WO2022032983A1 - 一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条及其应用 - Google Patents

一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条及其应用 Download PDF

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WO2022032983A1
WO2022032983A1 PCT/CN2021/071258 CN2021071258W WO2022032983A1 WO 2022032983 A1 WO2022032983 A1 WO 2022032983A1 CN 2021071258 W CN2021071258 W CN 2021071258W WO 2022032983 A1 WO2022032983 A1 WO 2022032983A1
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protein
quantum dot
sample
swine fever
african swine
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PCT/CN2021/071258
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French (fr)
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冯志新
刘斐
白昀
李佳豪
陈蓉
谢青云
张越
张磊
李悦
韦艳娜
刘蓓蓓
华利忠
熊祺琰
邵国青
王丽
单衍可
陆雨楠
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江苏省农业科学院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention belongs to the technical field of veterinary biological diagnostic products, and in particular relates to a quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody and its application.
  • African swine fever is an acute, febrile and highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV) infection. It is also an animal disease notifiable by the World Organization for Animal Health (OIE).
  • ASFV is a complex, enveloped, double-stranded DNA virus with a diameter of 170-190kb, with terminal cross-linking and inverted repeat regions, which encodes 151-167 kinds of proteins, and mature virions contain about 50 kinds structural protein.
  • ASFV mainly invades pigs through the respiratory tract and digestive tract.
  • pigs can produce specific sIgA antibodies in the mucosa of the respiratory tract and digestive tract in addition to specific IgG antibodies in serum.
  • sIgA antibodies are mainly found in respiratory secretions, saliva, tears, breast milk, gastrointestinal secretions and urogenital secretions. They are the main antibodies of the body's mucosal immunity and the first immune response after pathogens invade. They are often used for disease infection. Detection indicators for early diagnosis.
  • the first object of the present invention is to provide a quantum dot microsphere immunochromatographic test strip for detecting ASFV mucosal sIgA antibodies, which can be used for rapid, rapid detection of ASFV mucosal sIgA antibodies.
  • Qualitative detection with high specificity and high sensitivity.
  • the second object of the present invention is to provide a sample diluent for the above-mentioned quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody.
  • the third object of the present invention is to provide a non-diagnostic method for detecting African swine fever virus mucosal sIgA antibody using the quantum dot microsphere immunochromatographic test strip and the sample diluent. Simple operation, no professional training is required, and it can be well qualified for detection in various on-site, grass-roots laboratories and other environments; combined with fluorescence detectors that are cheaper than fluorescence microscopes and microplate readers, it can be timely and convenient. , get results in the field.
  • a quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody comprising a bottom plate, and a sample pad, a nitrocellulose membrane and a water absorption pad are arranged on the bottom plate in sequence; There is a detection line and a quality control line, the detection line is coated with mouse anti-pig SC protein monoclonal antibody, and the detection line is set near the side of the sample pad; the quality control line is coated with African pigs Plasma-positive serum purified protein, the quality control line is set near the side of the absorbent pad.
  • the purified protein of the African swine fever positive serum is the protein obtained by purifying the rabbit anti-African swine fever recombinant protein P30V and recombinant polypeptide tandem protein R10 hyperimmune serum using a protein G purification kit; the mouse anti-pig The SC protein monoclonal antibody is prepared by the hybridoma cell line 4H11 that secretes the anti-porcine SC protein monoclonal antibody, and the hybridoma cell line 4H11 has the deposit number: CCTCC NO: C201526.
  • the sample pad is obtained by soaking the glass fiber membrane with Tris-HCl buffer solution containing bovine serum albumin and Tween-20.
  • the present invention also provides a sample diluent for use with the test strip, which contains the recombinant polypeptide tandem protein R10 labeled with quantum dot microspheres and the recombinant protein P30V labeled with quantum dot microspheres; the sequence of the recombinant polypeptide tandem protein R10 is shown in SEQ ID NO: 2; the sequence of the recombinant protein P30V is shown in SEQ ID NO: 4.
  • the quantum dot microspheres are respectively coupled with recombinant polypeptide tandem protein R10 and recombinant protein P30V to obtain respectively the recombinant polypeptide tandem protein R10 labeled by quantum dot microspheres and the recombinant protein P30V labeled by quantum dot microspheres;
  • the quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 and the recombinant protein P30V are mixed in a volume ratio of 1:0.5-1.5 to obtain an African swine fever virus double protein-label; the African swine fever virus double protein-label is mixed with The PBS buffer containing BSA and Tween-20 was mixed in a volume ratio of 1:28-32 to obtain a sample dilution.
  • the mass ratio of the quantum dot microspheres to the recombinant polypeptide tandem protein R10 is 5-7:1
  • the mass ratio of the quantum dot microspheres to the recombinant protein P30V is 5-7:1;
  • the PBS contains 0.05-0.15% BSA and 0.2% Tween-20 in PBS buffer.
  • the present invention also provides a non-diagnostic method for detecting African swine fever virus mucosal sIgA antibody by using the quantum dot microsphere immunochromatographic test strip and the sample diluent, and adding the swab sample to the sample diluent After incubating in the medium, it was dropped on the sample pad of the test strip, and after the reaction, the C-line and the T-line were detected by an ultraviolet light emitter.
  • the detection sample is a swab sample or a mucosal sample.
  • the swab sample includes oral swab, nasal swab or anal swab, and the mucosal sample is bronchoalveolar lavage fluid.
  • the mucosal sIgA antibody in the sample is first combined with the quantum dot microspheres in the sample diluent.
  • the sphere-labeled recombinant protein P30V and the quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 were combined to obtain a mucosal sIgA antibody-quantum dot complex, which was added dropwise to the sample pad and chromatographed on a nitrocellulose membrane.
  • the dot complexes were combined with the mouse anti-pig SC protein monoclonal antibody on the detection line to be immobilized at the detection line.
  • the excess quantum dot microsphere-labeled recombinant protein P30V and quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 were chromatographed At the quality control line, it reacted with the purified protein from African swine fever positive serum, and was fixed on the quality control line. In this way, the African swine fever virus mucosal sIgA antibody immobilized at the detection line can be detected by a 365 nm ultraviolet light emitter.
  • the present invention has the following beneficial effects:
  • the quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody provided by the present invention, the quantum dot microsphere has the advantages of high uniformity, good monodispersity, strong stability, etc.
  • the batch-to-batch variation of test strips made with balls as markers is small;
  • the preparation method of the quantum dot microsphere immunochromatographic test strip for African swine fever virus mucosal sIgA antibody provided by the present invention is easy to operate, requires simple raw materials, can be mastered in a relatively short time, and has broad market prospects and greater economic benefits.
  • the detection method of the present invention is simple to operate, does not require professional training, and can be well qualified for detection in various on-site, grass-roots laboratories and other environments; combined with ultraviolet light emission that is cheaper than fluorescence microscopes and microplate readers The results can be obtained in the field in a timely and convenient manner.
  • Detecting swab samples and mucosal samples, compared with serum samples, is more convenient for sampling, reduces the stress response to pigs during sampling, and is more effective for animal welfare and biosecurity.
  • Figure 1 is the expression identification diagram of the recombinant polypeptide tandem protein R10 in Example 1 of the present invention, wherein lane 1 is the purified recombinant polypeptide tandem protein R10, and lane 2 is the purified recombinant polypeptide tandem protein R10 and African swine fever antibody positive reference port Western-bloting identification of swabs, M is protein maker.
  • Figure 2 is the expression identification diagram of recombinant protein P30V in Example 1 of the present invention, wherein lane 1 is the purified recombinant protein P30V, and lane 2 is the Western-bloting of the purified recombinant protein P30V and African swine fever antibody positive reference mouth swab Identification, M is the protein maker.
  • FIG. 3 is a schematic structural diagram of a quantum dot microsphere immunochromatographic test strip for early detection of African swine fever antibodies according to an embodiment of the present invention.
  • 1- Quantum dot microsphere immunochromatography test strip for detecting African swine fever virus mucosa sIgA antibody 2- bottom plate, 3- sample pad, 4- nitrocellulose membrane, 7- absorbent pad, 5- detection line, 6- Quality control line.
  • FIG. 4 is a cross-sectional view of a quantum dot microsphere immunochromatographic test strip for early detection of African swine fever antibodies according to an embodiment of the present invention.
  • 5 is a top view of the quantum dot microsphere immunochromatographic test strip for early detection of African swine fever antibodies according to an embodiment of the present invention.
  • Figure 6 is the SDS-PAGE identification chart of the purified protein from African swine fever positive serum, wherein lane 1 is the purified protein from African swine fever positive serum, and M is the protein maker.
  • FIG 8 Specificity map, in which ASFV is the African swine fever antibody positive reference oral swab, PRRS is the porcine reproductive respiratory syndrome virus mucosal antibody positive sample, PEDV is the porcine epidemic diarrhea virus mucosal antibody positive sample, APP is porcine pleuropneumoniae Line bacillus mucosal antibody positive sample, PCV is a porcine circo virus mucosal antibody positive sample, SIV is a swine influenza virus mucosal antibody positive sample.
  • ASFV is the African swine fever antibody positive reference oral swab
  • PRRS is the porcine reproductive respiratory syndrome virus mucosal antibody positive sample
  • PEDV is the porcine epidemic diarrhea virus mucosal antibody positive sample
  • APP porcine pleuropneumoniae Line bacillus mucosal antibody positive sample
  • PCV is a porcine circo virus mucosal antibody positive sample
  • SIV is a swine influenza virus mucosal antibody positive sample.
  • MES MES
  • EDC EDC
  • NHS Teween-20
  • sucrose sucrose
  • Freund's complete adjuvant and thimerosal
  • Tris-HCl BSA
  • goat anti-pig IgA enzyme-labeled antibody was purchased from Bethyl company products, ( Item No.
  • mouse anti-porcine SC protein monoclonal antibody (patent number: ZL201510239712.3, anti-porcine SC protein monoclonal antibody secreting hybridoma cell line 4H11, preservation number: CCTCC NO: C201526) by Jiangsu Academy of Agricultural Sciences Provided; glass fiber membrane was purchased from Ahlstrom; nitrocellulose membrane was purchased from Sartorius; ultrasonic instrument was purchased from Bandelin; absorbent filter paper and bottom plate were purchased from Shanghai Jinbiao Biotechnology Co., Ltd.; protein G purification kit was purchased from Nanjing GenScript Biotechnology Co., Ltd. Company; quantum dot microspheres (product number FM610C, hydrated particle size is 120nm) were purchased from Beijing Nano Crystal Biotechnology Co., Ltd.
  • pH 7.0 MES buffer preparation method Dissolve 4.265g MES in 1L of double distilled water, and then adjust the pH to 7.0.
  • PBS buffer (concentration 0.01M, pH 7.4) preparation method dissolve 3g of Na 2 HPO 4 .12H 2 O, 0.2g of KH 2 PO 4 , 8g of NaCl and 0.2g of KCl in 1L of double distilled water, and then adjust the pH to 7.4.
  • Tris-HCl buffer solution (50 mM, pH 8.0) preparation method dissolve 7.88 g of Tris in 1 L of double distilled water, and then adjust the pH to 8.0 with hydrochloric acid.
  • the gene of the recombinant polypeptide tandem protein R10 (SEQ ID NO: 1) was designed, and the amino acid sequence of the recombinant polypeptide tandem protein R10 was designed. As shown in SEQ ID NO:2.
  • the gene of the recombinant polypeptide tandem protein R10 is compatible with Escherichia coli tropism.
  • the gene of the recombinant polypeptide tandem protein R10 was obtained by gene synthesis (by Nanjing GenScript Biotechnology Co., Ltd.), and the gene of the recombinant polypeptide tandem protein R10 was inserted into the polyclonal restriction sites EcoRI and EcoRI of the vector pET-28a(+). Between HindIII, the recombinant plasmid pET-28a-R10 was obtained.
  • the recombinant plasmid pET-28a-R10 was transformed into E. coliBL21(DE3) competent cells to obtain recombinant strain pET-28a-R10(BL21).
  • the codons were optimized for E. coli tropism, and the gene sequence of the recombinant protein P30V was obtained, such as SEQ ID NO. : 3, the corresponding amino acid sequence is shown in SEQ ID NO: 4.
  • the gene of recombinant protein P30V was obtained by gene synthesis (Nanjing GenScript Biotechnology Co., Ltd.), and the gene sequence was inserted between the polyclonal restriction sites NdeI and XholI of the vector pET-21a(+) to obtain a recombinant plasmid pET-21a-P30V.
  • the recombinant plasmid pET-21a-P30V was transformed into E. coliBL21 (DE3) competent cells to obtain recombinant strain pET-21a-P30V (BL21).
  • the recombinant strain pET-28a-R10(BL21) was cultured in LB liquid medium containing 50 ⁇ g/mL kanamycin, and pET-21a-P30V(BL21) was cultured in LB liquid medium containing 100 ⁇ g/mL ampicillin
  • the bacterial cells were washed with buffer solution, resuspended and then added with PMSF (phenylmethylsulfonyl fluoride) for ultrasonic lysis, and centrifuged at 10,000 r/min for 30 minutes at 4°C. The supernatant was taken, and each recombinant protein was purified according to the instructions of Ni-NTA affinity chromatography medium (product of GenScript Biotechnology Co., Ltd., Cat. No: L00250). It can be seen from lane 1 of Figure 1 that the purified recombinant polypeptide tandem protein R10 has a specific band around 19KDa, which is consistent with the expectation.
  • PMSF phenylmethylsulfonyl fluoride
  • the recombinant protein P30V has a specific band around 43KDa, which is consistent with the expectation. Therefore, the recombinant polypeptide tandem protein R10 and the recombinant protein P30V were successfully expressed and stored at -70°C for future use.
  • the purified recombinant polypeptide tandem protein R10 and recombinant protein P30V were transferred to NC membrane after SDS-PAGE electrophoresis, and detected by Western-blotting respectively. After blocking with 5% skim milk overnight, a mixture of African swine fever antibody positive reference mouth swab and 5% skim milk in a volume ratio of 1:1 was used as the primary antibody, and incubated at 2-8°C for 12-18 hours. Wash 5 times with TBST, 5 min/time. Goat anti-pig IgA enzyme-labeled antibody (purchased from Bethyl Company, product number A100-102p) diluted 1:20000 was used as the secondary antibody, and incubated at 37°C for 1.0 hours.
  • the sample diluent of the present invention contains African swine fever virus double protein-marker.
  • African swine fever virus double protein-label is a mixture of quantum dot microsphere-labeled recombinant protein P30V and quantum dot microsphere-labeled recombinant polypeptide tandem protein R10.
  • the preparation method of the sample diluent of the present invention comprises the following steps:
  • Activation of quantum dot microspheres 50 ⁇ L of quantum dot microsphere suspension with a concentration of 1.2 ⁇ g/ ⁇ L was added to 50 ⁇ L of MES buffer with a concentration of 0.02 M and pH 7.0, and EDC (1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mg/mL of NHS (N-hydroxysuccinimide), incubate at 37°C in a shaker for 20 minutes. Then centrifuge at 10000g for 20 minutes in a centrifuge, discard the supernatant, add 50 ⁇ L of MES buffer with a concentration of 0.02M and pH 7.0 to resuspend to obtain activated quantum dot microspheres.
  • Coupling of quantum dot microspheres Add 10 ⁇ g of recombinant protein P30V (prepared in Example 1) to the above activated quantum dot microspheres, incubate at a constant temperature of 37°C in a shaker for 2 hours, and combine recombinant protein P30V with quantum dot microspheres Conjugate to obtain the conjugate of recombinant protein P30V and quantum dot microspheres.
  • Blocking of quantum dot microspheres In the obtained conjugate of recombinant protein P30V and quantum dot microspheres, add 2 ⁇ L of 10% BSA (bovine serum albumin) aqueous solution by mass, and incubate at 37°C in a shaker at constant temperature 15 minutes, then centrifuge at 10000g for 10 minutes in a centrifuge, discard the supernatant, add 100 ⁇ L of MES buffer with a concentration of 0.02M, pH 7.0 to resuspend to obtain a recombinant protein P30V solution labeled with quantum dot microspheres, which is placed in a refrigerator at 4°C save in.
  • BSA bovine serum albumin
  • the quantum dot microspheres were coupled to the recombinant polypeptide tandem protein R10 prepared in Example 1, except that 10 ⁇ g of recombinant protein P30V was replaced with 10 ⁇ g of recombinant polypeptide tandem protein R10, A solution of recombinant polypeptide tandem protein R10 labeled with quantum dot microspheres was obtained.
  • a 0.01M, pH7.4 PBS buffer containing 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) Tween-20 was prepared.
  • the quantum dot microsphere immunochromatography test strip 1 for detecting African swine fever virus mucosal sIgA antibody includes a bottom plate 2, and a sample pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 7 are arranged on the bottom plate in sequence.
  • the nitrocellulose membrane 4 is provided with a detection line 5 and a quality control line 6.
  • the detection line 5 is set near the side of the sample pad, and the detection line is coated with mouse anti-pig SC protein monoclonal antibody; the quality control line 6 is close to the absorbent pad 7 Set on one side, the quality control line is coated with purified protein from African swine fever positive serum.
  • recombinant protein P30V and recombinant polypeptide tandem protein R10 as immunogens, adult New Zealand white rabbits (1-2Kg/rabbit) were immunized by subcutaneous injection at multiple points on the back for 3 times, with an interval of 2 weeks for each immunization.
  • 1.0 ml of a solution containing 1.5 mg of recombinant protein P30V and 1.5 mg of recombinant polypeptide tandem protein R10 was mixed with an equal volume of Freund's complete adjuvant for immunization; the following two immunogens were used for immunization: 1.0 mg of A solution of recombinant protein P30V and 1.0 mg of recombinant polypeptide tandem protein 1.0 ml was mixed with an equal volume of incomplete Freund's adjuvant to obtain immunogens for the second and third immunity.
  • the dose of each immunization was 2.0ml, blood was collected 7 days after the third immunization, centrifuged to obtain African swine fever positive serum, and the antibody titer was detected by indirect ELISA.
  • the antibody titer of African swine fever positive serum against recombinant polypeptide tandem protein R10 was detected by indirect ELISA.
  • the specific method is as follows: use carbonate buffer (concentration 0.01M, pH 9.6) to prepare recombinant polypeptide tandem protein R10 containing 2 ⁇ g/mL The solution was coated with 100 ⁇ L per well of the ELISA plate, and coated overnight at 4°C; the liquid in the well was discarded, and each well was washed three times with 250 ⁇ L of PBST for 5 min each time, and patted dry.
  • HRP-goat anti-rabbit IgG enzyme-labeled antibody (brand name is BOSTER, product number is BA1054) was diluted with PBS buffer (concentration 0.01M, pH 7.4) at a dilution of 1:5000, and filtered with a 0.22 ⁇ m filter. Obtain the diluted HRP-goat anti-rabbit IgG enzyme-labeled antibody. Add 100 ⁇ L of diluted HRP-goat anti-rabbit IgG enzyme-labeled antibody to each well, and react at 37°C for 0.5 h.
  • the antibody titer of African swine fever-positive serum against recombinant protein P30V was detected by the same method as above, except that a solution containing 2.5 ⁇ g/mL recombinant protein P30V was prepared with carbonate buffer (concentration 0.01M, pH 9.6). , coat the microtiter plate with 100 ⁇ L per well. Calculated according to the conventional method, the result: the serum antibody titer against recombinant protein P30V is 1:4 ⁇ 10 5 .
  • the serum antibody titers of the African swine fever positive serum against the recombinant protein P30V and the recombinant polypeptide tandem protein R10 should be greater than or equal to 1:10 5 .
  • Wash Buffer aqueous solution containing 20 mM Na 2 HPO 4 and 0.15 M NaCl, pH 7.0
  • African swine fever positive serum were mixed at a volume ratio of 1:1 to obtain a diluent of African swine fever positive serum.
  • GenScript protein G purification kit (Item No. L00209) to isolate protein from African swine fever-positive serum, the specific method is as follows:
  • 1 Chromatography column packing Take 0.5mL of protein G purification medium in the protein G purification kit, add 0.5mL of Wash Buffer (same as above), shake well, and then add it to a chromatography column pre-filled with 1mL of Wash Buffer, let The packing settles naturally to the bottom of the column. Add 5mL of Wash Buffer to the column, let the Buffer slowly flow out, and the flow rate is about 1mL/min.
  • 2Purification through the column slowly add the sample (African swine fever positive serum dilution) to the upper end of the above column, and the flow rate of the sample is 0.5 mL/min. Collect the effluent. Use about 30 mL of Wash Buffer to wash impurities, and the flow rate of Wash Buffer is 2 mL/min, or wash with Wash Buffer until the absorbance (A280 value) of the effluent at 280 nm of the UV detector is stable. After washing the impurities, add 15 mL of Elution Buffer (aqueous solution of glycine with a pH of 2.5 and a concentration of 0.1 M) for elution.
  • Elution Buffer aqueous solution of glycine with a pH of 2.5 and a concentration of 0.1 M
  • Elution Buffer The flow rate of Elution Buffer is 1 mL/min, collect the eluate, and add 1/10 of the eluate volume.
  • Neutralization Buffer pH 8.5, 1 M Tris-HCl buffer
  • the pH of the eluate is 7.4 to obtain the purified protein of African swine fever-positive serum.
  • the identification results of the purified protein from African swine fever positive serum are shown in Figure 6. It can be seen that the purified protein has a band at 55kDa (heavy chain) and 25kDa (light chain) respectively, and the purity reaches 98%.
  • the glass fiber membrane was soaked for 20 min with 50 mM Tris-HCl buffer at pH 8.0 containing 3% (mass percent concentration) BSA (bovine serum albumin) and 1% (volume percent concentration) Tween-20, It is then dried to obtain a sample pad.
  • BSA bovine serum albumin
  • hybridoma cell line 4H11 (deposit number: CCTCC NO: C201526) that secretes anti-pig SC protein monoclonal antibody in Chinese patent ZL201510239712.3 to prepare anti-pig SC protein monoclonal antibody (ZL201510239712.3 Example 2 title 9), that is, mouse anti-pig SC protein monoclonal antibody.
  • the mouse anti-pig SC protein monoclonal antibody was diluted to 1.0 mg/mL with 0.01 M PBS buffer containing 3% (mass percent concentration) sucrose, pH 7.4, to obtain a detection line solution.
  • the PBS buffer containing 3% (mass percent concentration) sucrose is obtained by dissolving sucrose in the PBS buffer with a concentration of 0.01 M and pH 7.4.
  • Spray volume refers to the amount of protein sprayed per centimeter of T-line or C-line.
  • the widths of the T line and the C line are both 1 mm.
  • test strip in Figure 3-5 use the following method to assemble the test strip: on a clean operating table under normal humidity and temperature, put the treated sample pad 3, the nitric acid sprayed with the detection line and the quality control line.
  • the cellulose film 4 and the water-absorbing pad 7 are pasted on the bottom plate 2 with a 2-4mm overlap in sequence, and then sent to the slitter to obtain a test strip with a width of 4 ⁇ 0.5mm.
  • Pick the intact and tidy test strips put them into the card case, and seal them in an aluminum foil bag together with 1 desiccant after the lid is finished.
  • the bottom plate 2 is a PVC board.
  • test strip prepared in this example is denoted as the test strip of the present invention.
  • Embodiment 4 Test strip qualitative detection method and result judgment
  • the method for detecting African swine fever virus mucosal sIgA antibody using test strips of the present invention and sample diluent of the present invention comprises the following steps:
  • test sample Draw the test sample with a 1 mL Pasteur pipette, mix it with the sample diluent of the present invention at a volume ratio of 1:1, and incubate at room temperature (20° C.) for 10 minutes.
  • the reacted test strip is irradiated with ultraviolet light of a 365nm ultraviolet light emitter. If the C line of the test strip is colored and the T line is also colored, the result is judged to be positive, and the darker the color of the T line, the The higher the ASFV mucosa sIgA antibody titer in the sample. When the C line of the test strip is colored and the T line is not colored, the result is judged as negative, and there is no African swine fever virus mucosal sIgA antibody in the sample. If the C line of the test strip is not colored, the test result of the strip is invalid.
  • Embodiment 5 Characteristics of test strip detection
  • the sensitivity of detecting African swine fever virus mucosal sIgA antibody by using the test strip of the present invention and the sample diluent of the present invention was investigated.
  • the African swine fever antibody-positive reference oral swab (collected from the oral fluid of African swine fever seropositive pigs) was diluted with PBS (concentration 0.01M, pH 7.4) at 1:2, 1:4, 1:8 , 1:16, 1:32, 1:64, 1:128, 1:256 for dilution.
  • PBS concentration 0.01M, pH 7.4
  • the sample diluent of the present invention and the test strip of the present invention respectively, the diluent of different dilutions of the African swine fever antibody positive reference oral swab and the African swine fever antibody negative reference oral swab (collected from the oral fluid of healthy pigs) test.
  • the sample diluent of the present invention is combined with the test strip of the present invention, and the result of the dilution of the diluent of 1:128 for the African swine fever antibody positive reference mouth swab is positive, and the dilution of the African swine fever antibody positive reference mouth swab is positive.
  • the dilution of 1:256 and the negative reference mouth swab test for African swine fever antibody were negative, indicating that the sensitivity of the test strip for the detection of African swine fever virus mucosal sIgA antibody can reach 1:128 (dilution) .
  • 0.01M, 0.01M, 0.01M, 0.01M, 0.01M, 0.01M, 0.01M, 0.01M, 0.01M, 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) Tween-20 used in the preparation of the sample diluent of the present invention were prepared.
  • PBS buffer at pH 7.4 was replaced with PBS buffer (concentration 0.01M, pH 7.4) and PBST buffer, respectively, to obtain control sample dilution 1 and control sample dilution 2, respectively.
  • the PBST buffer is a 0.01M, pH7.4 PBS buffer containing 0.2% (volume percent concentration) Tween-20.
  • Example 4 Using the sample diluent of the present invention, the control sample diluent 1 or the control sample diluent 2, and the test strips of the present invention, the method in Example 4 was used to detect the positive reference mouth swab for African swine fever antibody in the title 1 of this example. Diluent for each dilution.
  • step (3) contains 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) 0.01M, pH7.4 PBS buffer of Tween-20 and quantum dot microsphere-labeled recombinant protein P30V solution were mixed at a volume ratio of 30:1 to obtain control sample diluent 3;
  • step (3) 0.01M, pH7.4 PBS buffer containing 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) Tween-20 was combined with the recombinant polypeptide tandem protein labeled with quantum dot microspheres.
  • the R10 solution was mixed at a volume ratio of 30:1 to obtain a control sample dilution 4.
  • the sample diluent of the present invention, the control sample diluent 3 and the control sample diluent 4 are respectively matched with the test strips of the present invention, and the positive reference mouth swab for the African swine fever antibody in the title 1 of this example is carried out according to the method in Example 4.
  • the dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and the African swine fever antibody negative reference mouth swab were tested , to conduct a sensitivity comparison test.
  • the specific results are shown in Table 1.
  • test strip of the present invention when used for detection, when the sample diluent of the present invention is used, the test result of the diluent with a dilution of 1:128 for the African swine fever antibody positive reference mouth swab is still positive;
  • control sample dilution 3 or 4 when the control sample dilution 3 or 4 was used, the dilution of the 1:32 dilution of the African swine fever antibody-positive reference mouth swab was positive, and the dilution of the African swine fever antibody-positive reference mouth swab was The 1:64 dilution tested negative.
  • the sample diluent of the present invention is combined with the test strip of the present invention to detect the dilution of each dilution of the African swine fever antibody positive reference oral swab in the title 1 of this example and the African swine fever antibody negative reference oral swab.
  • the sample diluent of the present invention is combined with the test strip of the present invention, and the method in Example 4 is used to detect the positive sample of porcine circovirus mucosal antibody, the positive sample of porcine reproductive respiratory syndrome virus mucosal antibody, and the positive mucosal antibody of Actinobacillus pleuropneumoniae.
  • test strip of the present invention has no cross-reaction with other swine viruses and has good specificity.
  • test strip of the present invention is sealed and stored in an aluminum foil bag containing 1 desiccant, it is stored at 20° C. and 37° C. for 7 days, 14 days, 21 days and 28 days, respectively, according to the title of this example. Sensitivity test was carried out by the method in 1, and specificity test was carried out according to title 5 of this example.
  • test strips stored at room temperature (20°C) and 37°C for 7 days, 14 days, 21 days and 28 days still have the same results for the dilution of 1:128 dilution of the African swine fever antibody positive reference oral swab.
  • Positive, porcine circovirus mucosal antibody positive samples, porcine reproductive respiratory syndrome virus mucosal antibody positive samples, Actinobacillus pleuropneumoniae mucosal antibody positive samples, swine influenza virus mucosal antibody positive samples and porcine epidemic diarrhea virus mucosal antibody positive samples The detection results of the samples are all negative, indicating that the test strip of the present invention has good stability.
  • Embodiment 6 Sensitivity comparison of test paper strip of the present invention and colloidal gold detection test paper
  • Preparation of colloidal gold particles reduce chloroauric acid with trisodium citrate reducing agent to make 20-40 nm colloidal gold particles.
  • the specific method is as follows: take 800 mL of chloroauric acid aqueous solution with a mass percentage concentration of 1% and use a constant temperature electromagnetic The stirrer was heated to boiling, and under the condition of continuous stirring, 1 mL of trisodium citrate aqueous solution with a concentration of 16% by mass was added, and the stirring and heating were continued for 5-10 minutes, and the solution was bright red. Cool at room temperature, make up the volume to 800 mL with deionized water to obtain 20-40 nm colloidal gold particles, and store at 4°C.
  • colloidal gold-labeled recombinant polypeptide tandem protein R10 The same method was used to prepare colloidal gold-labeled recombinant polypeptide tandem protein R10, except that the recombinant protein P30V was replaced with recombinant polypeptide tandem protein R10.
  • the colloidal gold-labeled recombinant protein P30V and the colloidal gold-labeled recombinant polypeptide tandem protein R10 were mixed in a volume ratio of 1:1 to obtain an African swine fever virus double protein-colloidal gold label.
  • Test strip assembly assemble the colloidal gold test strip according to the assembling method of the test strip of the present invention, the difference is that an above-mentioned colloidal gold pad (in the title 3 of this example) is set between the sample pad and the nitrocellulose membrane. preparation) to obtain colloidal gold test strips.
  • Detection method of colloidal gold test strip add 3 drops of the sample (about 30 microliters per drop) on the sample pad, observe directly with the naked eye, when the T line and C line are both colored, it is positive; when the C line develops color , the T line does not develop color is negative; when the C line does not develop color, the result is invalid.
  • the dilution of the reference mouth swab for the African swine fever antibody positive in Example 5 is 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256
  • Diluent and African swine fever antibody negative reference mouth swab were respectively detected with the test strip of the present invention (with the sample diluent of the present invention, the detection method was the same as that in Example 4) and the colloidal gold test strip, and the test results were shown in Table 4.
  • test strip of the present invention and the colloidal gold test paper respectively detect the dilutions of different dilutions of the African swine fever antibody positive reference mouth swab
  • test strip of the present invention detects the African swine fever antibody positive reference mouth.
  • the swab is positive at a 1:128 dilution.
  • Colloidal gold test strips are positive when the dilution of the reference mouth swab for detection of African swine fever antibody is 1:4 dilution, and the dilution of the reference mouth swab for detection of African swine fever antibody positive is 1:8 dilution Negative, the sensitivity is significantly lower than the test strip of the present invention.

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Abstract

一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条及其应用，属于兽医生物诊断制品领域。试纸条(1)包括底板(2)，底板(2)上依次设有样品垫(3)、硝酸纤维素膜(4)和吸水垫(7)；硝酸纤维素膜(4)上设有检测线(5)和质控线(6)，检测线(5)处包被有鼠抗猪SC蛋白单克隆抗体，检测线(5)靠近样品垫(3)一侧设置；质控线(6)处包被有非洲猪瘟阳性血清的纯化蛋白，质控线(6)靠近吸水垫(7)一侧设置。样本稀释液含有量子点微球标记的重组多肽串联蛋白R10和重组蛋白P30V；R10和P30V的序列分别如SEQ ID NO:2和4所示。将试纸条(1)配合样本稀释液，可用于非洲猪瘟病毒黏膜sIgA抗体的快速、高特异性、高灵敏度的定性检测。

Description

一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条及其应用 技术领域
本发明属于兽医生物诊断制品技术领域，具体涉及一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条及其应用。
背景技术
非洲猪瘟(African swine fever，ASF)是由非洲猪瘟病毒(African swine fever virus，ASFV)感染引起的家猪和野猪的一种急性、热性、高度接触性传染病，是我国一类动物疫病，也是世界动物卫生组织(OIE)法定报告动物疫病。
ASFV是一种复杂的、有囊膜的、双股DNA病毒，直径170～190kb，具有末端交联和反转重复区，其编码151-167种蛋白，成熟的病毒粒子包含有约50多种结构蛋白。
ASFV主要经呼吸道和消化道途径侵入猪体，猪体应答感染，除了血清中产生特异性的IgG抗体外，在呼吸道与消化道黏膜还可产生特异性的sIgA抗体。sIgA抗体主要存在于呼吸道分泌液、唾液、泪液、乳汁、胃肠分泌液和泌尿生殖道分泌液中，是机体黏膜免疫的主要抗体，也是病原体侵入后最先产生的免疫反应，常用作疾病感染早期诊断的检测指标。
目前针对ASFV防控尚无有效的商品化疫苗，因此疫情防控的监测与诊断变得至关重要。传统的诊断检测非洲猪瘟病毒抗体的方法有间接免疫荧光(IFA)、和酶联免疫法(ELISA)。其中IFA的敏感性偏低，需要荧光操作人员和荧光显微镜，成本高昂，限制了在现场快速检测中的应用。ELISA以抗原抗体特异性反应为原理检测动物具有非洲猪瘟病毒抗体，具有灵敏度高，特异性强等优点，但操作较为繁琐，耗时较长，加之需要酶标仪、恒温箱等设备，在基层养殖场难以展开实时检测。
发明内容
针对现有问题的不足，本发明的第一个目的是提供一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条，可用于非洲猪瘟病毒黏膜sIgA抗体的快速、高特异性、高灵敏度的定性检测。
本发明的第二个目的是提供配合上述检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条的样本稀释液。
本发明的第三个目的是提供一种以非诊断为目的的，采用所述量子点微球免疫层析试纸条和所述样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体的方法，该方法操作简单，无需经过专业的培训，可以很好地胜任各种现场、基层实验室等环境下的检测；结合较荧光显微镜和酶标仪价格更为低廉的荧光检测仪，可以及时的，方便的，在现场得到结果。
本发明解决其技术问题采用的技术方案是：
一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条，包括底板，所述底板上依次设有样品垫、硝酸纤维素膜和吸水垫；所述硝酸纤维素膜上设有检测线和 质控线，所述检测线处包被有鼠抗猪SC蛋白单克隆抗体，所述检测线靠近所述样品垫一侧设置；所述质控线处包被有非洲猪瘟阳性血清的纯化蛋白，所述质控线靠近所述吸水垫一侧设置。
在本发明中，所述非洲猪瘟阳性血清的纯化蛋白是将兔抗非洲猪瘟重组蛋白P30V和重组多肽串联蛋白R10高免血清采用protein G纯化试剂盒纯化后所得蛋白；所述鼠抗猪SC蛋白单克隆抗体是由分泌抗猪SC蛋白单克隆抗体的杂交瘤细胞株4H11所制备，所述杂交瘤细胞株4H11的保藏号为：CCTCC NO：C201526。
在本发明中，所述样品垫是将玻璃纤维膜采用含有牛血清白蛋白和Tween-20的Tris-HCl缓冲液浸泡处理后所得。
本发明还提供配合所述试纸条使用的样本稀释液，含有量子点微球标记的重组多肽串联蛋白R10和量子点微球标记的重组蛋白P30V；所述重组多肽串联蛋白R10的序列如SEQ ID NO:2；所述重组蛋白P30V的序列如SEQ ID NO:4所示。
在本发明中，将量子点微球分别与重组多肽串联蛋白R10和重组蛋白P30V偶联，分别得到量子点微球标记的重组多肽串联蛋白R10和量子点微球标记的重组蛋白P30V；然后将量子点微球标记的重组多肽串联蛋白R10和重组蛋白P30V按照体积比为1：0.5-1.5混合，得到非洲猪瘟病毒双蛋白-标记物；将所述非洲猪瘟病毒双蛋白-标记物与含有BSA和的Tween-20的PBS缓冲液按照体积比1:28-32混合，得到样本稀释液。
在本发明中，所述量子点微球与重组多肽串联蛋白R10的质量比为5-7:1，所述量子点微球与重组蛋白P30V的质量比为5-7:1；所述PBS缓冲液含有0.05-0.15％的BSA和0.2％的Tween-20的PBS缓冲液。
本发明还提供一种以非诊断为目的的采用所述量子点微球免疫层析试纸条和所述样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体的方法，将拭子样品加入样本稀释液中孵育后，滴加在所述试纸条的样品垫上，反应后，采用紫外光发射器检测C线和T线。
在本发明中，检测样品为拭子样品或黏膜样品。
在本发明中，所述拭子样品包括口拭子、鼻拭子或肛拭子，所述黏膜样品为肺泡灌洗液。
采用非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条配合样本稀释液，检测非洲猪瘟病毒黏膜sIgA抗体时，样品中的黏膜sIgA抗体首先与样本稀释液中的量子点微球标记的重组蛋白P30V、量子点微球标记的重组多肽串联蛋白R10结合得到黏膜sIgA抗体-量子点复合物，滴加至样品垫后，层析至硝酸纤维素膜时，黏膜sIgA抗体-量子点复合物与检测线上的鼠抗猪SC蛋白单克隆抗体结合从而被固定在检测线处，多余的量子点微球标记的重组蛋白P30V、量子点微球标记的重组多肽串联蛋白R10层析至质控线处与非洲猪瘟阳性血清的纯化蛋白反应，并被固定在了质控线上。这样，可以通过365nm的紫外光发射器可以检测到固定在检测线处的非洲猪瘟病毒黏膜sIgA抗体。
本发明与现有技术相比，具有以下有益效果：
1.本发明提供的检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条，量子点微球具有均一度高，单分散性好，稳定性强等优点，使用量子点微球作为标记物制作的试纸条批次间差异较小；
2.本发明提供的非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条的制 备方法，操作易上手，所需原料简单，可在较短时间内掌握，有广阔的市场前景和较大的经济收益。
3.由于黏膜抗体的产生早于血清抗体，因此采用本发明量子点微球免疫层析试纸条和样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体，可用于非洲猪瘟感染的早期检测，且采用本发明试纸条和样本稀释液检测，快速，特异性强，灵敏度高，稳定性好。
4.本发明的检测方法操作简单，无需经过专业的培训，可以很好地胜任各种现场、基层实验室等环境下的检测；结合较荧光显微镜和酶标仪价格更为低廉的紫外光发射器，可以及时的，方便的，在现场得到结果。
5.检测拭子样品和黏膜样品，较血清样品，采样方便，减少了采样时对猪的应激反应，更加有力于动物福利和生物安全。
附图说明
图1是本发明实施例1重组多肽串联蛋白R10的表达鉴定图，其中泳道1是纯化后的重组多肽串联蛋白R10，泳道2是纯化后的重组多肽串联蛋白R10和非洲猪瘟抗体阳性参考口拭子的Western-bloting鉴定，M为蛋白maker。
图2是本发明实施例1重组蛋白P30V的表达鉴定图，其中泳道1是纯化后的重组蛋白P30V，泳道2是纯化后的重组蛋白P30V和非洲猪瘟抗体阳性参考口拭子的Western-bloting鉴定，M为蛋白maker。
图3是本发明实施例所述的早期检测非洲猪瘟抗体的量子点微球免疫层析试纸条的结构示意图。其中，1-检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条，2-底板，3-样品垫、4-硝酸纤维素膜，7-吸水垫，5-检测线，6-质控线。
图4是本发明实施例所述的早期检测非洲猪瘟抗体的量子点微球免疫层析试纸条的剖面图。
图5是本发明实施例所述的早期检测非洲猪瘟抗体的量子点微球免疫层析试纸条的俯视图。
图6是非洲猪瘟阳性血清纯化蛋白的SDS-PAGE鉴定图，其中泳道1是非洲猪瘟阳性血清纯化蛋白，M为蛋白maker。
图7敏感性图。
图8特异性图，其中ASFV是非洲猪瘟抗体阳性参考口拭子、PRRS是猪繁殖呼吸综合症病毒黏膜抗体阳性样品，PEDV是猪流行性腹泻病毒黏膜抗体阳性样品，APP是猪胸膜肺炎放线杆菌黏膜抗体阳性样品，PCV是猪圆环病毒黏膜抗体阳性样品，SIV是猪流感病毒黏膜抗体阳性样品。
具体实施方式
下面将结合本发明的实施例，对本发明的优选实施方式进行详细说明。此外需要理解的是，以下的实施例仅是为了对本发明作进一步说明，帮助理解。而不是用于对本发明的范围进行限制，本发明的保护范围不受任何实施例形式上的限制。下述实施例中所使用的的实验方法如无特殊说明，皆为常规方法。
本发明中：MES、EDC、NHS、Tween-20、蔗糖、弗氏完全佐剂、硫柳汞购自Sigma； Tris-HCl、BSA购自ThermoFisher；羊抗猪IgA酶标抗体购自Bethyl公司产品，(货号A100-102p)；鼠抗猪SC蛋白单克隆抗体(专利号：ZL201510239712.3，抗猪SC蛋白单克隆抗体分泌杂交瘤细胞株4H11，保藏号为：CCTCC NO：C201526)由江苏省农业科学院提供；玻璃纤维膜购Ahlstrom；硝酸纤维素膜购自Sartorius；超声波仪购自Bandelin；吸水滤纸和底板购自上海金标生物科技有限公司；protein G纯化试剂盒购自南京金斯瑞生物科技有限公司；量子点微球(产品编号为FM610C，水合粒径是120nm)购自北京纳晶生物科技有限公司。
0.02M、pH 7.0的MES缓冲液配制方法：将4.265gMES溶于1L的双蒸水中，再调节pH至7.0。
PBS缓冲液(浓度为0.01M、pH7.4)配制方法：将3g的Na 2HPO 4.12H 2O、0.2g的KH 2PO 4、8g的NaCl和0.2g的KCl溶于1L的双蒸水中，再调节pH至7.4。
Tris-HCl缓冲液(浓度为50mM、pH8.0)配制方法：将7.88gTris溶于1L的双蒸水中，再采用盐酸调节pH至8.0。
实施例1制备重组多肽串联蛋白R10和重组蛋白P30V
1.P54E基因重组载体的构建
参考NCBI数据库公布的我国首例非洲猪瘟P54全基因序列(MH766894，E183L，162222-162776)，设计了重组多肽串联蛋白R10的基因(SEQ ID NO:1)，重组多肽串联蛋白R10的氨基酸序列如SEQ ID NO:2所示。重组多肽串联蛋白R10的基因是符合大肠杆菌嗜性的。采用基因合成的方式(由南京金斯瑞生物科技有限公司)获得重组多肽串联蛋白R10的基因，将重组多肽串联蛋白R10的基因***载体pET-28a(+)的多克隆酶切位点EcoRⅠ和HindⅢ之间，得到重组质粒pET-28a-R10。
通过“热激”法，将重组质粒pET-28a-R10转化E.coliBL21(DE3)感受态细胞，获得重组菌pET-28a-R10(BL21)。
2.P30V基因重组载体的构建
参考NCBI数据库公布的我国首例非洲猪瘟CP204L基因序列(MH766894，CP204L，124770～125375)，对密码子进行了针对大肠杆菌的嗜性优化，得到了重组蛋白P30V的基因序列，如SEQ ID NO：3所示，对应的氨基酸序列如SEQ ID NO：4所示。采用基因合成的方式(南京金斯瑞生物科技有限公司)获得重组蛋白P30V的基因，将该基因序列***载体pET-21a(+)的多克隆酶切位点NdeⅠ和XholⅠ之间，得到重组质粒pET-21a-P30V。
通过“热激”法，将重组质粒pET-21a-P30V转化E.coliBL21(DE3)感受态细胞，获得重组菌pET-21a-P30V(BL21)。
3.重组蛋白的表达及纯化
将重组菌pET-28a-R10(BL21)在含有50μg/mL卡那霉素的LB液体培养基中培养，将pET-21a-P30V(BL21)在含有100μg/mL氨苄青霉素的LB液体培养基中培养，培养条件均如下：培养温度为37℃、摇床转速为180r/min，待OD 600＝0.6～0.8时降温至20℃，30min后加入终浓度为1.0mmol/L的IPTG诱导表达，然后在20℃培养17小时，收集菌体。用缓冲液洗涤菌体，菌体重悬后加入PMSF(苯甲基磺酰氟)进行超声波裂解，4℃下10000r/min离心30分钟。取上清，按照Ni-NTA亲合层析介质(金斯瑞生物科技有限公司产品，Cat.No：L00250)的说明书纯化各重组蛋白。从图1的泳道1可见，纯化后的重组多肽串联蛋白R10在 19KDa左右出现特异性的条带，与预期一致。从图2的泳道1可见，重组蛋白P30V在43KDa左右出现特异性的条带，与预期一致。因此重组多肽串联蛋白R10和重组蛋白P30V成功表达，置于-70℃以下保存备用。
4.重组蛋白的抗原性检测:
将纯化的重组多肽串联蛋白R10和重组蛋白P30V经SDS-PAGE电泳后转印至NC膜，分别进行Western-bloting检测。用5％脱脂乳封闭过夜后，以体积比为1:1的非洲猪瘟抗体阳性参考口拭子和5％脱脂乳混合物作为一抗，2～8℃孵育12～18小时。TBST洗涤5次，5分钟/次。用1:20000稀释的羊抗猪IgA酶标抗体(购自Bethyl公司产品，货号A100-102p)作为二抗，37℃孵育1.0小时。TBST洗涤5次，5分钟/次。ECL避光显色5min。结果：重组多肽串联蛋白R10在19KDa左右出现了特异性反应条带(如图1泳道2)；重组蛋白P30V在43KDa左右出现了特异性反应条带(如图2泳道2)。上述结果说明，重组多肽串联蛋白R10和重组蛋白P30V均可与非洲猪瘟抗体阳性参考口拭子发生特异性反应，具有良好的抗原性。
实施例2本发明样本稀释液的制备
本发明样本稀释液：含有非洲猪瘟病毒双蛋白-标记物。非洲猪瘟病毒双蛋白-标记物是量子点微球标记的重组蛋白P30V和量子点微球标记的重组多肽串联蛋白R10的混合物。
本发明样本稀释液的制备方法包括如下步骤：
(1)制备量子点微球标记的重组蛋白P30V
量子点微球的活化：取50μL浓度为1.2μg/μL的量子点微球悬浮液加入至50μL浓度为0.02M、pH 7.0的MES缓冲液中，加入终浓度为10mg/mL的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和10mg/mL的NHS(N-羟基丁二酰亚胺)，在37℃摇床中恒温孵育20分钟。再至离心机中10000g离心20分钟，弃上清，加入50μL浓度为0.02M、pH 7.0的MES缓冲液重悬，得到活化的量子点微球。
量子点微球的偶联：在上述活化的量子点微球中，加入10μg重组蛋白P30V(实施例1制备)，于37℃摇床中恒温孵育2小时，将重组蛋白P30V与量子点微球进行偶联，得到重组蛋白P30V和量子点微球的偶联物。
量子点微球的封闭：在所得重组蛋白P30V和量子点微球的偶联物中，加入2μL质量百分浓度为10％的BSA(牛血清白蛋白)水溶液，在37℃摇床中恒温孵育15分钟，然后在离心机中10000g离心10分钟，弃上清，加入100μL浓度为0.02M、pH 7.0的MES缓冲液重悬，得到量子点微球标记的重组蛋白P30V溶液，置于4℃冰箱中保存。
(2)制备量子点微球标记的重组多肽串联蛋白R10
按照本实施例标题(1)中相同方法，将量子点微球与实施例1制备的重组多肽串联蛋白R10偶联，不同之处在于，将10μg重组蛋白P30V替换为10μg重组多肽串联蛋白R10，得到量子点微球标记的重组多肽串联蛋白R10溶液。
(3)混合
配置含有0.1％(质量百分浓度)BSA和0.2％(体积百分浓度)Tween-20的0.01M、pH7.4的PBS缓冲液。将本实施例标题(1)制备的量子点微球标记的重组蛋白P30V溶液和本实施例标题(2)制备的量子点微球标记的重组多肽串联蛋白R10溶液以体积比为1:1混匀，得到非洲猪瘟病毒双蛋白-标记物。将含有0.1％(质量百分浓度)BSA和0.2％(体积百分浓 度)Tween-20的0.01M、pH7.4的PBS缓冲液和非洲猪瘟病毒双蛋白-标记物以体积比为30:1混匀，得到样本稀释液，于4℃避光保存。
实施例3本发明试纸条的制备
如图3-5，检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条1，包括底板2，在底板上依次设置样品垫3、硝酸纤维素膜4和吸水垫7。硝酸纤维素膜4上设有检测线5和质控线6，检测线5靠近样品垫一侧设置，检测线处包被有鼠抗猪SC蛋白单克隆抗体；质控线6靠近吸水垫7一侧设置，质控线处包被有非洲猪瘟阳性血清的纯化蛋白。
1.制备非洲猪瘟阳性血清的纯化蛋白
(1)样品准备
将重组蛋白P30V和重组多肽串联蛋白R10作为免疫原，背部皮下多点注射免疫成年新西兰大白兔(1-2Kg/只)，共免疫3次，每次免疫间隔2周。首次免疫，将含有1.5mg重组蛋白P30V和1.5mg重组多肽串联蛋白R10的溶液1.0ml与等体积弗氏完全佐剂混合后，进行免疫；后两次使用如下免疫原进行免疫：将含有1.0mg重组蛋白P30V和1.0mg重组多肽串联蛋白的溶液1.0ml与等体积弗氏不完全佐剂混合，得到二免和三免所用免疫原。每次免疫剂量为2.0ml，第三次免疫后7d采血，离心分离，得到非洲猪瘟阳性血清，利用间接ELISA的方法检测抗体效价。
采用间接ELISA方法检测非洲猪瘟阳性血清针对重组多肽串联蛋白R10的抗体效价，具体方法如下：用碳酸盐缓冲液(浓度0.01M、pH9.6)配置含有2μg/mL重组多肽串联蛋白R10溶液，以每孔100μL包被酶标板，4℃包被过夜；弃去孔内液体，每孔再用250μL的PBST洗涤3次，每次5min，拍干。每孔加入200μL含5％脱脂乳的0.01M、pH为7.4的PBS缓冲液，于37℃封闭2h，弃去孔内液体，每孔用250μLPBST洗涤3次，每次5min，拍干。加入10倍梯度稀释的待检血清，每孔100μL，于37℃孵育1h。弃去孔中液体，每孔用250μLPBST洗涤3次，每次5min，拍干。用PBS缓冲液(浓度0.01M、pH为7.4)对HRP-羊抗兔IgG酶标抗体(品牌是BOSTER，货号是BA1054)以稀释度为1:5000进行稀释，用0.22μm的滤膜过滤，得到稀释后的HRP-羊抗兔IgG酶标抗体。每孔加入100μL稀释好的HRP-羊抗兔IgG酶标抗体，37℃反应0.5h。每孔加入100μL底物显色液TMB，37℃避光作用15min；再加入50μL浓度为2mol/L的硫酸水溶液终止反应。在酶标仪上测定OD 450读值，按照常规方法计算效价，结果：非洲猪瘟阳性血清针对重组多肽串联蛋白R10的抗体效价为1：10 5。采用上述相同方法检测非洲猪瘟阳性血清针对重组蛋白P30V的抗体效价，不同之处在于，用碳酸盐缓冲液(浓度0.01M、pH9.6)配置含有2.5μg/mL重组蛋白P30V的溶液，以每孔100μL包被酶标板。按照常规方法计算，结果：针对重组蛋白P30V的血清抗体效价为1：4×10 5。
在生产过程中，非洲猪瘟阳性血清针对重组蛋白P30V和重组多肽串联蛋白R10的血清抗体效价均大于等于1：10 5即可。
将Wash Buffer(含有20mMNa 2HPO 4和0.15MNaCl的水溶液，pH7.0)与非洲猪瘟阳性血清以体积比为1:1混合，得到非洲猪瘟阳性血清稀释液。
(2)采用金斯瑞protein G纯化试剂盒(货号为L00209)从非洲猪瘟阳性血清中分离蛋白，具体方法如下：
①层析柱填充：在protein G纯化试剂盒中取0.5mL的protein G纯化介质，加入0.5mLWash Buffer(同上)，充分摇匀，然后加入到一个预先装有1mLWash Buffer的层析 柱中，让填料自然沉降到柱子底部。加入5mLWash Buffer到柱中，让Buffer缓慢的流出，流速大约为1mL/min。
②过柱纯化：在上述层析柱上端缓慢地加入样品(非洲猪瘟阳性血清稀释液)，样品的流速为0.5mL/min。收集流出液。使用大约30mL的Wash Buffer洗涤杂质，Wash Buffer流速为2mL/min，或者采用WashBuffer洗涤到流出液在紫外检测仪280nm下的吸光值(A280值)稳定为止。洗杂完毕后，加入15mLElution Buffer(pH为2.5、浓度为0.1M的甘氨酸水溶液)进行洗脱，Elution Buffer流速为1ml/min，收集洗脱液，加入洗脱液体积1/10的中和Buffer(pH8.5、浓度为1M的Tris-HCl缓冲液)调节洗脱液的pH到7.4，得到非洲猪瘟阳性血清的纯化蛋白。
非洲猪瘟阳性血清的纯化蛋白的鉴定结果如图6所示，可以看到纯化蛋白在55kDa(重链)和25kDa(轻链)处分别有一个条带，纯度达到98％。
2.样品垫的制备
用含有3％(质量百分浓度)BSA(牛血清白蛋白)、1％(体积百分浓度)Tween-20的50mM、pH8.0的Tris-HCl缓冲液，将玻璃纤维膜浸泡处理20min，然后干燥，得到样品垫。
3.硝酸纤维素膜的制备
(1)采用中国专利ZL201510239712.3中分泌抗猪SC蛋白单克隆抗体的杂交瘤细胞株4H11(保藏号为：CCTCC NO：C201526)制备抗猪SC蛋白单克隆抗体(ZL201510239712.3实施例2标题9)，也就是鼠抗猪SC蛋白单克隆抗体。将鼠抗猪SC蛋白单克隆抗体用含有3％(质量百分浓度)蔗糖的0.01M、pH7.4的PBS缓冲液稀释至1.0mg/mL，得到检测线溶液。含有3％(质量百分浓度)蔗糖的PBS缓冲液，是将蔗糖溶解在浓度为0.01M、pH7.4的PBS缓冲液中所得。
(2)将非洲猪瘟阳性血清纯化蛋白用含有3％(质量百分浓度)蔗糖的0.01M、pH7.4的PBS缓冲液稀释至0.5mg/mL，得到质控线溶液。
(3)在硝酸纤维素膜上，用划膜仪喷涂检测线溶液形成T线，喷涂量为1ug/cm；喷涂质控线溶液形成C线，喷涂量为1ug/cm。喷涂量是指每厘米长的T线或C线上喷涂的蛋白质量。T线和C线的宽度均是1mm。
(4)过夜干燥，得到喷涂了检测线(T线)和质控线(C线)的硝酸纤维素膜。
4.试纸条的组装
按照图3-5中试纸条的结构，采用如下方法组装试纸条：在正常湿度和温度下的洁净操作台上，将处理好的样品垫3、喷涂了检测线和质控线的硝酸纤维素膜4、吸水垫7(材质为吸水滤纸)依次以2-4mm重叠黏贴在底板2上，然后送入切条机，得到宽为4±0.5mm的试纸条。挑取完好整齐的试纸条，放入卡壳中，压盖完成后连同1粒干燥剂放入铝箔袋中密封保存。其中底板2为PVC板。
将本实施例制备的试纸条记为本发明试纸条。
实施例4试纸条定性检测方法及结果判定
1.采用本发明试纸条和本发明样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体的方法，包括如下步骤：
(1)样品处理：用1根医用脱脂棉签伸入猪口腔，充分吸取口腔液后，放入含有1ml PBS缓冲液(浓度为0.01M、pH7.4，含0.01％硫柳汞)的EP管中，反复挤压混匀，所得溶液即 为口拭子。将口拭子或口拭子采用PBS缓冲液稀释后所得的稀释液作为检测样本。
(2)取出本发明试纸条(实施例3制备)，将其放于洁净操作台上。
(3)用1mL巴氏吸管吸取检测样本，以体积比为1:1与本发明样本稀释液混合，室温(20℃)孵育10min。
(4)孵育完成后，用1mL巴氏吸管吸取3滴(每滴约30微升)，于样品垫上方，垂直缓慢滴加。
(5)滴加完成后，等待10～15min，判定结果。
2.定性判定结果(目视法)
将反应后的试纸条采用365nm的紫外光发射器的紫外光照射，如果试纸条的C线显色，T线也显色时，结果判定为阳性，且T线的颜色越深，则样品中的非洲猪瘟病毒黏膜sIgA抗体效价越高。当试纸条的C线显色，T线不显色时，结果判定为阴性，样品中无非洲猪瘟病毒黏膜sIgA抗体。若试纸条的C线不显色，则该纸条的检测结果无效。
实施例5试纸条检测的特性
1.试纸条检测时的灵敏度
考察采用本发明试纸条和本发明样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体时灵敏度情况。
1)方法
将非洲猪瘟抗体阳性参考口拭子(采集自非洲猪瘟血清抗体阳性猪的口腔液)用PBS(浓度0.01M、pH为7.4)按照稀释度为1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256进行稀释。用本发明样本稀释液配合本发明试纸条，分别对非洲猪瘟抗体阳性参考口拭子的不同稀释度的稀释液和非洲猪瘟抗体阴性参考口拭子(采集自健康猪的口腔液)进行检测。
2)结果
本发明样本稀释液配合本发明试纸条，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:128的稀释液检测的结果为阳性，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:256的稀释液和非洲猪瘟抗体阴性参考口拭子检测的结果为阴性，表明试纸条对非洲猪瘟病毒黏膜sIgA抗体的检出敏感性可达到1:128(稀释度)。
2.样本稀释液中缓冲液对检测灵敏度的影响
1)对照样本稀释液1和对照样本稀释液2的配制
为了考察样本稀释液中组分对检测结果的影响，将本发明样本稀释液配制时使用的含有0.1％(质量百分浓度)BSA和0.2％(体积百分浓度)Tween-20的0.01M、pH7.4的PBS缓冲液，分别更换成PBS缓冲液(浓度0.01M、pH7.4)和PBST缓冲液，分别得到对照样本稀释液1和对照样本稀释液2。其中，PBST缓冲液是含有0.2％(体积百分浓度)Tween-20的0.01M、pH7.4的PBS缓冲液。
2)检测方法
分别采用本发明样本稀释液、对照样本稀释液1或对照样本稀释液2，配合本发明试纸条，以实施例4中方法检测本实施例标题1中非洲猪瘟抗体阳性参考口拭子的各稀释度的稀释液。
3)检测结果
在使用PBS缓冲液或PBST缓冲液时，在检测非洲猪瘟阴性参考口拭子时均出现了 假阳性，因此，检测结果无效。采用本发明样本稀释液，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:128的稀释液检测的结果为阳性，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:256的稀释液和非洲猪瘟抗体阴性参考口拭子检测的结果为阴性，因此检出灵敏度可达1:128(稀释度)。
3.样本稀释液中非洲猪瘟病毒重组蛋白标记物对检测灵敏度的影响
(1)按照本发明实施例2样本稀释液的制备方法，制备对照样本稀释液3和对照样本稀释液4，不同点仅在于：步骤(3)中含有0.1％(质量百分浓度)BSA和0.2％(体积百分浓度)Tween-20的0.01M、pH7.4的PBS缓冲液与量子点微球标记的重组蛋白P30V溶液以体积比为30:1混匀，得到对照样本稀释液3；步骤(3)中将含有0.1％(质量百分浓度)BSA和0.2％(体积百分浓度)Tween-20的0.01M、pH7.4的PBS缓冲液与量子点微球标记的重组多肽串联蛋白R10溶液以体积比为30:1混匀，得到对照样本稀释液4。
(2)将本发明样本稀释液、对照样本稀释液3和对照样本稀释液4分别配合本发明试纸条，按实施例4中方法对本实施例标题1中非洲猪瘟抗体阳性参考口拭子的稀释度为1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256的稀释液和非洲猪瘟抗体阴性参考口拭子进行检测，进行灵敏度对比试验。具体结果见表1。
表1各种方法对样品检测的结果
Figure PCTCN2021071258-appb-000001
由检测结果可以得出：配合本发明试纸条进行检测，采用本发明样本稀释液时，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:128的稀释液检测结果仍然为阳性；采用对照样本稀释液3或4时，均对非洲猪瘟抗体阳性参考口拭子的稀释度为1:32的稀释液检测结果为阳性，对非洲猪瘟抗体阳性参考口拭子的稀释度为1:64的稀释液检测结果为阴性。可见样本稀释液中单独使用量子点微球标记的重组蛋白P30V或量子点微球标记的重组多肽串联蛋白R10时，其灵敏度显著低于使用非洲猪瘟病毒双蛋白-标记物。
4.体外孵育温度和时间的影响
将本发明样本稀释液配合本发明试纸条，对本实施例标题1中非洲猪瘟抗体阳性参考口拭子的各稀释度的稀释液和非洲猪瘟抗体阴性参考口拭子进行检测，考察拭子样品与样本稀释液的孵育温度和孵育时间，对检测结果的影响。结果：当孵育温度在4～37℃之间，孵育时间在7～20min时结果均成立。选室温(20℃)作为孵育温度及10min作为孵育时间的最适条件。
5.试纸条特异性的检测
1)检测方法：
将本发明样本稀释液配合本发明试纸条，采用实施例4中方法，检测猪圆环病毒黏膜抗体阳性样品、猪繁殖呼吸综合症病毒黏膜抗体阳性样品、猪胸膜肺炎放线杆菌黏膜抗体阳性样品、猪流感病毒黏膜抗体阳性样品、猪流行性腹泻病毒黏膜抗体阳性样品。
2)检测结果：
判定结果均为阴性，说明本发明试纸条与猪的其他病毒无交叉反应，特异性良好。
6.稳定性检测
1)检测方法：
将本发明试纸条在放入含有1粒干燥剂的铝箔袋中密封保存后，置于20℃和37℃下分别保存7日、14日、21日和28日，分别按照本实施例标题1中方法进行灵敏度的检验，按照本实施例标题5进行特异性的检验。
2)检测结果：
在常温(20℃)和37℃下保存7日、14日、21日和28日的试纸条对非洲猪瘟抗体阳性参考口拭子的稀释度为1:128的稀释液检测结果仍然均为阳性，猪圆环病毒黏膜抗体阳性样品、猪繁殖呼吸综合症病毒黏膜抗体阳性样品、猪胸膜肺炎放线杆菌黏膜抗体阳性样品、猪流感病毒黏膜抗体阳性样品和猪流行性腹泻病毒黏膜抗体阳性样品的检测结果均为阴性，说明本发明试纸条稳定性良好。
实施例6本发明试纸条与胶体金检测试纸的灵敏度对比
1.制备胶体金颗粒：用柠檬酸三钠还原剂将氯金酸还原，制成20-40nm胶体金颗粒，具体方法如下：取质量百分浓度为1％的氯金酸水溶液800mL用恒温电磁搅拌器加热至沸腾，持续搅拌的情况下加入质量百分浓度为16％的柠檬酸三钠水溶液1mL，继续搅拌加热5-10min，溶液呈透亮的红色。室温冷却，用去离子水补足体积至800mL，得到20-40nm胶体金颗粒，4℃保存。
2.非洲猪瘟病毒双蛋白-胶体金标记物的制备：取步骤1制备的胶体金颗粒1mL，在其中加入3.5μL浓度为0.1mol/L的K 2CO 3溶液以调节pH值，然后加入1.3mg的重组蛋白P30V，混匀后静置5min，加入10μL质量百分浓度为10％的聚乙二醇2000溶液，12000rpm离心7min，弃上清，加入100μL复溶液(含有0.05M三羟甲基氨基甲烷和5％蔗糖的水溶液)，混合均匀，得到胶体金标记的重组蛋白P30V。采用相同方法，制备胶体金标记的重组多肽串联蛋白R10，不同之处在于将其中的重组蛋白P30V替换为重组多肽串联蛋白R10。将胶体金标记的重组蛋白P30V和胶体金标记的重组多肽串联蛋白R10以体积比为1:1混合，得到非洲猪瘟病毒双蛋白-胶体金标记物。
3.胶体金垫的制备：将非洲猪瘟病毒双蛋白-胶体金标记物与含有0.3％BSA、0.5％Tween-20和4％蔗糖的0.01M、pH7.4的PBS缓冲液按照体积比为1:100混合均匀，然后用于浸泡玻璃纤维膜30分钟，37℃干燥30分钟，得到胶体金垫。胶体金垫制备中，使用的含有0.3％BSA、0.5％Tween-20和4％蔗糖的0.01M、pH7.4的PBS缓冲液及其与非洲猪瘟病毒双蛋白-胶体金标记物的体积比均是经过优化的条件。
4.试纸条组装：按照本发明试纸条的组装方法组装胶体金试纸条，不同之处在于 在样品垫和硝酸纤维素膜之间设置一个上述胶体金垫(本实施例标题3中制备)，得到胶体金试纸条。胶体金试纸条的检测方法：将样品滴加3滴(每滴约30微升)在样品垫上，直接肉眼观察，当T线、C线均有显色时为阳性；当C线显色，T线不显色为阴性；当C线不显色时，结果无效。
5.本发明试纸条与胶体金试纸条灵敏度对比试验
对实施例5中非洲猪瘟抗体阳性参考口拭子的稀释度为1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256的稀释液和非洲猪瘟抗体阴性参考口拭子分别用本发明试纸条(配合本发明样品稀释液，检测方法同实施例4)与胶体金试纸条进行检测，检测结果表4所示。
表4本发明试纸条与胶体金试纸条灵敏度的对比
Figure PCTCN2021071258-appb-000002
由检测结果可以看出：本发明试纸条与胶体金试纸分别对非洲猪瘟抗体阳性参考口拭子的不同稀释度的稀释液进行检测，本发明试纸条检测非洲猪瘟抗体阳性参考口拭子的稀释度为1:128的稀释液时为阳性。胶体金试纸条检测非洲猪瘟抗体阳性参考口拭子的稀释度为1:4的稀释液时为阳性，检测非洲猪瘟抗体阳性参考口拭子的稀释度为1:8的稀释液时为阴性，灵敏度显著低于本发明试纸条。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (9)

  1. 一种检测非洲猪瘟病毒黏膜sIgA抗体的量子点微球免疫层析试纸条，其特征在于，包括底板，所述底板上依次设有样品垫、硝酸纤维素膜和吸水垫；所述硝酸纤维素膜上设有检测线和质控线，所述检测线处包被有鼠抗猪SC蛋白单克隆抗体，所述检测线靠近所述样品垫一侧设置；所述质控线处包被有非洲猪瘟阳性血清的纯化蛋白，所述质控线靠近所述吸水垫一侧设置。
  2. 根据权利要求1所述量子点微球免疫层析试纸条，其特征在于所述非洲猪瘟阳性血清的纯化蛋白采用如下方法制备：将重组蛋白P30V和重组多肽串联蛋白R10免疫兔后所得非洲猪瘟阳性血清，采用protein G纯化试剂盒纯化，得到所述纯化蛋白；所述鼠抗猪SC蛋白单克隆抗体是由分泌抗猪SC蛋白单克隆抗体的杂交瘤细胞株4H11所制备，所述杂交瘤细胞株4H11的保藏号为：CCTCC NO：C201526。
  3. 根据权利要求1或2所述量子点微球免疫层析试纸条，其特征在于：所述样品垫是将玻璃纤维膜采用含有牛血清白蛋白和Tween-20的Tris-HCl缓冲液浸泡处理后所得。
  4. 配合权利要求1-3之一所述试纸条使用的样本稀释液，其特征在于含有量子点微球标记的重组多肽串联蛋白R10和量子点微球标记的重组蛋白P30V；所述重组多肽串联蛋白R10的序列如SEQ ID NO:2；所述重组蛋白P30V的序列如SEQ ID NO:4所示。
  5. 根据权利要求4所述样本稀释液，其特征在于将量子点微球分别与重组多肽串联蛋白R10和重组蛋白P30V偶联，分别得到量子点微球标记的重组多肽串联蛋白R10和量子点微球标记的重组蛋白P30V；然后将量子点微球标记的重组多肽串联蛋白R10和重组蛋白P30V按照体积比为1：0.5-1.5混合，得到非洲猪瘟病毒双蛋白-标记物；将所述非洲猪瘟病毒双蛋白-标记物与含有BSA和Tween-20的PBS缓冲液按照体积比1:28-32混合，得到样本稀释液。
  6. 根据权利要求5所述样本稀释液，其特征在于所述量子点微球与重组多肽串联蛋白R10的质量比为5-7:1，所述量子点微球与重组蛋白P30V的质量比为5-7:1；所述PBS缓冲液含有质量百分浓度为0.05-0.15％的BSA和体积百分浓度为0.2％的Tween-20。
  7. 一种以非诊断为目的的采用权利要求1所述量子点微球免疫层析试纸条和权利要求5所述样本稀释液检测非洲猪瘟病毒黏膜sIgA抗体的方法，其特征在于将拭子样品加入样本稀释液中孵育后，滴加在所述试纸条的样品垫上，反应后，采用紫外光发射器检测C线和T线。
  8. 根据权利要求7所述方法，其特征在于检测样品为拭子样品或黏膜样品。
  9. 根据权利要求8所述方法，其特征在于所述拭子样品包括口拭子、鼻拭子或肛拭子，所述黏膜样品为肺泡灌洗液。
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