阅读说明：本技术 一种柯萨奇病毒a16型病毒抗原检测试剂盒 (Coxsackie virus A16 type virus antigen detection kit ) 是由 梁争论 崔博沛 高强 蔡芳 毛群颖 武瑞霞 戈小琴 吴星 高帆 卞莲莲 于 2019-10-30 设计创作，主要内容包括：本发明提供一种柯萨奇病毒A16型病毒抗原检测试剂盒,所述检测试剂盒是基于ELISA方法的检测试剂盒,所述试剂盒含有兔抗CA16多克隆抗体和柯萨奇病毒A16型病毒单克隆抗体16E1。本发明的试剂盒特异性强、灵敏度好、重复性佳,与肠道病毒地其他型别不存在交叉反应,又可以和CA16的多个亚型的抗原反应,具备广谱性,具有重要的实用意义。(The invention provides a coxsackie virus A16 type virus antigen detection kit, which is based on an ELISA method and contains a rabbit anti-CA 16 polyclonal antibody and a coxsackie virus A16 type virus monoclonal antibody 16E 1. The kit has strong specificity, good sensitivity and good repeatability, does not have cross reaction with other types of enteroviruses, can react with antigens of a plurality of subtypes of CA16, has broad spectrum and has important practical significance.)

1. A coxsackievirus A16 type virus antigen detection kit is characterized in that the detection kit is based on an ELISA method, and the kit contains a rabbit anti-CA 16 polyclonal antibody and a coxsackievirus A16 type virus monoclonal antibody 16E 1.

2. The detection kit according to claim 1, wherein the monoclonal antibody is produced by a hybridoma cell line 16E1 which has a preservation number of CCTCC number C2019167 and a preservation date of 2019, 8 months and 1 days, and is preserved in the unit of China center for type culture Collection, the preservation number of CCTCC No. C2019167 and the preservation address of Wuhan university, Wuhan, China.

3. The test kit according to claim 1, characterized in that the coating antibody 16E1 is used at a concentration of 3.7 μ g/ml.

4. The test kit according to claim 1, wherein the rabbit anti-CA 16 polyclonal antibody contains IgG heavy chain and light chain content of not less than 80%.

5. The test kit according to claim 1, characterized in that the rabbit anti-CA 16 polyclonal antibody has a titer of 10⁶The use concentration is 1: 4000.

6. the detection kit according to claim 1, wherein the detection kit further comprises a sample diluent, a concentrated washing solution, hydrolyzed casein, an enzyme diluent, a developing solution A, a developing solution B and an ELISA plate.

7. The test kit according to claim 6, characterized in that the sample diluent comprises 100ml of calf serum, 900ml of 0.01M PBS, 3000.5ml of Proclin;

the concentrated wash solution (20X) comprises NaH₂PO₄·2H₂O 2.96g、Na₂HPO₄·12H₂O29.0 g, NaCl 234g, Tween-2020ml, and purified water to 1000 ml;

the hydrolyzed casein comprises 100g of casein and 3001ml of Proclin, and purified water is added to reach 1000 ml;

the enzyme diluent comprises 100ml of calf serum, 100ml of hydrolyzed casein, 300100 ul of Proclin, Tween-2050 ul of purified water and 1000ml of purified water;

the stop solution comprises 112ml of concentrated sulfuric acid and 888ml of purified water;

the color development liquid A comprises 27.2g of sodium acetate, 3.2g of citric acid and 30% of H₂O₂0.6ml, adding double distilled water to 1000 ml;

the color developing solution B comprises 0.4g of TMB and EDTA-Na₂0.4g, 1.9g of citric acid, 100ml of glycerol and double distilled water to 1000 ml.

8. The rabbit anti-CA 16 polyclonal antibody according to claim 5, further characterized in that said polyclonal antibody is labeled with horseradish peroxidase, pyruvate kinase, or glucose oxidase, or fluorescein, biotin, colloidal gold, or alkaline phosphatase.

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of virology and antibody medicines, in particular to a coxsackie virus A16 type virus antigen detection kit.

[ background of the invention ]

The hand-foot-and-mouth disease is one of class-C infectious diseases in China, the first occurrence of the disease is fever, rash, herpes and small ulcer of hands, feet and oral cavities, and part of patients can develop aseptic encephalitis, meningitis and other serious disease manifestations, so that a large disease burden is brought to China. Currently, most areas around the world have reports related to hand-foot-and-mouth diseases, and the asia-pacific area is regarded as a high-incidence area of hand-foot-and-mouth diseases and receives enough attention in recent years. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are main pathogens causing hand-foot-and-mouth disease, and although the hand-foot-and-mouth disease caused by coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) is increasing in recent years, EV71 and CA16 infection has obvious association with severe hand-foot-and-mouth disease.

Through 8 years of efforts of domestic scholars and biological product companies, EV71 vaccine independently developed by China in 2016 is successfully marketed, which provides a foundation for development of hand-foot-and-mouth disease prevention and treatment work. However, CA16 is another important pathogen in severe hand-foot-and-mouth disease, and co-infection of EV71 and CA16 is likely to cause aggravation of disease symptoms and prolong the disease course, and is more likely to cause severe nervous system symptoms. And EV71 and CA16 mixed or alternate epidemics are easy to cause virus recombination, and the complexity of the hand-foot-and-mouth disease epidemics is increased. The vaccine is an important means for controlling the epidemic of the hand-foot-and-mouth disease, and after the EV71 whole virus inactivated vaccine comes into the market successfully in 2016, an important technical support is provided for the research and development of a CA16 vaccine and an EV71/CA16 combined vaccine, but at present, a vaccine capable of preventing CA16 is lacked.

CA16 is a member of Enterovirus of picornaviridae, has a diameter of 27-30 nm, is non-enveloped, and has an icosahedral structure. The single-strand positive-strand RNA virus has a 7400bp genome with an open reading frame, encodes a polyprotein precursor which is cleaved into a structural protein P1 and non-structural proteins P2 and P3 by self-produced hydrolases. The structural protein P1 mainly encodes capsid protein composed of VP1, VP0 and VP3, VP0 is hydrolyzed into VP2 and VP4 in the virus maturation process, wherein VP4 is located in the capsid protein, and VP1, VP2 and VP3 are distributed on the surface of capsid virus to form pentamer. The open reading frame coding region is flanked on both sides by 5 'and 3' non-coding regions, respectively, the 5 'non-coding region consisting of 740 nucleotides and being associated with replication and translation functions of the viral genome, and the 3' non-coding region comprising a PolyA tail, which is essential for successful infection of the virus.

At present, the CA16 vaccine of the national research in China is mainly divided into whole virus inactivated vaccine and virus-like particle vaccine. The effectiveness of a vaccine depends primarily on the humoral (i.e., neutralizing) and cellular immune responses that it induces. A large amount of experimental data indicate that both the formalin inactivated EV71/CA16 vaccine and the EV71/CA16 VLPs vaccine can cause better neutralizing antibody response, and then the generated neutralizing antibody can be combined with the corresponding epitope of the virus, thereby blocking the combination of the virus and a receptor on the cell surface. Besides, VLP vaccine can induce cell immune response, which is mainly manifested by the increase of Th1 type and Th2 type cytokines such as IFN-gamma, IL-2, IL-4, IL-6, IL-10, etc. This suggests that both inactivated vaccines and VLPs may be candidates for combination vaccines.

Meanwhile, researches show that enteroviruses have cross reactivity between a plurality of types and between types, and recombination is easy to occur between different types, for example, genome similarity of EV71 and CA16 is as high as 80%, and the enteroviruses have a plurality of identical and similar epitopes, so that screening of a neutralizing monoclonal antibody specific to CA16 is difficult, and establishment of an antigen detection system of CA16 is greatly restricted.

At present, a plurality of enterprises in China research the CA16 vaccine, and each vaccine research and development enterprise establishes an antigen detection kit aiming at the strain generation and process thereof, the result difference of each enterprise is large, and a set of uniform antigen detection kit needs to be established urgently to meet the detection requirements of intermediate products and vaccine finished products of most enterprises.

[ summary of the invention ]

The invention aims to overcome the defect of the blank CA16 antigen detection system, and provides a CA16 monoclonal antibody, and an in-vitro detection method and a detection kit for a CA16 antigen are established by applying the monoclonal antibody, wherein the kit has broad spectrum, can react with other types of antigens of CA16, has good specificity and does not have cross reaction with viruses such as EV71, CA6, CA10 and the like and other types of viruses.

In order to achieve the above object, the present invention provides a coxsackievirus A16 type virus antigen detection kit, which is an ELISA method-based detection kit comprising a rabbit anti-CA 16 polyclonal antibody and a coxsackievirus A16 type virus monoclonal antibody 16E 1.

The monoclonal antibody 16E1 of the coxsackie virus A16 is generated by a hybridoma cell strain 16E1, the hybridoma cell strain is preserved in a China center for type culture collection at 8 months and 1 days in 2019, the preservation number is CCTCC No. C2019167, and the preservation address is Wuhan university, Wuhan, China.

In the present invention, the method for constructing hybridoma cell 16E1 comprises the following steps:

(1) immunization of mice

Immunizing a BALB/c female mouse with the age of 6-8 weeks by using a CA16 virus immunogen, strengthening the immunization once every 2 weeks, detecting the serum titer of the mouse before each immunization, stopping the immunization when the serum titer of the mouse reaches a platform period, and preparing for fusion;

(2) preparation and screening of fused hybridomas

(2.1) preparation of mouse macrophage

Killing BALB/c mice, and dissolving the abdominal cavity cell sap in HAT culture solution or HT culture solution until the final concentration of abdominal cavity cells in the culture solution is 2 x 105 cells/mL; adding the cells into a 96-hole cell culture plate, and culturing in an incubator to obtain mouse macrophages;

(2.2) preparation of mouse thymocytes

Killing BALB/c mice, taking out thymus, grinding the thymus, and sieving through a 200-mesh cell sieve to obtain thymus feeder cell liquid;

(2.3) preparation of mouse myeloma cells

Taking mouse myeloma cell strain Sp2/0-Ag14(Sp2/0), culturing with 20% FBS RPMI-1640 culture solution, selecting cells in logarithmic growth phase, transferring myeloma cells from a culture bottle into a centrifuge tube before fusion, washing with RPMI-1640 culture solution for 3 times, resuspending the cells with the RPMI-1640 culture solution, and counting;

(2.4) preparation of immune spleen cells

Taking a BALB/C mouse to be fused, and collecting blood after the mouse is sacrificed to prepare antiserum serving as a positive control of antibody detection; taking mouse spleen, sieving with 200 mesh cell sieve, squeezing and grinding with grinding rod, dripping RPMI-1640 culture solution, standing for 3-5min, taking 2/3 part of suspension, and transferring into 50mL plastic centrifuge tube; repeating the steps for 2-3 times, washing the cells for 3 times by using RPMI-1640 culture solution, then re-suspending the cells by using the RPMI-1640 culture solution and counting to obtain immune spleen cells;

(2.5) preparation of hybridomas Using PEG fusion promoters

1mL of PEG-1500, 10mL of RPMI-1640 serum-free medium, and 200mL of complete medium were equilibrated to 37 ℃; mixing the myeloma cells prepared in the step (2.3) and the spleen cells prepared in the step (2.4) in a 50mL centrifugal tube, wherein the number ratio of the spleen cells to the myeloma cells is 10:1, centrifuging at 1500rpm for 8min, and lightly tapping the bottom of the tube to loosen the cells into paste; sucking 0.8mL of PEG, adding into a centrifugal tube, adding 10mL of RPM-1640 complete culture solution at 37 ℃, stirring gently, finally supplementing the RPMI-1640 culture solution to 40mL, and centrifuging at 1000RPM for 5 min; discarding the supernatant, taking the HT culture solution to blow the cells away, and then adding the HT culture solution; the resulting cells were added to a 96-well cell culture plate at 100. mu.L per well, and CO was added₂After 12 hours of incubation in an incubator, 100. mu.L of HAT was added dropwise to each well for complete incubationA group; after 5 days, using an HT complete culture medium to exchange liquid for cell supernatant in the holes, wherein the volume ratio of the liquid exchange is 50-100%; after 9-14 days, sucking the supernatant for detection;

(2.6) screening of hybridomas

Screening by using an indirect ELISA method, coating 100 ng/hole of purified CA16 virus antigen, adding 50 mu L of cell supernatant for detection, and selecting a positive cloning hole;

(2.7) cloning of hybridoma cells

And selecting hybridoma cells by using a clone culture method, and repeatedly cloning for 2-3 times until the hybridoma cells are 100% positive to obtain the hybridoma cells 16E 1.

According to a preferred embodiment, the coating concentration of the coating antibody is 3.7. mu.g/ml.

In the present invention, the monoclonal antibody 16E1 is either biomarker or chemical (e.g., enzyme-labeled or fluorescent-labeled), for example, the label may be horseradish peroxidase, pyruvate kinase or glucose oxidase or fluorescein, biotin, colloidal gold, alkaline phosphatase.

In the present invention, the detection kit based on the ELISA method further comprises a sample diluent, an enzyme diluent, a developing solution a, a developing solution B, and a stop solution and/or instructions. Those skilled in the art can select a suitable sample diluent, enzyme diluent, developing solution A, developing solution B and/or stop solution according to the ELISA detection method, which is not described herein.

The kit adopts the 16E1 monoclonal antibody and the rabbit polyclonal antibody as enzyme-labeled secondary antibodies, aims at the CA16 neutralizing epitope, can better correlate the measured antigen content with immunogenicity, is convenient for establishing an in vitro detection method, is used for replacing in vivo experiments, and reduces the use of experimental animals.

Experiments confirm that the kit disclosed by the invention is coated by the 16E1 monoclonal antibody, and the antibody does not have cross reaction with other types of enteroviruses, so that the specificity of the kit is ensured.

The kit adopts rabbit polyclonal antibody as enzyme-labeled secondary antibody, and can capture antigen to the maximum extent.

The kit can react with antigens of a plurality of subtypes of CA16, and has broad spectrum.

Due to the very significant intra-and inter-type cross-reactivity of enteroviruses, the genome similarity of e.g. EV71 and CA16 reaches 80%. The monoclonal antibody 16E1 does not have cross reaction with other enteroviruses of other types such as EV71, CA6, CA10 and the like, so that the restriction of the cross reaction on the establishment of the kit can be well avoided; in addition, the kit can be further developed and used for screening hand-foot-and-mouth diseases, epidemiological investigation and the like, and has important practical significance.

The monoclonal antibody 16E1 of the coxsackie virus A16 is generated by a hybridoma cell strain 16E1, the hybridoma cell strain is preserved in a China center for type culture collection at 8 months and 1 days in 2019, the preservation number is CCTCC No. C2019167, and the preservation address is Wuhan university, Wuhan, China.

[ description of the drawings ]

FIG. 1 is an SDS-PAGE electrophoresis of CA16 rabbit polyclonal antiserum from example 1 after purification, wherein lane 1: pre-staining protein marker; lane 2: and (5) purifying the polyclonal antibody.

Figure 2 is the sensitivity and linear range results of example 6.

FIG. 3 shows the accuracy results of example 6.

FIG. 4 shows the results of the applicability study of example 7.

[ detailed description ] embodiments

The following examples serve to illustrate the technical solution of the present invention without limiting it.

The following examples relate to the following materials or devices, which are also directly available to the skilled person in the light of the prior art teachings:

the sources of the CA16 virus were:

(1) the strain CA16V-24 is provided by Beijing Kexing biological products, Inc., and can be obtained by the same method by those skilled in the art, and the strain specimen is from Hangzhou city, Zhejiang province.

(2) CA16/00190/B1B (GenBank No. MF177223) virus was isolated from Xiamen university in 2011 and specimens were obtained from Taiwan clinical samples positive for HFMD.

The RPMI-1640 culture solution comprises the following components: dry powder of medium (GIBCO-31800-.

FBS：GIBCO-10099-141

The 20% FBS RPMI-1640 culture solution is prepared by adding 20% FBS by total volume on the basis of the RPMI-1640 culture solution.

The HT culture solution is prepared by adding hypoxanthine (SIGMA-ALBRICH-H9377) and thymidine (SIGMA-ALBRICH-T9250) to RPMI-1640 culture solution.

HAT culture medium is HT culture medium supplemented with aminopterin (SIGMA-ALBRICH-A5159).

The RPMI-1640 complete culture solution is prepared by adding 10% FBS (FBS) into the RPMI-1640 culture solution according to the total volume.

HT complete medium is obtained by adding 10% FBS to HT medium in total volume.

HAT complete medium is prepared by adding 10% FBS to HAT medium.

In the present invention, "%" used for explaining the concentration is mass percent, ": all the terms are by weight.