WO2022032983A1 - Quantum dot microsphere immunochromatography test strip for detection of african swine fever virus mucous membrane siga antibody, and use thereof - Google Patents

Abstract

A quantum dot microsphere immunochromatography test strip for detection of an African swine fever virus mucous membrane sIgA antibody and a use thereof, relating to the field of veterinary biology diagnostic products. The test strip (1) comprises a bottom panel (2), and a sample pad (3), a nitric acid cellulose membrane (4), and a water absorption pad (7) are provided in succession on the bottom panel (2); a detection line (5) and a quality control line (6) are provided on the nitric acid cellulose membrane (4), the detection line (5) is coated with a mouse anti-swine SC protein monoclonal antibody, and the detection line (5) is arranged on the side near to the sample pad (3); the quality control line (6) is coated with a purified protein of a positive serum of African swine fever, and the quality control line (6) is arranged on the side near to the water absorption pad (7). A sample diluent contains recombinant polypeptide tandem protein R10 and recombinant protein P30V labeled by a quantum dot microsphere; the sequences of R10 and P30V are as shown in SEQ ID NO: 2 and 4, respectively. Use the test strip (1) in coordination with the sample diluent, for use in rapid, high-specificity, and high-sensitivity qualitative detection of an African swine fever virus mucous membrane sIgA antibody.

Description

A quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody and its application technical field

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.

Background technique

African swine fever (ASF) 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. In response to infection, 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.

At present, there is no effective commercial vaccine for ASFV prevention and control, so the monitoring and diagnosis of epidemic prevention and control has become very important. Traditional diagnostic methods for detecting African swine fever virus antibodies include indirect immunofluorescence (IFA) and enzyme-linked immunosorbent assay (ELISA). Among them, the sensitivity of IFA is low, requiring a fluorescence operator and a fluorescence microscope, and the cost is high, which limits its application in rapid on-site detection. ELISA is based on the principle of antigen-antibody specific reaction to detect African swine fever virus antibodies in animals. It has the advantages of high sensitivity and strong specificity, but the operation is cumbersome and time-consuming. In addition, it requires microplate reader, incubator and other equipment. It is difficult for grass-roots farms to carry out real-time detection.

SUMMARY OF THE INVENTION

In view of the deficiencies of the existing problems, 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.

The technical scheme adopted by the present invention to solve the technical problem is:

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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, the mass ratio of the quantum dot microspheres to the recombinant polypeptide tandem protein R10 is 5-7:1, and the mass ratio of the quantum dot microspheres to the recombinant protein P30V is 5-7:1; the PBS The buffer 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.

In the present invention, the detection sample is a swab sample or a mucosal sample.

In the present invention, the swab sample includes oral swab, nasal swab or anal swab, and the mucosal sample is bronchoalveolar lavage fluid.

When detecting ASFV mucosal sIgA antibody using quantum dot microsphere immunochromatography test strips with ASFV mucosal sIgA antibody, 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.

Compared with the prior art, the present invention has the following beneficial effects:

1. 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;

2. 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.

3. Because the generation of mucosal antibodies is earlier than that of serum antibodies, the use of quantum dot microsphere immunochromatography test strips and sample diluents of the present invention to detect African swine fever virus mucosal sIgA antibodies can be used for early detection of African swine fever infection, and The test strip and the sample diluent of the invention are used for detection, which is fast, has strong specificity, high sensitivity and good stability.

4. 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.

5. 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.

Description of drawings

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.

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. Among them, 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.

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.

Figure 7 Sensitivity map.

Figure 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.

detailed description

The preferred embodiments of the present invention will be described in detail below with reference to the embodiments of the present invention. In addition, it should be understood that the following embodiments are only to further illustrate the present invention and to help understanding. Rather than limiting the scope of the present invention, the protection scope of the present invention is not limited by any embodiment form. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

In the present invention: MES, EDC, NHS, Tween-20, sucrose, Freund's complete adjuvant, and thimerosal were purchased from Sigma; Tris-HCl, BSA were purchased from ThermoFisher; goat anti-pig IgA enzyme-labeled antibody was purchased from Bethyl company products, ( Item No. A100-102p); 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.

0.02M, 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.

Example 1 Preparation of recombinant polypeptide tandem protein R10 and recombinant protein P30V

1. Construction of P54E gene recombinant vector

Referring to the complete gene sequence (MH766894, E183L, 162222-162776) of the first case of African swine fever in my country published in the NCBI database, 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.

By the "heat shock" method, the recombinant plasmid pET-28a-R10 was transformed into E. coliBL21(DE3) competent cells to obtain recombinant strain pET-28a-R10(BL21).

2. Construction of P30V gene recombinant vector

Referring to the first African swine fever CP204L gene sequence (MH766894, CP204L, 124770～125375) published in the NCBI database, 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.

Through the "heat shock" method, the recombinant plasmid pET-21a-P30V was transformed into E. coliBL21 (DE3) competent cells to obtain recombinant strain pET-21a-P30V (BL21).

3. Expression and purification of recombinant protein

The recombinant strain pET-28a-R10(BL21) was cultured in LB liquid medium containing 50 μg/mL kanamycin, and pET-21a-P30V(BL21) was cultured in LB liquid medium containing 100 μg/mL ampicillin The culture conditions were as follows: the culture temperature was 37 °C, the shaking speed was 180 r/min, the temperature was lowered to 20 °C when OD ₆₀₀ = 0.6 to 0.8, and IPTG with a final concentration of 1.0 mmol/L was added after 30 min to induce expression, and then The cells were cultured at 20°C for 17 hours, and the cells were collected. 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. It can be seen from lane 1 of Figure 2 that 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.

4. Antigenicity detection of recombinant protein:

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. Wash 5 times with TBST, 5 min/time. ECL was protected from light for 5min. Results: The recombinant polypeptide tandem protein R10 showed a specific reaction band at about 19KDa (lane 2 in Figure 1); the recombinant protein P30V showed a specific reaction band at about 43KDa (lane 2 in Figure 2). The above results indicate that both the recombinant polypeptide tandem protein R10 and the recombinant protein P30V can specifically react with the African swine fever antibody-positive reference oral swab, and have good antigenicity.

Example 2 Preparation of the sample diluent of the present invention

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:

(1) Preparation of recombinant protein P30V labeled with quantum dot microspheres

Activation of quantum dot microspheres: 50 μL of quantum dot microsphere suspension with a concentration of 1.2 μg/μL was added to 50 μL 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 μL 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 μg 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 μL 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 μL 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.

(2) Preparation of recombinant polypeptide tandem protein R10 labeled with quantum dot microspheres

According to the same method in the title (1) of this example, the quantum dot microspheres were coupled to the recombinant polypeptide tandem protein R10 prepared in Example 1, except that 10 μg of recombinant protein P30V was replaced with 10 μg of recombinant polypeptide tandem protein R10, A solution of recombinant polypeptide tandem protein R10 labeled with quantum dot microspheres was obtained.

(3) Mixed

A 0.01M, pH7.4 PBS buffer containing 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) Tween-20 was prepared. Mix the quantum dot microsphere-labeled recombinant protein P30V solution prepared in the title (1) of this example and the quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 solution prepared in the title (2) of this example at a volume ratio of 1:1. Homogenize to obtain African swine fever virus double protein-marker. The 0.01M, pH7.4 PBS buffer containing 0.1% (mass percent concentration) BSA and 0.2% (volume percent concentration) Tween-20 and African swine fever virus double protein-label in a volume ratio of 30: 1 Mix well to obtain sample diluent and store at 4°C in the dark.

The preparation of embodiment 3 test strips of the present invention

As shown in Figure 3-5, 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.

1. Preparation of purified protein from African swine fever-positive serum

(1) Sample preparation

Using 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. For the first 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 μg/mL The solution was coated with 100 μL 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 μL of PBST for 5 min each time, and patted dry. Add 200 μL of 0.01M PBS buffer containing 5% skim milk, pH 7.4 to each well, block at 37°C for 2 h, discard the liquid in the wells, wash each well with 250 μL PBST 3 times, 5 min each time, and pat dry. Add 10-fold serially diluted serum to be tested, 100 μL per well, and incubate at 37°C for 1 h. The liquid in the wells was discarded, and each well was washed three times with 250 μL 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 μm filter. Obtain the diluted HRP-goat anti-rabbit IgG enzyme-labeled antibody. Add 100 μL of diluted HRP-goat anti-rabbit IgG enzyme-labeled antibody to each well, and react at 37°C for 0.5 h. Add 100 μL of substrate chromogenic solution TMB to each well, protect from light at 37°C for 15 min; then add 50 μL of sulfuric acid aqueous solution with a concentration of 2 mol/L to terminate the reaction. The OD ₄₅₀ reading value was measured on a microplate reader, and the titer was calculated according to the conventional method. The result: the antibody titer of the African swine fever positive serum against the recombinant polypeptide tandem protein R10 was 1:10 ⁵ . 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 μg/mL recombinant protein P30V was prepared with carbonate buffer (concentration 0.01M, pH 9.6). , coat the microtiter plate with 100 μL per well. Calculated according to the conventional method, the result: the serum antibody titer against recombinant protein P30V is 1:4×10 ⁵ .

In the production process, 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) and African swine fever positive serum were mixed at a volume ratio of 1:1 to obtain a diluent of African swine fever positive serum.

(2) Using GenScript’s 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. 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) to adjust the pH of the eluate to 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%.

2. Preparation of Sample Pads

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.

3. Preparation of Nitrocellulose Membrane

(1) Use the 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.

(2) The purified protein of African swine fever positive serum was diluted to 0.5 mg/mL with 0.01 M PBS buffer containing 3% (mass percent concentration) sucrose, pH 7.4, to obtain a quality control line solution.

(3) On the nitrocellulose membrane, spray the detection line solution with a scriber to form a T line, and the spray amount is 1ug/cm; spray the quality control line solution to form a C line, and the spray amount is 1ug/cm. 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.

(4) Dry overnight to obtain a nitrocellulose membrane sprayed with a detection line (T line) and a quality control line (C line).

4. Assembly of test strips

According to the structure of the 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 (the material is water-absorbing filter paper) 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.

The 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

1. 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:

(1) Sample processing: Insert a medical absorbent cotton swab into the pig's oral cavity, fully absorb the oral fluid, and put it into an EP tube containing 1 ml of PBS buffer (concentration 0.01M, pH 7.4, containing 0.01% thimerosal), Repeated squeezing and mixing, the obtained solution is the mouth swab. The oral swab or the dilution obtained by diluting the oral swab with PBS buffer was used as the test sample.

(2) Take out the test strip of the present invention (prepared in Example 3) and place it on a clean operating table.

(3) 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.

(4) After the incubation is completed, use a 1 mL Pasteur pipette to suck up 3 drops (about 30 microliters per drop), and drop them vertically and slowly on the top of the sample pad.

(5) After the dropping is completed, wait for 10 to 15 minutes to judge the result.

2. Qualitative judgment results (visual method)

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

1. Sensitivity 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.

1) Method

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. Using 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.

2) Results

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) .

2. Influence of buffer in sample diluent on detection sensitivity

1) Preparation of Control Sample Diluent 1 and Control Sample Diluent 2

In order to investigate the influence of the components in the sample diluent on the detection results, 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.

2) Detection method

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.

3) Test results

False positives were detected on ASF-negative reference mouth swabs when using either PBS buffer or PBST buffer, therefore, the test results were invalid. Using the sample diluent of the present invention, the result of the dilution for the ASF antibody-positive reference oral swab with a dilution of 1:128 is positive, and the dilution for the African swine fever antibody-positive reference oral swab is 1:256 The results of the reference mouth swab test were negative for the dilution of the ASF and African swine fever antibodies, so the detection sensitivity can reach 1:128 (dilution).

3. The influence of African swine fever virus recombinant protein marker in sample dilution on detection sensitivity

(1) According to the preparation method of the sample diluent in Example 2 of the present invention, prepare the control sample diluent 3 and the control sample diluent 4, the difference is only that: 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; In 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.

(2) 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.

Table 1 Results of sample detection by various methods

From the test results, it can be concluded that when the test strip of the present invention is 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; 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. It can be seen that when the quantum dot microsphere-labeled recombinant protein P30V or the quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 is used alone in the sample diluent, the sensitivity is significantly lower than that using the African swine fever virus double protein-label.

4. Influence of in vitro incubation temperature and time

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 effect of incubation temperature and incubation time of subsamples and sample diluents on detection results. Results: When the incubation temperature was between 4 and 37 °C and the incubation time was between 7 and 20 min, the results were all established. Room temperature (20°C) was selected as the optimal conditions for incubation temperature and 10 min as incubation time.

5. Test strip specificity detection

1) Detection method:

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. Samples, swine influenza virus mucosal antibody positive samples, porcine epidemic diarrhea virus mucosal antibody positive samples.

2) Test results:

The judgment results are all negative, indicating that the test strip of the present invention has no cross-reaction with other swine viruses and has good specificity.

6. Stability test

1) Detection method:

After the 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.

2) Test results:

The 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

1. 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.

2. Preparation of African swine fever virus double protein-colloidal gold label: Take 1 mL of the colloidal gold particles prepared in step 1, add 3.5 μL of K ₂ CO ₃ solution with a concentration of 0.1 mol/L to it to adjust the pH value, and then add 1.3 mg of recombinant protein P30V, mixed and left for 5 minutes, added 10 μL of polyethylene glycol 2000 solution with a concentration of 10% by mass, centrifuged at 12000 rpm for 7 minutes, discarded the supernatant, and added 100 μL of the reconstituted solution (containing 0.05M trihydroxymethane). aminomethane and 5% sucrose in water), and mixed evenly to obtain colloidal gold-labeled recombinant protein P30V. 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.

3. Preparation of colloidal gold pads: The African swine fever virus double protein-colloidal gold marker was mixed with 0.01M, pH7.4 PBS buffer containing 0.3% BSA, 0.5% Tween-20 and 4% sucrose according to the volume ratio of Mix well at 1:100, then soak the glass fiber membrane for 30 minutes, and dry at 37°C for 30 minutes to obtain a colloidal gold pad. In the preparation of colloidal gold pads, 0.01M PBS buffer containing 0.3% BSA, 0.5% Tween-20 and 4% sucrose, pH 7.4 and its volume ratio to African swine fever virus double protein-colloidal gold marker were used All are optimized conditions.

4. 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.

5. Sensitivity comparison test of test strip of the present invention and colloidal gold test strip

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.

The comparison of the sensitivity of table 4 test strip of the present invention and colloidal gold test strip

It can be seen from the test results that the 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, and the 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.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (9)

1. A quantum dot microsphere immunochromatographic test strip for detecting African swine fever virus mucosal sIgA antibody, characterized in that it comprises a bottom plate, and the bottom plate is provided with a sample pad, a nitrocellulose membrane and a water-absorbing pad in sequence; the nitric acid A detection line and a quality control line are arranged on the cellulose membrane, the detection line is coated with a 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 a monoclonal antibody. For the purified protein with African swine fever positive serum, the quality control line is set near the side of the absorbent pad.
2. The quantum dot microsphere immunochromatographic test strip according to claim 1, wherein the purified protein of the African swine fever positive serum is prepared by the following method: the recombinant protein P30V and the recombinant polypeptide tandem protein R10 obtained after immunizing rabbits with African swine fever The swine fever-positive serum was purified by a protein G purification kit to obtain the purified protein; the mouse anti-pig SC protein monoclonal antibody was prepared by a hybridoma cell line 4H11 that secreted anti-pig SC protein monoclonal antibody, and the The deposit number of the hybridoma cell line 4H11 is: CCTCC NO: C201526.
3. The quantum dot microsphere immunochromatographic test strip according to claim 1 or 2, wherein the sample pad is made by soaking the glass fiber membrane in Tris-HCl buffer containing bovine serum albumin and Tween-20 later income.
4. The sample diluent used in conjunction with the test strip described in any one of claims 1 to 3 is characterized in that it contains the recombinant polypeptide tandem protein R10 labeled with quantum dot microspheres and the recombinant protein P30V labeled with quantum dot microspheres; the recombinant polypeptide tandem protein The sequence of protein R10 is shown in SEQ ID NO:2; the sequence of the recombinant protein P30V is shown in SEQ ID NO:4.
5. The sample diluent according to claim 4, characterized in that the quantum dot microspheres are respectively coupled with recombinant polypeptide tandem protein R10 and recombinant protein P30V to obtain recombinant polypeptide tandem protein R10 and quantum dot microspheres labeled with quantum dot microspheres, respectively. The labeled recombinant protein P30V; then the quantum dot microsphere-labeled recombinant polypeptide tandem protein R10 and the recombinant protein P30V were mixed in a volume ratio of 1:0.5-1.5 to obtain an African swine fever virus double protein-label; the African pig Pestivirus double protein-label was mixed with PBS buffer containing BSA and Tween-20 at a volume ratio of 1:28-32 to obtain a sample dilution.
6. The sample diluent according to claim 5, wherein the mass ratio of the quantum dot microspheres to the recombinant polypeptide tandem protein R10 is 5-7:1, and the mass ratio of the quantum dot microspheres to the recombinant protein P30V is 5 -7:1; the PBS buffer contains BSA with a concentration of 0.05-0.15% by mass and Tween-20 with a concentration of 0.2% by volume.
7. A non-diagnostic method for detecting African swine fever virus mucosal sIgA antibody using the quantum dot microsphere immunochromatographic test strip described in claim 1 and the sample diluent described in claim 5, characterized in that the swab After the sample is added to the sample diluent for incubation, it is dropped on the sample pad of the test strip, and after the reaction, the C-line and the T-line are detected by an ultraviolet light emitter.
8. The method according to claim 7, wherein the detection sample is a swab sample or a mucosal sample.
9. The method according to claim 8, wherein the swab sample comprises an oral swab, a nasal swab or an anal swab, and the mucosal sample is bronchoalveolar lavage fluid.

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