005] Coxsackievirus A16 is one of the main pathogens of HFMD. In recent years, there has been a gradual increase in the number of severe infections caused by non-enterovirus 71 (EV71) and enterovirus infections, mainly including CA16. Therefore, the development of a kit to distinguish between EV71 and CA16 virus infections causing hand-foot-mouth disease is of great significance in diagnosis and treatment. SUMMARY

[0006] Therefore, the present application provides a kit and a preparation process thereof, which has excellent sensitivity, specificity, and precision, and can distinguish between EV71 and coxsackievirus A16 in hand-foot-mouth disease, with high inter-test reproducibility and easy-to-control standards.

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

[0008] The present application provides a composition comprising an enterovirus 71 recombinant antigen and an enzyme-labeled coxsackievirus A16 recombinant antigen;

[0009] The enterovirus 71 recombinant antigen comprises enterovirus 71 polyprotein P1 and / or enterovirus 71 3CD protease; and / or

[0010] The recombinant antigen of Coxsackievirus A16 type includes a Coxsackievirus A16 type polyprotein P1 and / or a Coxsackievirus A16 type 3CD protease;

[0011] The nucleic acid molecule encoding the Coxsackievirus A16 type polyprotein P1 has:

[0012] (1) the nucleotide sequence as shown in SEQ ID NO. 5; or

[0013] (2) a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence as shown in (1), and having the same or similar function as (1); or

[0014] (3) a nucleotide sequence having at least 70% homology with the nucleotide sequence as shown in (1) or (2);

[0015] and / or

[0016] The nucleic acid molecule encoding the Coxsackievirus A16 type 3CD protease has:

[0017] (4) the nucleotide sequence as shown in SEQ ID NO. 6; or

[0018] (5) a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence as shown in (4), and having the same or similar function as (4); or

[0019] (6) a nucleotide sequence having at least 70% homology with the nucleotide sequence as shown in (4) or (5);

[0020] and / or

[0021] The nucleic acid molecule encoding the Coxsackievirus A16 type polyprotein P1 has:

[0022] (7) the nucleotide sequence as shown in SEQ ID NO. 2; or

[0023] (8) a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence as shown in (7), and having the same or similar function as (7); or

[0024] (9) a nucleotide sequence having at least 70% homology with the nucleotide sequence as shown in (7) or (8);

[0025] and / or

[0026] The nucleic acid molecule encoding the Coxsackievirus A16 type 3CD protease has:

[0027] (10) the nucleotide sequence as shown in SEQ ID NO. 3; or

[0028] (11) the nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence of (10), and having the same or similar function as (10); or

[0029] (12) a nucleotide sequence having at least 70% homology with the nucleotide sequence of (10) or (11);

[0030] the plurality is 2 to 500.

[0031] In some embodiments of the present application, the composition described above comprises:

[0032] the coxsackievirus A16 includes one or more of coxsackievirus A16 R37 / YN / CHN / 2013 strain, coxsackievirus A16 Tainan strain, coxsackievirus A16 G10 strain, coxsackievirus A16 BJ09 / 06 strain or coxsackievirus A16 107 / Toyama / 1990 strain; and / or

[0033] the enterovirus EV71 includes one or more of enterovirus 71 BRCR strain, enterovirus 71 Fuyang.Anhui.P.R.C / 17.08 / 3 strain, enterovirus 71 7423 / MS / 87 strain, enterovirus 71 STH / MCN / 2006 strain or enterovirus 71 BJ08-Z025-5 strain.

[0034] In some embodiments of the present application, the composition described above comprises:

[0035] The preparation method of the enzyme-labeled coxsackievirus A16 recombinant antigen comprises: mixing the coxsackievirus A16 recombinant antigen with sodium periodate-activated horseradish peroxidase at a molar ratio of 1:5, incubating, and then mixing with PBS buffer containing glycerol at a final concentration of 50% to obtain the horseradish peroxidase-labeled coxsackievirus A16 recombinant antigen.

[0036] and / or

[0037] The preparation method of the coxsackievirus A16 recombinant antigen or the enterovirus 71 recombinant antigen comprises: inserting the gene encoding the coxsackievirus A16 recombinant antigen or the gene encoding the enterovirus 71 recombinant antigen into an insect baculovirus expression vector to obtain a recombinant vector, expressing the recombinant vector to obtain the coxsackievirus A16 recombinant antigen or the enterovirus 71 recombinant antigen.

[0038] In some embodiments of the present application, the method for preparing the expression vector of the recombinant antigen of Coxsackie virus A16 or the recombinant antigen of enterovirus 71 in the kit comprises: constructing the polyprotein P1 of Coxsackie virus A16 or enterovirus 71 to the rear of the PH promoter of pFBDCMV, and controlling the expression by the PH promoter; constructing the corresponding 3CD protease gene to the rear of the CMV promoter, and controlling the expression by the CMV promoter, to obtain the expression vector.

[0039] In some embodiments of the present application, the method for preparing the recombinant antigen of Coxsackie virus A16 or the recombinant antigen of enterovirus 71 in the kit comprises: preparing P3 generation insect baculovirus liquid by using SF9 cells, infecting SF9 cells, and the multiplicity of infection MOI being 1; after 64 hours of virus infection, collecting the cell culture supernatant, and purifying by using several methods of tangential flow filtration, PEG6000 precipitation, anion exchange chromatography, complex mode chromatography, and density gradient centrifugation, to obtain the recombinant antigen of Coxsackie virus A16 or the recombinant antigen of enterovirus 71.

[0040] In some embodiments of the present application, the insect baculovirus expression vector of the composition has:

[0041] (13) the nucleotide sequence shown in SEQ ID NO. 1; or

[0042] (14) a nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (13), and having the same or similar function as (13); or

[0043] (15) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in (13) or (14);

[0044] The plurality is 2 to 500.

[0045] The present application also provides the use of the composition in excluding the false positive of enterovirus 71 in the detection of Coxsackie virus A16.

[0046] The present application also provides the use of the composition in preparing a reagent or a kit for detecting anti-Coxsackie virus A16 IgM.

[0047] The present application also provides the use of the composition in the following aspects:

[0048] (a) preparing a diagnostic reagent or a diagnostic kit for Coxsackie virus A16 infection; and / or

[0049] (b) preparing a diagnostic reagent or a diagnostic kit for hand-foot-mouth disease.

[0050] The present application also provides the above-mentioned reagent, including the above-mentioned composition, and acceptable adjuvant or auxiliary agent.

[0051] The present application also provides the above-mentioned kit, including the above-mentioned reagent, and acceptable adjuvant or auxiliary agent.

[0052] In some specific embodiments of the present application, the above-mentioned kit further includes one or more of magnetic particles coated with mouse anti-human IgM monoclonal antibody, magnetic particle storage solution, sample diluent, enzyme conjugate diluent, washing solution, substrate A solution, substrate B solution, negative control or positive control.

[0053] In some specific embodiments of the present application, the above-mentioned kit includes:

[0054] The preparation method of the magnetic particles coated with mouse anti-human IgM monoclonal antibody includes: mixing 0.2 μg per human portion of magnetic particles from Merck and 0.5 μg per human portion of mouse anti-human IgM monoclonal antibody, incubating for 2 hours, mixing with PBS buffer with a final concentration of 1% Casein, to obtain the magnetic particles coated with mouse anti-human IgM monoclonal antibody; and / or

[0055] The positive control includes mouse anti-cosxackievirus A16 antibody with chimeric human IgM Fc fragment and PBS buffer containing 20% bovine serum; and / or

[0056] The negative control includes PBS buffer containing 20% bovine serum; and / or

[0057] The sample diluent includes 0.05M Tris-HCl buffer containing 2% BSA and having a pH of 7.4; and / or

[0058] The washing solution includes PBS phosphate buffer containing 0.1% Tween20; and / or

[0059] The enzyme conjugate diluent includes 0.05M Tris-HCl buffer containing 20% bovine serum and having a pH of 7.4; and / or

[0060] The substrate A solution and the substrate B solution include a buffer containing luminol.

[0061] The kit of the present application has the following effects:

[0062] 1. By using EV71 blocking antigen, it is realized to distinguish hand-foot-mouth disease caused by EV71 and CA16 infection, and to avoid cross infection caused by EV71 infection;

[0063] 2. By preparing virus-like particle recombinant antigen, the potential infection risk caused by using natural virus source material is avoided.

[0064] 3. The recombined antigen labeled by horseradish peroxidase is assembled into a coxsackie virus A16 IgM antibody capture kit, which can specifically recognize coxsackie virus A16 IgM positive serum. DETAILED DESCRIPTION

[0065] The kit and its preparation process are disclosed, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0066] The purpose of the present application is to develop a coxsackie virus A16 IgM antibody detection kit for specific detection and differentiation of EV71 infection in response to the demand for detection of hand-foot-mouth disease pathogens;

[0067] The second purpose of the present application is to provide a preparation method of recombined antigen for specific detection of coxsackie virus A16 IgM antibody;

[0068] The third purpose of the present application is to provide a preparation method of EV71 recombined blocking antigen for specific detection of coxsackie virus A16 IgM antibody;

[0069] The fourth purpose of the present application is to provide a vector for expressing coxsackie virus A16 recombined antigen and EV71 recombined blocking antigen;

[0070] The fifth purpose of the present application is to provide a method for labeling horseradish peroxidase of coxsackie virus A16 recombined antigen;

[0071] The sixth purpose of the present application is to provide a kit for specific detection of coxsackie virus A16 IgM antibody;

[0072] To achieve the first purpose, the technical scheme adopted by the present application is that the sequence of the recombinant antigen of coxsackievirus A16 is derived from one or more of the following: coxsackievirus A16 virus Tainan strain, G10 strain, R37 / YN / CHN / 2013 strain, BJ09 / 06 strain, Human coxsackievirus A16 107 / Toyama / 1990; the sequence of the blocking antigen of the recombinant virus of coxsackievirus EV71 is derived from one or more of the following: coxsackievirus 71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain, BRCR strain, 7423 / MS / 87 strain, STH / MCN / 2006 strain, BJ08-Z025-5 strain.

[0073] The sequences of coxsackievirus A16 and EV71 virus are highly similar (capsid protein similarity is 80%), and there is a possibility of EV71 cross when coxsackievirus A16 antigen is used alone for detection, so when designing the coxsackievirus A16 IgM antibody detection kit, relevant measures need to be introduced to avoid false positives caused by EV71 cross.

[0074] To achieve the second purpose, the technical scheme adopted by the present application is that the recombinant antigen of coxsackievirus A16 is purified by using an insect baculovirus expression system and combining several methods selected from the following: tangential flow filtration, PEG6000 precipitation, anion exchange chromatography, complex mode chromatography, and density gradient centrifugation.

[0075] To achieve the third purpose, the technical scheme adopted by the present application is that the recombinant blocking antigen of EV71 is purified by using an insect baculovirus expression system and combining several methods selected from the following: tangential flow filtration, PEG6000 precipitation, anion exchange chromatography, complex mode chromatography, and density gradient centrifugation.

[0076] To achieve the fourth purpose, the technical scheme adopted by the present application is a DNA vector, and the sequence of the DNA vector is SEQ ID NO. 1.

[0077] To achieve the fifth purpose, the technical scheme adopted by the present application is a sodium periodate labeling method.

[0078] To achieve the sixth purpose, the technical scheme adopted by the present application is a capture method kit for detecting coxsackievirus A16 IgM, and the kit contains any one of the polypeptides or any one of the molecules described above.

[0079] To achieve the sixth purpose, the technical scheme adopted by the present application is a capture method kit for detecting coxsackievirus A16 IgM antibody, and the kit contains any one of the molecules described above.

[0080] Method for obtaining the attached calibrator (640 AU / mL of Coxsackie virus A16 virus IgM antibody high value serum):

[0081] Collect Coxsackie virus A16 virus IgM antibody positive high value serum, test with the kit provided by the application, and mix the serum with detection luminescence value greater than 50 million for standby.

[0082] Collect Coxsackie virus A16 virus IgM antibody negative serum, test with the kit provided by the application, and mix the serum with detection luminescence value less than 20,000 for standby.

[0083] The preparation method of the calibrator of 640 AU / mL is as follows: first dilute the Coxsackie virus A16 virus IgM antibody high value serum with negative serum, and the dilution ratio is 4 times, 8 times, 16 times, 32 times, 64 times, 128 times, 256 times, 512 times, 1024 times, 2048 times, 4096 times, and 8192 times; use the kit provided by the application to detect the mixed serum after dilution, and when the detection value of the mixed serum is close to the detection value of the luminescent substrate solution, the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum after dilution is defined as 0 AU / mL; for example, when the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum diluted 8192 times is defined as 0 AU / mL, the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum at the next highest concentration (diluted 4096 times) is defined as 5 AU / mL, the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum diluted 2048 times is defined as 10 AU / mL, and the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum diluted 1024 times is defined as 20 AU / mL; in turn, the concentration of Coxsackie virus A16 virus IgM antibody in the mixed serum diluted 32 times is defined as 640 AU / mL. Use the kit provided by the application to detect 10 positive sera and 10 negative sera with the antibody mixed serum of 20-640 AU / mL as the calibrator, and if the detection is accurate, it meets the requirements; if it meets the requirements, dilute the mixed serum of this batch number by 32 times, and the calibrator of Coxsackie virus A16 virus IgM antibody with a concentration of 640 AU / mL is obtained.

[0084] Calibrator preparation method:

[0085] Preparation of the calibration diluent: 2.5 g of BSA was added into a container with 1 L of PBS buffer, stirred until completely dissolved, then the pH value was adjusted to 7.10-7.30, filtered with a 0.2 μm filter, and the obtained calibration diluent was stored at 2-8℃ for later use; the high-value serum of coxsackievirus A16 virus IgM antibody was prepared into 20, 40, 80, 160, 320, 640 AU / mL for a total of 6 concentration points with the above calibration diluent, and the calibration was obtained.

[0086] The following is the sequence information involved in the present application:

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary commercially available products, which can be purchased from the market.

[0098] The present application will be further described below in combination with examples:

[0099] Preparation Example

[0100] 1. Preparation of coxsackievirus A16 virus recombinant vector:

[0101] (1) Synthesis of coxsackievirus A16 virus R37 / YN / CHN / 2013 strain P1 gene, such as SEQ ID NO. 2;

[0102] (2) Synthesis of coxsackievirus A16 virus R37 / YN / CHN / 2013 strain 3CD gene, such as SEQ ID NO. 3;

[0103] (3) Synthesize the P1 gene of the coxsackievirus A16 virus R37 / YN / CHN / 2013 strain and clone it to the rear of the PH promoter of pFBDCMV, synthesize the 3CD gene of the coxsackievirus A16 virus R37 / YN / CHN / 2013 strain and clone it to the rear of the CMV promoter of pFBDCMV, and the DNA sequence of the obtained recombinant is as shown in SEQ ID NO. 4.

[0104] 2. Preparation of an enterovirus EV71 virus recombinant vector:

[0105] (1) Synthesize the P1 gene of the enterovirus EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain, as shown in SEQ ID NO. 5;

[0106] (2) Synthesize the 3CD gene of the enterovirus EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain, as shown in SEQ ID NO. 6;

[0107] (3) Synthesize the P1 gene of the enterovirus EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain and clone it to the rear of the PH promoter of pFBDCMV, synthesize the 3CD gene of the enterovirus EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain and clone it to the rear of the CMV promoter of pFBDCMV, and the DNA sequence of the obtained recombinant is as shown in SEQ ID NO. 7.

[0108] 3. Preparation of a P3 generation recombinant baculovirus containing the P1 gene of the coxsackievirus A16 virus R37 / YN / CHN / 2013 strain

[0109] The preparation method of the P3 generation recombinant baculovirus containing the P1 gene of the coxsackievirus A16 virus R37 / YN / CHN / 2013 strain refers to the bac-to-bac baculovirus expression system instruction manual of Thermo-fisher company; the preparation method of the P3 generation recombinant baculovirus containing the P1 gene of the enterovirus EV71 virus Fuyang.Anhui.P.R.C / 17.08 / 3 strain refers to the bac-to-bac baculovirus expression system instruction manual of Thermo-fisher company.

[0110] 4. Preparation method of recombinant coxsackievirus A16 antigen and EV71 type recombinant blocking antigen

[0111] The SF9 cells are suspended and cultured on a shaker to a cell density of 2*10 6The recombinant baculovirus liquid of P3 generation was added to the culture system at MOI = 1, and the volume of the virus liquid was not more than 5% of the culture volume, and the cell survival rate was greater than 98%.

[0112] The culture was continued for 64 hours or the cell survival rate was not less than 70%, and the cells were removed by centrifugation at 1000 rpm, and the culture cell supernatant was filtered through a 0.45 μm filter membrane to remove cell debris.

[0113] The culture cell supernatant was exchanged into PB buffer using a tangential flow filtration device with a molecular weight cut-off of not more than 300 kD, and further concentrated 20 times.

[0114] 1M NaCl and 10% PEG6000 were added to the concentrated liquid to a final concentration, and the mixture was incubated at 4°C for 16 hours. Centrifugation was performed at 10000 rpm for 40 minutes, and the supernatant was discarded.

[0115] The precipitate was resuspended in PB buffer at pH 7.5, and the virus antigen was purified by centrifugation with a 40% sucrose cushion, with the centrifugation conditions being 100000g, 4°C, and centrifugation for 5 hours. The sucrose layer was carefully removed, the bottom of the tube was reselected with PB buffer, and dialysis was performed in PB.

[0116] The virus antigen was further purified using the capto core 400 filler of cytiva company.

[0117] 5. Preparation method of horseradish peroxidase-labeled recombinant antigen stock solution of coxsackie virus A16 type

[0118] The coxsackie virus A16 type virus antigen was dialyzed into carbonate buffer CB at pH 9.6, and the antigen concentration was adjusted to 2 mg / mL.

[0119] The required amount of HRP was calculated according to the molar ratio of 1:5, dissolved in an appropriate amount of distilled water to make the final concentration 5 mg / mL, and then 1 / 5 volume of 0.1M NaIO4 solution was added. Stir at room temperature for 20 minutes in the dark. Dialyze the above system into 1 mM sodium acetate pH 4.4.

[0120] 0.2M CB, pH 9.6 solution was added to the activated HRP solution to raise the pH value to about 9.5, and mixed with the previously dialyzed coxsackie virus A16 type virus antigen, and stirred at 4°C for 12 hours.

[0121] 0.1 mL of freshly prepared 4 mg / mL NaBH4 solution was added and mixed well, and the reaction was carried out at 4°C for 2 hours.

[0122] The reaction solution was dialyzed into PBS buffer, and a final concentration of 50% glycerol was added.

[0123] The method for preparing the recombinant antigen blocking solution of coxsackievirus A16 type provided by the application is as follows:

[0124] The coxsackievirus EV71 virus antigen is added into 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 the antigen concentration is adjusted to 0.1 mg / mL; the solution is defined as the antigen blocking solution.

[0125] 6. A magnetic microparticle chemiluminescence kit for coxsackievirus A16 type IgM

[0126] The kit comprises magnetic microparticles coupled with anti-mu secondary antibodies, 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, calibration product and instructions.

[0127] In the formula, R is a C1-C6 alkyl group, and n is 1-3.

[0128] The magnetic microparticles coupled with anti-mu secondary antibodies are prepared by Zhengzhou Imenno Biotechnology Co., Ltd.

[0129] The enzyme-labeled antigen stock solution is the horseradish peroxidase-labeled coxsackievirus A16 type recombinant antigen mentioned above.

[0130] The antigen blocking solution is the coxsackievirus A16 type recombinant antigen blocking solution in item 5.

[0131] The positive control is a 20% calf serum PBS buffer solution containing mouse anti-coxsackievirus A16 type antibody with chimeric human IgM Fc fragment, wherein the mouse anti-coxsackievirus A16 type antibody with chimeric human IgM Fc fragment is prepared by Zhengzhou Imenno Biotechnology Co., Ltd.

[0132] The negative control is a 20% calf serum PBS buffer solution.

[0133] Sample diluent (10 mM PBS buffer solution, 0.6% BSA, 0.2% casein, 0.1% ProClin300, 20% calf serum, pH 7.2)

[0134] The substrate A solution and the substrate B solution are prepared by Zhengzhou Antuo Biotechnology Co., Ltd.

[0135] Enzyme-labeled antigen diluent (10 mM PBS buffer solution, 0.6% BSA, 0.2% casein, 0.1% ProClin300, 0.1% Triton X-100, pH 7.2)

[0136] Specifically, the enzyme-labeled antigen stock solution is diluted to 5000 times with the enzyme-labeled antigen diluent.

[0137] The detection instrument uses A2000 plus chemiluminescence instrument of Zhengzhou Antu Biological Co., Ltd.

[0138] 7. The use method of the magnetic particle chemiluminescence kit for coxsackievirus A16 type IgM:

[0139] The above-mentioned coxsackievirus A16 type IgM magnetic particle chemiluminescence kit is balanced at room temperature for 15-30 minutes.

[0140] The calibration curve is performed on the instrument software, the calibrator is placed in the instrument test position, and the fitting curve output by the full-automatic chemiluminescence immunoassay analyzer is obtained.

[0141] If the instrument prompts that the fitting is passed, the quality control sample test application is performed on the instrument software, the quality control sample is placed in the instrument designated position, 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.

[0142] 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 test luminescence value and the concentration value obtained by fitting the standard curve are output by the full-automatic chemiluminescence analyzer.

[0143] The result determination: the sample concentration value <20 AU / mL is judged as negative; the concentration value ≥25 AU / mL is judged as positive; 20 AU / mL ≤ concentration value <25 AU / mL is judged as suspicious, and it is recommended to re-sample detection after 1-2 weeks or to be confirmed by other detection methods.

[0144] Comparative example

[0145] 1. The kit of the present comparative example comprises magnetic particles coupled with anti-mu 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 instruction.

[0146] Among them:

[0147] The magnetic particles coupled with anti-mu secondary antibody are prepared by Zhengzhou Yimeno Biological Technology Co., Ltd.

[0148] The enzyme-labeled antigen stock solution is the horseradish peroxidase-labeled coxsackievirus A16 type recombinant antigen mentioned above.

[0149] The antigen blocking solution is 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 the antigen blocking solution does not contain the aforementioned EV71 recombinant antigen.

[0150] Positive control: 20% calf serum PBS buffer containing chimeric human IgM Fc fragment of mouse anti-cocci virus A16 antibody. The chimeric human IgM Fc fragment of mouse anti-cocci virus A16 antibody is prepared by Zhengzhou Imuno Biotechnology Co., Ltd.

[0151] Negative control: 20% calf serum PBS buffer.

[0152] Sample diluent (10 mM PBS buffer, 0.6% BSA, 0.2% casein, 0.1% ProClin300, 20% calf serum, pH 7.2)

[0153] Substrate A and substrate B are prepared by Zhengzhou Antuo Biotechnology Co., Ltd.

[0154] Enzyme-labeled antigen diluent (10 mM PBS buffer, 0.6% BSA, 0.2% casein, 0.1% ProClin300, 0.1% Triton X-100, pH 7.2)

[0155] Specifically, the enzyme-labeled antigen stock solution is diluted 5000 times with the enzyme-labeled antigen diluent.

[0156] The detection instrument uses A2000 plus chemiluminescence instrument of Zhengzhou Antuo Biotechnology Co., Ltd.

[0157] Effect example 1: sensitivity determination

[0158] 10 cases of positive serum are stored by the company (cocci virus A16 IgM antibody positive serum verified by hospital clinic), and the cocci virus A16 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.

[0159] 10 positive quality controls are detected respectively using the kit provided in the preparation example, and the results are shown in Table 1 according to the interpretation of the Antuo magnetic microparticle cocci virus A16 IgM detection program. The positive reference product compliance rate is 10 / 10.

[0160] Table 1: Positive reference product compliance rate data statistics table (RFU)

[0161]

[0162] Effect example 2: specificity determination

[0163] 10 cases of negative serum are kept by the company (Coxsackie virus A16 IgM antibody negative serum verified by hospital clinical), and the coxsackie virus A16 IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Technology Co., Ltd. is used for re-screening and confirmation of negative.

[0164] 10 negative quality control samples are detected by using the kit provided by the preparation example, and the results are shown in Table 2 according to the interpretation of the Antu magnetic particle coxsackie virus A16 IgM detection program. The negative reference product coincidence rate is 10 / 10.

[0165] Table 2

[0166]

[0167] Example 3: EV71 positive sample blocking ability verification

[0168] 1. The kit provided by the preparation example is used for EV71 positive sample blocking ability verification

[0169] 10 cases of EV71 positive and CA16 negative serum are kept by the company (Coxsackie virus A16 IgM antibody negative serum verified by hospital clinical, Antu biological EV71 IgM antibody detection kit detection positive), and the coxsackie virus A16 IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Technology Co., Ltd. is used for re-screening and confirmation of negative.

[0170] The above 10 samples are detected by using the kit provided by the preparation example, and the results are shown in Table 3 according to the interpretation of the Antu magnetic particle coxsackie virus A16 IgM detection program. The CA16 negative coincidence rate is 10 / 10, and the results show that the kit of the application can effectively block the interference of EV71 infection on detection.

[0171] Table 3

[0172]

[0173] 2. EV71 positive sample specificity determination of the kit provided by the comparative example

[0174] 10 cases of EV71 positive and CA16 negative serum are kept by the company (Coxsackie virus A16 IgM antibody negative serum verified by hospital clinical, Antu biological EV71 IgM antibody detection kit detection positive), and the coxsackie virus A16 IgM antibody detection kit (enzyme-linked immunoassay) produced by Beijing Wantai Biological Technology Co., Ltd. is used for re-screening and confirmation of negative.

[0175] The above 10 samples were detected using the kit provided in the comparative example, respectively, according to the Antu magnetic particle coxsackie virus A16 IgM detection procedure for interpretation, and the results are shown in Table 4:

[0176] Table 4

[0177]

[0178] The above results show that without the addition of blocking antigen, the detection will be interfered by the sample EV71 virus infection.

[0179] Example 4: Repetitive test

[0180] The 3 positive sera for sensitivity determination in Example 1 were taken, and the kit provided in the preparation example was used for 5 times of repetitive detection within the batch and between batches according to its use method, and the results were interpreted according to the Antu magnetic particle coxsackie virus A16 IgM detection procedure, as shown in Table 5:

[0181] Table 5

[0182]

[0183] The above results show that the coefficient of variation of 5 times of detection within the batch is within 3%, the coefficient of variation of 5 times of detection between batches is within 4%, and the coefficient of variation between batches and within the batch is within 5%.

[0184] Example 5: PCR identification coincidence

[0185] The magnetic particle chemiluminescence kit for coxsackie virus A16 IgM prepared in the preparation example was used to detect 90 normal human blood samples and 10 samples of hand-foot-mouth disease clinically diagnosed as coxsackie virus A16 infection (PCR identification), and the detection procedure was strictly according to the use method provided in the preparation example.

[0186] The results are shown in Table 6:

[0187] Table 6: Coincidence of the kit of the application and PCR identification

[0188]

[0189] The data in Table 6 show that the coincidence of the kit of the application and PCR identification is 100%.

[0190] The data in Table 6 show that the magnetic particle chemiluminescence kit for coxsackie virus A16 IgM provided by the application can effectively detect coxsackie virus A16 IgM positive samples in hand-foot-mouth disease.

[0191] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A composition, characterized in that, It consists of an antigen blocking solution and an enzyme-labeled recombinant Coxsackievirus A16 antigen; The blocking antigen in the antigen blocking solution is a recombinant antigen of enterovirus 71. The recombinant antigen of enterovirus 71 is enterovirus 71 polyprotein P1 and enterovirus 71 3CD protease; The recombinant antigen of Coxsackievirus A16 is Coxsackievirus A16 polyprotein P1 and Coxsackievirus A16 3CD protease; in: The nucleotide sequence of the nucleic acid molecule encoding the enterovirus 71 polyprotein P1 is shown in SEQ ID NO.5; The nucleotide sequence of the nucleic acid molecule encoding the enterovirus 71 3CD protease is shown in SEQ ID NO. 6; The nucleotide sequence of the nucleic acid molecule encoding the Coxsackievirus A16 polyprotein P1 is shown in SEQ ID NO.2; The nucleotide sequence of the nucleic acid molecule encoding the Coxsackievirus A16 type 3CD protease is shown in SEQ ID NO.3; The preparation method of the enzyme-labeled Coxsackievirus A16 recombinant antigen is as follows: the Coxsackievirus A16 recombinant antigen is mixed with horseradish peroxidase activated by sodium periodate at a molar ratio of 1:5, incubated, and then mixed with PBS buffer containing 50% glycerol to obtain horseradish peroxidase-labeled Coxsackievirus A16 recombinant antigen. The preparation method of the Coxsackievirus A16 recombinant antigen or the Enterovirus 71 recombinant antigen is as follows: insert the gene encoding the Coxsackievirus A16 recombinant antigen or the gene encoding the Enterovirus 71 recombinant antigen into an insect baculovirus expression vector to obtain a recombinant vector, express the recombinant vector, and obtain the Coxsackievirus A16 recombinant antigen or the Enterovirus 71 recombinant antigen. The nucleotide sequence of the insect baculovirus expression vector is shown in SEQ ID NO.

1.

2. The use of the composition as described in claim 1 in excluding false positives for enterovirus 71 in the detection of Coxsackievirus A16.

3. The use of the composition of claim 1 in the preparation of reagents or kits for detecting anti-Coxsackievirus A16 IgM.

4. The use of the composition according to claim 1 in the following aspects: (a) Preparation of diagnostic reagents or kits for Coxsackievirus A16 infection; and / or (b) Preparation of diagnostic reagents or kits for hand-foot-mouth disease.

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 coated with mouse anti-human IgM monoclonal antibodies, magnetic particle sealing solution, sample diluent, enzyme conjugate diluent, washing solution, substrate A solution, substrate B solution, negative control or positive control.

8. The kit according to claim 7, characterized in that, The magnetic particles coated with the mouse anti-human IgM monoclonal antibody are prepared as follows: 0.2 μg / person of Merck magnetic microparticles and 0.5 μg / person of mouse anti-human IgM monoclonal antibody are mixed, incubated for 2 hours, and then mixed with PBS buffer with a final casein concentration of 1% to obtain the magnetic particles coated with the mouse anti-human IgM monoclonal antibody; and / or The positive control was a mouse anti-Coxsackievirus A16 antibody chimeric with human IgM Fc fragment and PBS buffer containing 20% ​​fetal bovine serum; and / or The negative control is a PBS buffer containing 20% ​​fetal bovine serum; and / or The sample diluent is a 0.05 M Tris-HCl buffer solution with pH 7.4 containing 2% BSA; and / or The washing solution is PBS phosphate buffer containing 0.1% Tween 20; and / or The enzyme conjugate dilution solution is a 0.05M Tris-HCl buffer solution with a pH of 7.4 containing 20% ​​fetal bovine serum; and / or Substrate A and substrate B are buffer solutions containing luminol.

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