阅读说明：本技术 一种非洲猪瘟病毒磁微粒化学发光抗体检测试剂盒及其应用 (Magnetic particle chemiluminescence antibody detection kit for African swine fever virus and application thereof ) 是由 郑海学 �田宏 罗俊聪 石正旺 王丽娟 万颖 宋锐 杨波 张克山 李丹 茹毅 于 2020-07-30 设计创作，主要内容包括：本发明属于免疫检测分析技术领域,具体涉及一种非洲猪瘟病毒磁微粒化学发光抗体检测试剂盒及其应用,该试剂盒以ASFV P30重组蛋白为磁微粒偶联物,以化学发光标记物标记的羊抗猪IgG为检测抗体。具体由偶联有ASFV P30重组蛋白的磁微粒混悬液、试剂R、定标品、质控品、样品稀释液、洗涤液及发光液组成,所述试剂R为稀释后的化学发光标记物标记的羊抗猪IgG抗体工作液。本发明试剂盒以磁微粒化学发光法为检测技术,同时结合碱性磷酸酶标记技术,具有特异性高、灵敏性好、重复性优、稳定性强等特点,可广泛应用于基层ASFV检测。(The invention belongs to the technical field of immunodetection and analysis, and particularly relates to a magnetic particle chemiluminescence antibody detection kit for African swine fever virus and application thereof. The kit specifically comprises a magnetic particle suspension coupled with ASFV P30 recombinant protein, a reagent R, a calibration product, a quality control product, a sample diluent, a washing solution and a luminescent solution, wherein the reagent R is a diluted working solution of goat anti-pig IgG antibody marked by a chemiluminescent marker. The kit takes a magnetic particle chemiluminescence method as a detection technology, is combined with an alkaline phosphatase labeling technology, has the characteristics of high specificity, good sensitivity, excellent repeatability, strong stability and the like, and can be widely applied to basic ASFV detection.)

1. The kit is characterized by consisting of magnetic particle suspension coupled with ASFVP30 recombinant protein, a reagent R, a calibration product, a quality control product, a sample diluent, a washing solution and a luminescent solution, wherein the reagent R consists of an antibody diluent and a goat anti-pig IgG antibody marked by alkaline phosphatase.

2. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the preparation of the ASFV P30 recombinant protein magnetic particle suspension comprises the following steps: activating the magnetic beads with EDC and NHS, mixing the two according to the proportion of 10 mu g of recombinant protein/mg of magnetic beads, and coating for 3h at 37 ℃; the supernatant was removed by magnetic adsorption, blocked with 10% ethanolamine hydrochloride aqueous solution for 3 hours, and the beads were washed with a washing solution 3 times and diluted to 0.5mg/mL with a storage solution containing 2% BSA.

3. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 2, wherein the preservation solution containing 2% BSA consists of 2% BSA, 1% trehalose, 0.1% EDTA, 0.9% NaCl, 0.05% Tris-HCl, 0.5% casein, 0.05M PMSF.

4. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the chemiluminescent marker is alkaline phosphatase.

5. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the preparation of reagent R comprises the steps of filling an antibody diluent and goat anti-pig IgG antibody labeled with alkaline phosphatase into a box, performing plastic packaging, placing into a magnetic bead cup, and performing secondary sealing.

6. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 5, wherein the antibody diluent comprises 1% BSA, 0.9% NaCl, 0.5% Tween-20, 0.05M MOPS with pH7.0, 1mM ZnCl₂、1mM MgCl₂And (4) forming.

7. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the calibrator comprises calibrator C1 and calibrator C2; the calibration sample C1 is ASFV antibody negative serum, and the calibration sample C2 is prepared by diluting ASFV antibody strong positive inactivated serum to working concentration by using preservation solution.

8. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the quality control product is prepared by diluting ASFV antibody strong positive inactivated serum with ASFV antibody negative serum.

9. The African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, wherein the sample diluent comprises 1% BSA, 0.9% NaCl, 0.5% Tween-20, 0.05M MOPS at pH 7.0.

10. The application of the African swine fever virus magnetic particle chemiluminescence antibody detection kit of claim 1, which is characterized by comprising the following steps: adding a sample to be detected into a reaction cup, adding an ASFV P30 recombinant protein magnetic particle suspension, uniformly mixing, reacting for 10min at 37 ℃, adding a washing solution to wash off the supernatant, adding a proper amount of alkaline phosphatase-labeled goat anti-pig IgG antibody working solution, reacting for 10min at 37 ℃, adding a washing solution to wash off the supernatant, adding a luminescent solution, and determining the corresponding luminescent value.

Technical Field

The invention belongs to the technical field of immunodetection and analysis, and particularly relates to a magnetic particle chemiluminescence antibody detection kit for African swine fever virus and application thereof.

Background

African Swine Fever Virus (ASFV), a single-stranded double-stranded DNA virus, which infects pigs causes hyperpyrexia, cyanosis of the skin and severe bleeding of lymph nodes and internal organs, with an acute mortality rate of 100%, and African swine fever does not always show complete clinical symptoms, which are easily confused with other epidemic symptoms when infected in the early stages of the disease or in a small number of animals. After 8 months in 2018 and outbreak in China, the disease not only seriously troubles the prevention and control of animal epidemic diseases in China, but also seriously harms the development of the pig industry, and has huge economic loss.

ASFV is about 200nm in diameter, has a capsule coat capsid, is in icosahedral symmetry, has a complex multilayer structure, has about 151 Open Reading Frames (ORFs), and the structure and function of 131 viral proteins in the encoded protein have been reported, so far, researches show that the main structural components of the virion, such as p72, p30, p54, p12, are respectively involved in the functions of virus adsorption, invasion of host target cells, virus replication, early mRNA synthesis and processing, and the like, and the proteins are also widely applied to various serological diagnosis methods.

The African swine fever virus has multiple genotypes, is easy to mutate, can not effectively generate a neutralizing antibody after infecting a pig body, and the African swine fever immunity relates to complex cellular immunity and innate non-specific immunity, thereby hindering the development of the African swine fever vaccine. To date, no commercial vaccine is available for ASFV, and only the virus spreading can be prevented by killing and disinfecting measures after the epidemic situation is discovered. Therefore, the establishment of a rapid and effective detection method for large-scale detection makes early discovery, early diagnosis and early isolation have important practical significance.

Summarizing the existing diagnostic detection technology, the colloidal gold technology and the enzyme-linked immunosorbent technology are applied more on the market. The colloidal gold detection technology is simple and convenient to operate, but has low sensitivity and does not meet the requirement of high-precision detection; the enzyme-linked immunosorbent assay mainly adsorbs antibodies or antigens onto solid-phase or liquid-phase carriers to detect corresponding antigens or antibodies, and the adsorption process can greatly reduce the action area of the antigens or antibodies, thereby reducing the sensitivity.

Disclosure of Invention

In order to overcome the problems of low sensitivity of the detection technology and reduction of the action area of the antigen or the antibody in the adsorption process, the invention provides an excellent carrier capable of enlarging the action area of the antigen and the antibody. The invention establishes a magnetic particle chemiluminescence antibody detection kit for African swine fever virus by using magnetic particles as carriers and alkaline phosphatase as a chemiluminescence marker. The kit has good specificity, sensitivity and repeatability, is simple and rapid to operate, and is more favorable for the detection technology of the African swine fever virus serum antibody popularized and applied at the basic level.

In order to achieve the purpose, the invention adopts the following technical scheme:

the kit for detecting the ASFV serum antibody by the magnetic particle chemiluminescence method provided by the invention takes the ASFV 30 recombinant protein as a magnetic particle conjugate and takes goat anti-pig IgG labeled by a chemiluminescence label as a detection antibody. The kit comprises a magnetic particle suspension coupled with ASFV P30 recombinant protein, a reagent R, a calibration product, a quality control product, a sample diluent, a washing solution and a luminescent solution, wherein the reagent R is a diluted working solution of goat anti-pig IgG antibody marked by a chemiluminescent marker.

As a preferable scheme of the invention, the preparation of the ASFV P30 recombinant protein magnetic particle suspension comprises the following steps: activating the magnetic beads with EDC and NHS, mixing the two according to the proportion of 10 mu g of recombinant protein/mg of magnetic beads, and coating for 3h at 37 ℃; the supernatant was removed by magnetic adsorption, blocked with 10% ethanolamine hydrochloride aqueous solution for 3 hours, and the beads were washed with a washing solution 3 times and diluted to 0.5mg/mL with a storage solution containing 2% BSA.

Specifically, the magnetic beads were diluted to 10mg/mL with 0.1M aqueous MES solution at pH5.0 and washed 2 times with an equal volume of 0.1M MES. EDC and NHS were rewarming, weighed, dissolved in 0.1M MES to 10mg/mL, mixed immediately with 1mg magnetic beads 10. mu. LEDC and 10. mu.L NHS, and reacted at 37 ℃ for 30 min. Removing supernatant after magnetic adsorption, adding equal volume of 0.1MMES, adding p30 recombinant protein according to determined optimal dosage, mixing uniformly, and reacting at 37 ℃ for 3 h. Removing supernatant after magnetic adsorption, adding equal volume of confining liquid, mixing uniformly, and reacting at 37 ℃ for 3 h. The magnetic beads were washed 3 times with a washing solution and diluted to 0.5mg/mL with a magnetic bead storage solution. Mixing, packaging, sealing, and storing at 4 deg.C.

In a preferred embodiment of the present invention, the preservation solution containing 2% BSA consists of 2% BSA, 1% trehalose, 0.1% EDTA, 0.9% NaCl, 0.05% Tris-HCl, 0.5% casein, 0.05M PMSF.

In a preferred embodiment of the present invention, the chemiluminescent label is alkaline phosphatase.

As a preferred scheme of the invention, the preparation of the reagent R comprises the following steps of filling an antibody diluent and the goat anti-pig IgG antibody marked by alkaline phosphatase into a box, performing plastic package, putting into a magnetic bead cup, and performing secondary sealing.

As a preferred embodiment of the present invention, the antibody dilution solution is composed of 1% BSA, 0.9% NaCl, 0.5% Tween-20, 0.05M MOPS at pH7.0, 1mM ZnCl₂、1mM MgCl₂And (4) forming.

As a preferable scheme of the invention, the calibration sample consists of calibration sample C1 and calibration sample C2; the calibration sample C1 is ASFV antibody negative serum, and the calibration sample C2 is prepared by diluting ASFV antibody strong positive inactivated serum to working concentration by using preservation solution.

As a preferable scheme of the invention, the quality control product is prepared by diluting ASFV antibody strong positive inactivated serum by ASFV antibody negative serum.

As a preferred embodiment of the present invention, the sample diluent consists of 1% BSA, 0.9% NaCl, 0.5% Tween-20, 0.05M MOPS at pH 7.0.

The application of the magnetic particle chemiluminescence antibody detection kit for the African swine fever virus comprises the following steps: adding a sample to be detected into a reaction cup, adding an ASFV P30 recombinant protein magnetic particle suspension, uniformly mixing, reacting for 10min at 37 ℃, adding a washing solution to wash off the supernatant, adding a proper amount of alkaline phosphatase-labeled goat anti-pig IgG antibody working solution, reacting for 10min at 37 ℃, adding a washing solution to wash off the supernatant, adding a luminescent solution, and determining the corresponding luminescent value.

The invention has the following beneficial effects:

the invention adopts the magnetic particle chemiluminescence technology in combination with the alkaline phosphatase labeling technology, can rapidly, accurately and massively detect the ASFV antibody, and provides a new technical support for the rapid diagnosis of the ASFV.

The preservation solution (2% BSA, 1% trehalose, 0.1% EDTA, 0.9% NaCl, 0.05% Tris-HCl, 0.5% casein, 0.05M PMSF) has a good preservation effect, can preserve antigen magnetic beads for a long time without influencing the reactivity of the antigen magnetic beads, and endows the detection kit with high stability.

According to the application, the recombinant protein magnetic beads are prepared from the ASFV P30 recombinant protein, the P30 protein is generated in the early stage of ASFV infection, the generation time is short, the immunogenicity is good, and the P30 protein continuously exists in cytoplasm in the whole infection cycle of the ASFV.

Drawings

FIG. 1 is a graph showing ROC values of ASFV serum samples.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the scope of the present invention is not limited to the following examples, and any technical solutions that can be conceived by those skilled in the art based on the present invention and the common general knowledge in the art are within the scope of the present invention.