A porcine circovirus type 3 antigen detection reagent strip and a preparation method and application thereof

Abstract

The application discloses an anti-PCV3 Cap protein monoclonal antibody, which is a monoclonal antibody 1D4-10-16 and a monoclonal antibody 2B6-1-1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1D4-10-16 are shown as SEQ ID NO. 3 and SEQ ID NO. 4 respectively. The application further discloses a porcine circovirus type 3 antigen detection test strip, which comprises a detection test strip and a matching sample diluent, wherein the detection test strip comprises a PVC bottom plate, and a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption paper are sequentially fixed on the PVC bottom plate; the latex microsphere pad is a latex microsphere marked with an effective amount of a PCV3 Cap protein polyclonal antibody; and the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, wherein the detection line is an effective amount of the monoclonal antibody 1D4-10-16, and the quality control line is an effective amount of the PCV3 Cap protein. The test strip of the application has good sensitivity and specificity, and provides a new product for rapid clinical detection of PCV3 infection.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biotechnology, in particular to a porcine circovirus type 3 antigen detection reagent strip and a preparation method and application thereof. BACKGROUND

[0002] Porcine circovirus (PCV) is a single-stranded DNA virus (ssDNA), and is one of the smallest self-replicating viruses known. So far, four types of PCV have been found, namely PCV1, PCV2, PCV3 and PCV4.

[0003] At present, the PCV3 isolation and culture technology is not mature, and there is no safe and effective PCV3 vaccine product. In the prevention and control of PCV3, it is still necessary to rely on biological safety prevention and control measures. In the biological safety prevention and control, the pig farm should do a good job in the monitoring of PCV3, avoid the introduction of PCV3, timely eliminate the pigs carrying PCV3, and eliminate the source of infection; do a good job in physical isolation, prevent the introduction of carriers into the pig farm; implement the disinfection work well, and cut off the transmission route.

[0004] At present, the diagnosis technology of PCV3 and other new animal diseases is insufficient, mainly the molecular biology detection technology based on conventional PCR and qPCR. These detection methods are time-consuming, high-cost, and require high equipment, which is not convenient for rapid diagnosis and application in pig farms. There are few researches on the serological detection technology of PCV3, and only a few reports on indirect ELISA using recombinant PCV3 Cap protein. At present, there is no rapid detection method or product of PCV3 antigen on the market.

[0005] In view of the fact that there is no commercialized PCV3 vaccine available at present, it is urgent to establish corresponding pathogen monitoring and evaluation methods, and to comprehensively carry out systematic monitoring and risk assessment of PCV3, so as to timely master the epidemic situation and scientifically judge the trend, and effectively prevent and control the outbreak and prevalence of such epidemic.

[0006] Based on this, in the present application, the PCV3 Cap gene is cloned, and then connected to the pET-32a vector to construct a recombinant prokaryotic expression vector. After transforming the competent cells of Escherichia coli, the recombinant PCV3 Cap protein is obtained by IPTG induction and nickel column chromatography purification. The purified recombinant PCV3 Cap protein is used as an antigen to immunize New Zealand white rabbits and BALB / c mice to prepare polyclonal antibodies and monoclonal antibodies. Further, the prepared recombinant PCV3 Cap protein, polyclonal antibodies and monoclonal antibodies are used as raw materials to develop a PCV3 antigen rapid detection reagent strip product. It is proved that the rapid detection reagent strip can be applied to clinical rapid detection by detecting clinical samples. SUMMARY

[0007] In order to make up for the deficiencies of the prior art, the purpose of the present application is to provide a porcine circovirus type 3 antigen detection reagent strip and a preparation method and application thereof.

[0008] Therefore, in one aspect, the present application discloses an anti-PCV3 Cap protein monoclonal antibody, which is monoclonal antibody 1D4-10-16 and monoclonal antibody 2B6-1-1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1D4-10-16 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0009] In another aspect, the present application further discloses a porcine circovirus type 3 antigen detection test strip, which comprises a detection test strip and a matching sample diluent, wherein the detection test strip comprises a PVC bottom plate, on which a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption paper are sequentially fixed in order; the latex microsphere pad is a latex microsphere labeled with an effective amount of PCV3 Cap protein polyclonal antibody; the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, wherein the detection line is an effective amount of monoclonal antibody 1D4-10-16, and the quality control line is an effective amount of PCV3 Cap protein.

[0010] Preferably, the concentration of the PCV3 Cap protein polyclonal antibody labeled latex microspheres on the latex microsphere pad of the present application is 1 mg / mL.

[0011] Preferably, the PCV3 Cap protein polyclonal antibody of the present application is a rabbit anti-PCV3 Cap protein polyclonal antibody, and the ELISA titer thereof is 1:80000.

[0012] Preferably, the concentration of the monoclonal antibody 1D4-10-16 on the detection line of the present application is 1 mg / mL.

[0013] Preferably, the concentration of the PCV3 Cap protein on the detection line of the present application is 1 mg / mL.

[0014] Preferably, the amino acid sequence of the PCV3 Cap protein of the present application is shown in SEQ ID NO. 1.

[0015] Preferably, the nucleotide sequence of the codon-optimized PCV3 Cap protein of the present application is shown in SEQ ID NO. 2.

[0016] In another aspect, the present application further discloses the use of the monoclonal antibody 1D4-10-16 in the preparation of a PCV3 or PCV3 Cap protein detection reagent.

[0017] The monoclonal antibody 1D4-10-16 and the monoclonal antibody 2B6-1-1 prepared by the application have good sensitivity and specificity, and are suitable for detection of PCV3 and PCV3 Cap protein, such as ELISA method or Western blot method or colloidal gold method or chemiluminescence method for detecting PCV3 antigen or PCV3 Cap protein antibody.

[0018] Meanwhile, the PCV3 Cap protein prepared by the application has good immunogenicity and high expression amount, and the PCV3 Cap protein polyclonal antibody prepared by the application also has good sensitivity and specificity, and is suitable for detection of PCV3 and PCV3 Cap protein, such as ELISA method or Western blot method or colloidal gold method or chemiluminescence method for detecting PCV3 antigen or PCV3 Cap protein antibody.

[0019] On the basis of the monoclonal antibody 1D4-10-16, the monoclonal antibody 2B6-1-1, the PCV3 Cap protein and the PCV3 Cap protein polyclonal antibody, the swine circovirus type 3 antigen detection test strip developed by the application has good sensitivity and specificity, and is suitable for rapid detection of PCV3 virus and PCV3 Cap protein, and provides a good product for rapid detection of PCV3 in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Schematic diagram of construction of recombinant plasmid pET-32a-Cap.

[0021] Figure 2 SDS-PAGE identification of induced expression of recombinant plasmid pET32a-Cap transformed expression bacteria Rossatta.

[0022] Figure 3 Purification identification result of recombinant PCV3 Cap protein inclusion body.

[0023] Figure 4 Western Blot (His primary antibody) identification result of recombinant PCV3 Cap protein.

[0024] Figure 5 Western Blot (polyclonal antibody) identification result of recombinant PCV3 Cap protein.

[0025] Figure 6 ELISA titer detection result of immune mouse serum.

[0026] Figure 7 ELISA titer determination of positive monoclonal antibody (undiluted).

[0027] Figure 8SDS-PAGE electrophoresis result chart of 1D4-10-16 monoclonal antibody after purification.

[0028] Figure 9 Figure of identification of recombinant PCV3 Cap protein Western Blot (1D4-10-16 monoclonal antibody).

[0029] Figure 10 1D4-10-16 and 2B6-1-1 monoclonal antibody ascites titer determination result.

[0030] Figure 11 1D4-10-16 and 2B6-1-1 monoclonal antibody specificity identification result.

[0031] Figure 12 Figure of screening result of labeled antibody and system.

[0032] Figure 13 Figure of screening result of labeled antibody corresponding to point membrane antibody.

[0033] Figure 14 Labeled antibody sensitivity test result.

[0034] Figure 15 Figure of test result of test strip prepared by the same antibody as point membrane antibody and labeled antibody.

[0035] Figure 16 Figure of best combination of different point membrane antibody and labeled antibody.

[0036] Figure 17 Product packaging diagram.

[0037] Figure 18 Assembly diagram of test strip. Wherein A: sample pad, B: latex microsphere pad, C: detection line, D: quality control line.

[0038] Figure 19 Test strip detection judgment diagram.

[0039] Figure 20 Clinical serum sample detection result. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0041] Unless otherwise indicated, the reagents, methods, and apparatuses employed in the present application are of a type commonly employed in the art. Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0042] Example 1: Preparation of recombinant PCV3 Cap protein

[0043] 1. Acquisition of target gene and construction of expression vector

[0044] The PCV3 Cap protein sequence was searched in the NCBI database, and the conserved protein sequence was selected by sequence alignment. The amino acid sequence of the PCV3 Cap protein is shown in SEQ ID NO. 1. The target gene was synthesized after codon optimization by E. coli expression system (the sequence after codon optimization is shown in SEQ ID NO. 2). After double digestion with restriction enzymes BamHI and Hind III, it was connected to the pET32a prokaryotic expression vector to construct a recombinant prokaryotic expression vector. The construction schematic is shown in Figure 1 The recombinant plasmid was transformed into TG1 cloning bacteria, and the recombinant vector obtained after screening and identification was named pET-32a-Cap.

[0045] 2. Expression, purification and identification of target protein

[0046] The recombinant plasmid pET-32a-Cap was transformed into E. coli Rossatta (DE3) expression bacteria, and the recombinant Cap protein was obtained by IPTG induction expression and His nickel column purification, and identified by SDS-PAGE electrophoresis and Western Blot (His primary antibody). The specific steps are as follows:

[0047] (1) Protein expression: The recombinant expression bacteria were inoculated into LB medium at a ratio of 1:100, and 1 mM Amp antibiotic was added. The bacteria were shaken at 37°C and 220 rpm for 3-4 h, and then induced by adding 1 mM IPTG with a final concentration. The bacteria were induced to grow overnight at 16°C and 220 rpm.

[0048] (2) Bacterial disruption: The bacteria solution induced overnight was collected and centrifuged at 4000 rpm for 40 min to obtain the bacterial cells. The bacterial cells were resuspended and washed with an appropriate amount of sterile PBS solution, and centrifuged at 12000 rpm and 4°C for 10 min to discard the supernatant. The bacterial cells were resuspended with a small amount of pre-cooled purification washing solution or PBS, and then broken in an ultrasonic disrupter with the condition of 3 s on and 3 s off, and ice bath ultrasonic for 20-30 min. Finally, the supernatant and the precipitate were collected by centrifugation at 12000 rpm and 4°C for 15 min. The samples of pre-induction, post-induction, and the supernatant and the precipitate after disruption were prepared for SDS-PAGE electrophoresis, and the results are shown in Figure 2 As can be seen from the figure, the PCV3 Cap protein is expressed in the form of inclusion body.

[0049] (3) Purification of inclusion body protein: collect the precipitate after the bacteria are broken, dissolve the inclusion body with inclusion body lysis solution, centrifuge the dissolved inclusion body suspension at 4°C, 12000 rpm for 15 min, collect the supernatant and dialyze in 6M, 4M, 2M urea refolding buffer for 2h, and then dialyze in 0M urea refolding buffer overnight. Collect the protein solution after dialysis at 4°C, 12000 rpm for 10 min, separate the dialysis supernatant and dialysis precipitate, and collect the dialysis supernatant and dialysis precipitate for SDS-PAGE electrophoresis analysis.

[0050] (4) SDS-PAGE electrophoresis and Western Blot identification: after the purified PCV3 Cap is prepared, 12% SDS-PAGE gel electrophoresis is performed, and the results are as shown in Figure 3 Western Blot identification is performed using His antibody, and the results are as shown in Figure 4 .

[0051] The results show that the target protein is expressed in E. coli Rossatta (DE3) expression bacteria, and the expression form is inclusion body. According to the conventional denaturation and renaturation method of inclusion body protein and nickel column chromatography, the recombinant protein is obtained, which is identified as recombinant PCV3 Cap protein by SDS-PAGE and Western Blot. Further detection of purity (SDS-PAGE purity > 90%) and concentration (accounting for the expression amount after purification to reach 1 g / L or more) confirms that it meets the requirements of antigen for preparing monoclonal and polyclonal antibodies.

[0052] Example 2: Preparation of anti-PCV3 Cap protein polyclonal antibody

[0053] 1. Animal immunization and preparation of antisera: The recombinant PCV3 Cap protein prepared above was quantified by BCA protein quantification kit, and diluted to a concentration of 1 mg / mL. Animal immunization was performed by subcutaneous injection at multiple points on the back, and immunization was performed at 0, 10, 20 and 30 d, respectively. Each time of immunization used equal volume of Freund's adjuvant to emulsify the antigen, i.e. 1 mL of adjuvant + 1 mL of antigen (concentration of 1 mg / mL), a total of 2 mL of emulsified vaccine was used for each immunization, and the antigen content was 1 mg. The first immunization used Freund's complete adjuvant, and the remaining three booster immunizations used Freund's incomplete adjuvant. Before immunization at 0 d, the ear vein blood of each group of experimental rabbits was collected to separate the serum as negative control serum. The experimental rabbits were heart blooded at 40 d after the first immunization, and the immune serum was separated.

[0054] 2. Purification of antiserum: The antiserum to be purified was filtered with a 0.45 μm filter to remove impurities, then applied to a Protein A Agarose gravity column and combined for 30 min to allow the IgG in the serum to fully bind to Protein A. The liquid in the column was discarded, and 10 times the column volume of TBS solution was used to wash and remove non-specifically bound proteins. The eluted liquid was measured for absorbance at 280 nm using a NanoDrop, and the washing was continued until no impurities were eluted. After washing, 10 mL of 50 mmol / L glycine (pH 1.9) solution was used as an eluent to elute the specific antibodies bound to the column. The eluate was collected in separate tubes, and the target antibody elution was determined according to the protein concentration in which several collection tubes were used. Finally, the eluted antibody solution was neutralized with 1 mol / L NaHCO3 solution, glycerol was added to a final concentration of 50%, and it was stored at -80°C.

[0055] 3. Determination of the titer of polyclonal antibodies: The purified rabbit anti-PCV3 Cap protein polyclonal antibodies were detected by indirect ELISA. When the sample absorbance value / negative serum absorbance value > 2.1, it was considered positive. The specific method steps are as follows:

[0056] (1) Coating antigen: Dilute the antigen to 1 μg / mL with the antigen coating buffer, add 100 μL of the diluted antigen to each well of the ELISA plate, and incubate at 4°C overnight; (2) Washing: Shake off the antigen coating liquid, and wash 3 times with washing solution, each time shaking on the 96-well plate shaker for 1 min; (3) Blocking: Add 300 μL of blocking solution to each well, cover the lid, and incubate in a 37°C biochemical incubator for 2 h; (4) Washing: Remove the blocking solution and wash 3 times in the same manner; (5) Incubate the primary antibody: Add 100 μL of the measured antibody diluted with antibody diluent to each well, cover the lid, and incubate at 37°C for 1 h. The serum taken on day 0 is used as a negative control. (6) Washing: Remove the measured antibody and wash 4 times; (7) Incubate the secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody (1:1000 dilution) to each well, cover the lid, and incubate at 37°C for 1 h; (8) Washing: Remove the secondary antibody and wash 4 times; (9) Color development: Add 200 μL of TMB color developing solution to each well, develop the color at room temperature in the dark for 15 min; (10) Stop color development: Add 50 μL of stop solution to each well to stop the color development reaction; (11) Absorbance value determination: Wipe the bottom of the ELISA plate with alcohol cotton to remove stains, and place it in the ELISA reader to read the absorbance value at 450 nm wavelength. If the OD 450 value of the measured antibody well is 2.1 times greater than that of the negative control well, it is considered positive. 450

[0057] ​The purified rabbit anti-PCV3 Cap protein polyclonal antibody was tested by indirect ELISA. The titer of the prepared rabbit anti-PCV3 Cap protein polyclonal antibody was calculated to be 1:80,000. The results are shown in Table 1.

[0058] Table 1 Anti-PCV3 Cap polyclonal antibody titer determination table

[0059]

[0060] The polyclonal antibody prepared above was used as the primary antibody and the recombinant PCV3 Cap protein was used as the antigen to verify its binding specificity with the antigen protein. The results showed that the prepared polyclonal antibody could specifically recognize the recombinant PCV3 Cap protein. Figure 5 shown.

[0061] Example 3: Preparation of monoclonal antibodies against PCV3 Cap protein

[0062] 1. Mouse immunization

[0063] (1) Immunize BALB / c mice with the purified antigen at a dose of 100 μg / mouse for the first immunization and 50 μg / mouse for the second to fourth immunizations. During the immunization process, Freund's adjuvant and antigen were emulsified and mixed at a volume ratio of 1:1. Freund's complete adjuvant was used for the first immunization; in the subsequent booster immunization stage, Freund's incomplete adjuvant was used. (2) Immunization was performed by subcutaneous multi-point injection in mice, ensuring that the interval between each immunization was 2 weeks. Starting from the third immunization, blood samples were collected from the mouse eye sockets on the 7th day after each immunization for the preparation of serum for subsequent evaluation and analysis. (3) On the 7th day after the fourth immunization, blood samples were collected from the mouse eye sockets again. After the blood samples were centrifuged, the supernatant was taken for subsequent analysis.

[0064] 2. Determination of immune serum titer and screening of hybridoma cells

[0065] The serum of the immunized mice was tested by indirect ELISA. The spleen of the mice with higher titers was removed aseptically. B lymphocytes were isolated and fused with sp2 / 0 myeloma cells. After screening in HAT medium, hybridoma cell lines capable of producing antibodies were obtained. The results of ELISA titer test of serum antibodies of immunized mice are shown in Figure 6 shown.

[0066] According to the ELISA detection results of the serum of the immunized mice, the spleen of the 13# mouse was finally selected for cell fusion. After the primary screening, 145 positive hybridoma cell strains were obtained, and the positive hybridoma cell culture supernatant was further screened by indirect ELISA to obtain 20 positive hybridoma cell strains with high titer for monoclonal expansion. At the same time, 20 hybridoma culture supernatants were subjected to ELISA and Western blot verification screening, and the results are shown in Figure 7 As shown in the figure, finally, two hybridoma cells with high titer (1D4-10-16 and 2B6-1-1) were selected to prepare ascites by intraperitoneal injection of mice, and the antibodies in the ascites were purified.

[0067] 3. Preparation, purification and identification of monoclonal antibodies

[0068] The two selected hybridoma cell strains (1D4-10-16 and 2B6-1-1) were respectively expanded and cultured according to 1×10 7 The hybridoma cells were intraperitoneally injected into mice to prepare ascites, the collected ascites was centrifuged at 4000 rpm for 10 min, the precipitate was discarded, and the antibody was purified after filtration through 0.45 μm / 0.22 μm filter membrane. The purified monoclonal antibody was subjected to SDS-PAGE and Western blot identification, and the results are shown in Figure 8 and 9 .

[0069] On the basis of the above identification, the titer of the purified ascites was further detected by ELISA method, and the results showed that the two monoclonal antibodies had high titer, indicating that the two monoclonal antibodies had good sensitivity, as shown in Figure 10 .

[0070] At the same time, the two monoclonal antibodies prepared by the application were used for Western blot detection of PCV2 Cap protein (prepared by the inventor), and the results showed that the two monoclonal antibodies did not specifically bind to PCV2 Cap protein, indicating that the two monoclonal antibodies had good specificity, as shown in Figure 11 .

[0071] Example 4, hybridoma monoclonal antibody gene variable region sequence determination

[0072] The total RNA was extracted from the lysed hybridoma cells (1D4-10-16), and after detection, the RNA was reversely transcribed into cDNA using the RACE technology; the heavy chain and light chain variable region sequences were obtained by PCR amplification; the target fragments were connected to the vector using a ligase, and the ligation product was transformed into competent E. coli cells, and then a single colony was picked for sequencing; finally, the variable region sequence of the single-chain antibody gene was obtained by analyzing and annotating the sequencing results, as shown in Table 2 and SEQ ID NO. 3 and SEQ ID NO. 4.

[0073] Table 2: Amino acid sequence information of the variable region of the monoclonal antibody 1D4-10-16

[0074]

[0075] The recombinant PCV3 Cap protein was immunized in mice, and the spleen lymphocytes were separated, fused and screened to obtain hybridoma cell strains. The culture supernatant of the hybridoma cell strains was determined for titer and specificity by ELISA and Western blot experiments. Finally, two high-titer monoclonal cells (1D4-10-16 and 2B6-1-1) were injected into the abdominal cavity of mice to prepare ascites. After affinity purification of the ascites, the titer, concentration and subtype were detected, and the results confirmed that the 1D4-10-16 and 2B6-1-1 monoclonal hybridoma cells obtained after injection into the abdominal cavity of mice had a titer of more than 1:1024000. Further sequencing obtained the variable region sequence of the 1D4-10-16 hybridoma cell monoclonal antibody gene, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0076] Example 5: Preparation of a porcine circovirus type 3 antigen detection test strip (latex method)

[0077] 1. Experimental materials: PCV3 Cap monoclonal antibody (2B6-1-1) is denoted as antibody 1, monoclonal antibody (1D4-10-16) is denoted as antibody 2, PCV3 Cap polyclonal antibody is denoted as antibody 3, recombinant PCV3 Cap protein (quality control), pig and dog serum, NC membrane, BSA blocking solution, Tris-BSA resuspension solution (high sugar), sample pad (glass fiber), marker pad (glass fiber), etc.

[0078] 2. Experimental method

[0079] (1) Antibody labeling: Antibodies 1, 2 and 3 were labeled according to the following steps.

[0080] ① Washing: Take 200 μL latex, add 800 μL borax washing solution, centrifuge at 12000 rpm for 15 min, discard the supernatant; add 800 μL borax washing solution, ultrasonic mixing (large super power 4%, time 2 min), centrifuge at 12000 rpm for 15 min, discard the supernatant; add 800 μL 0.05M MES (pH 5.5) buffer solution, ultrasonic mixing (large super power 4%, time 2 min).

[0081] ② Activation: Take 800 μL of the solution obtained in step (1) above, ultrasonic mixing, add 20% of the original latex volume of 10 mg / mL EDAC (freshly prepared) to activate for 0.5 h; then add 10% of the original latex volume of 10 mg / mL NHS (freshly prepared) to activate for 0.5 h; after activation, divide into 12 parts, centrifuge at 12000 rpm for 15 min, discard the supernatant, and add 4 times the original latex volume of 100 mM borax cleaning solution / 0.05M MES / 0.05M PB to each part and ultrasonic mixing.

[0082] ③ Labeling: Dilute the raw material to 1 mg / mL with 100 mM borax cleaning solution / 0.05M MES / 0.05M PB respectively, add the solution obtained in step (2) above, ultrasonic mixing, label for 1.5 h, centrifuge at 12000 rpm for 15 min, and discard the supernatant.

[0083] ④ Blocking: Add 800 μL of 1# blocking solution to the precipitate obtained in the above step, ultrasonic mixing, block for 0.5 h, centrifuge at 12000 rpm for 15 min, and discard the supernatant.

[0084] ⑤ Resolubilization: Add 1600 μL of 1# resolubilization solution to the precipitate, ultrasonic mixing.

[0085] ⑥ Gold spraying: Spray 1.0, 1.5 and 2.0 μL / cm onto K75 glass cellulose, dry at 45°C overnight.

[0086] (2) Screening of labeled antibodies and system: After diluting the above labeled antibodies 1, antibody 2 and antibody 3 with PB (0.05M PB solution), MES (0.05M MES solution) and PS (100 mM borax solution) respectively, spot the film (concentration 1.0 mg / mL), add 100-fold and 500-fold diluted recombinant PCV3 Cap protein solution to the sample wells as the quality control, and at the same time, use Ag3 special Buffer (1) as the negative control to test and screen the labeled antibodies and the diluent system. The test system is shown in Table 3, and the detection results are shown in Figure 12 Fig. 2. As can be seen from the figure, the labeled antibody 2 has bands in the 100-fold and 500-fold diluted quality control in the PB, MES and borax solution diluents, indicating that the antibody 2 can be used as the labeled antibody for testing the test strip.

[0087] Table 3 Labeling antibody and dilution screening system

[0088]

[0089] Note: PB is 0.05M PB solution, MES is 0.05M MES solution, PS is 100mM borax solution.

[0090] (3) Screening of point membrane antibody: The above latex method labeled antibodies 1, 2 and 3 are used as labeled antibodies, and 100-fold and 500-fold diluted recombinant PCV3 Cap protein is used as quality control, and Buffer (1) and Buffer (2) are used as negative controls, respectively, to screen the corresponding point membrane antibodies. The screening system is shown in Table 4, and the test results are shown in Figure 13 , in which X and Y of mAb 1 correspond to antibodies 2 and 3, respectively, X and Y of mAb 2 correspond to antibodies 1 and 3, respectively, and X and Y of mAb 3 correspond to antibodies 1 and 2, respectively. As can be seen from the figure, the labeled antibody 2 corresponds to the point membrane antibody 1, the labeled antibody 2 corresponds to the point membrane antibody 3, and the labeled antibody 3 corresponds to the point membrane antibody 2, which has good effect.

[0091] Table 4 Point membrane antibody screening system

[0092]

[0093] (4) Sensitivity test of labeled antibody: The recombinant PCV3 Cap protein was diluted 500, 2500, 10000, 400000 and 160000 times with MES and PS solutions respectively to test the sensitivity of labeled antibody 2, and Buffer (1) was used as negative control. The labeled antibody sensitivity test system is shown in Table 5, and the test results are shown in Figure 14 , in which X and Y of mAb 1 correspond to antibodies 2 and 3, respectively, X and Y of mAb 2 correspond to antibodies 1 and 3, respectively, and X and Y of mAb 3 correspond to antibodies 1 and 2, respectively. As can be seen from the figure, the labeled antibody 2 corresponds to the point membrane antibody 1, the labeled antibody 2 corresponds to the point membrane antibody 3, and the labeled antibody 3 corresponds to the point membrane antibody 2, which has good effect.

[0094] Table 5 Sensitivity test system of labeled antibody

[0095]

[0096] (5) Test of labeled antibody 2 + point membrane antibody 2 test strip serum sample: Antibody 2 is used as point membrane antibody (1.0mg / mL), and test strip is prepared with latex method labeled antibody 2, and 18 clinical pig serum samples are tested, and the results are shown in Figure 15As can be seen from the figure, all serum samples show a band, indicating that there are false positive results, indicating that the combination of labeled antibody 2 and dot membrane antibody 2 prepared test strip has false positive. Similarly, the combination of labeled antibody 1 + dot membrane antibody 1 and labeled multimer 3 + dot membrane antibody 3 prepared test strips are verified by negative pig serum and dog serum, and the results also have false positive bands, indicating that the same antibody as a dot membrane antibody and a labeled antibody prepared test strip has false positive.

[0097] (6) Different antibodies were used as labeled antibodies and dot membrane antibodies to prepare test strips: On the basis of the above, in order to screen the best combination of different dot membrane antibodies and labeled antibodies, antibodies 1, 2 and 3 were combined respectively to prepare test strips, and 3 pig sera (S15, S23 and S24), 500-fold diluted positive control (recombinant PCV3 Cap protein) and negative control (buffer) were used to test their detection effect, and the test system is shown in Table 6. The results are as follows Figure 16 As can be seen from the figure, the combination of antibody 3 and antibody 2 meets the requirements and can be used for the preparation of test strips.

[0098] Table 6 Test system of labeled antibody and dot membrane antibody combination

[0099]

[0100] (7) Product preparation: According to the test strip process screened above, PCV3 antigen test strips were prepared, as shown in Table 7, the product specification is 10 / box, which is mainly used for rapid screening of PCV3 virus and PCV3 Cap protein in pig serum / plasma, tissue samples and other samples, and the developed product provides a reagent product for rapid detection of PCV3 infection in clinic. The specific process is as follows: Figure 17

[0101] ① Preparation of nitrocellulose membrane

[0102] Preparation of coating buffer: 1g sucrose was added to 100mL PBS (0.01mol / L, pH 7.2) to prepare coating buffer, 0.22μm filter membrane was used for sterilization, and stored at 2-8℃ for standby.

[0103] ​Preparation of nitrocellulose membrane: The nitrocellulose membrane was pasted on the corresponding position of the PVC base plate, and the antibody 2 (monoclonal antibody 1D4-10-16) was diluted to 1 mg / mL with coating buffer. The line position and height of the membrane marker were adjusted, and the line was T line, which was the detection line. The PCV3 Cap protein was diluted to 1 mg / mL with coating buffer, and the line position and height of the membrane marker were adjusted. The line was C line, which was the quality control line. The distance between the two lines was 5-8 mm. The sample was dried in a 37°C oven for 15 h, sealed in an aluminum foil bag containing a desiccant, and stored at 2-30°C for standby.

[0104] ②Preparation of latex pad

[0105] Labeling: The antibody 3 (PCV3 Cap polyclonal antibody) was added to the latex microspheres at a concentration of 1 mg / mL, and rotated for 2 h. BSA was added to a final concentration of 1%, and rotated for 30 min. The mixture was centrifuged at 10,000 rpm for 30 min at 2-8°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 1 mL of storage solution (storage solution preparation: Tris 0.25 g, BSA 1 g, sucrose 2 g were weighed and added to 90 mL of double distilled water, dissolved completely, mixed, adjusted to pH 8.0, and then double distilled water was added to 100 mL) and ultrasonicated for 1 min, and stored at 2-8°C.

[0106] Preparation of latex microsphere pad: The resuspended latex microsphere labeled antibody was evenly spread on the treated latex microsphere pad, dried in a 37°C oven for 15 h, sealed in an aluminum foil bag, and stored at 2-30°C for standby.

[0107] ③Treatment of sample pad

[0108] The sample pad (300 mm x 20 mm) was soaked in blocking solution (blocking solution preparation: BSA 1 g was weighed and added to 10 mL of purified water, and dissolved completely) for 30 min, dried in a 37°C oven for 15 h, sealed in an aluminum foil bag, and stored at 2-30°C for standby.

[0109] ④Assembly of test strip

[0110] As shown in Figure 18 The sample pad, latex microsphere pad, and absorbent pad were sequentially pasted to the corresponding position of the PVC base plate with the nitrocellulose membrane already pasted, so that the latex microsphere pad and the water-absorbing pad were partially in contact with the nitrocellulose membrane, and the sample pad was partially in contact with the latex microsphere pad, to form a large plate.

[0111] ⑤Packaging

[0112] Cut the large plate into 3mm wide test paper strips with a slitter, put it into the shell, seal it with an aluminum foil bag, containing one test paper strip, one straw, and one package of desiccant, store it at 2-30℃ away from light, and the shelf life is 24 months.

[0113] Preparation of sample buffer

[0114] 1xPBS (0.01mol / L PBS pH 7.4) was filtered through a 0.22μm microporous filter to remove bacteria. Sterile packaging, 10mL / tube, 2-30℃ storage for standby.

[0115] Assembly and testing of test paper strips: Assemble the test paper strips according to the following table.

[0116] Components Quantity Specification Test strips 50 bags 1 strip / bag Sample buffer 1 tube 10 mL / tube Instructions 1 copy /

[0117] Use and determination of test paper strips

[0118] 1 Sample processing

[0119] 1.1 Processing method of tissue sample: Grind the tissue sample with PBS at a ratio of 1:1, then centrifuge to obtain the supernatant for direct detection.

[0120] 1.2 Processing method of blood test sample: Directly used for detection.

[0121] 2 Operation steps: Take an appropriate amount of test sample (30-40μL, 1 drop of dropper), slowly drop it into the sample well, then add 2 drops of sample buffer. After adding the sample, place the test paper strip on the table, and observe the results within 5-10min.

[0122] 3 Determine (as shown in Figure 19

[0123] 3.1 Two bands appear on the test paper strip (T: detection line, C: quality control line), judged as positive.

[0124] 3.2 Only one blue band appears on the test paper strip (C: quality control line), judged as negative.

[0125] 3.3 No band appears at the quality control line of the test paper strip, judged as invalid.

[0126] (8) Application of the product

[0127] ① Use the above prepared product to test PCV2 virus liquid (containing 10 6.0 TCID 50 / mL), PCV2 Cap protein, PEDV virus liquid (containing 10 7.0 TCID 50 / mL), PRV (containing 10 8.0 TCID​50 / mL) were detected, and the results showed that the test strip detection was negative, indicating that the test strip had good specificity.

[0128] ②The product prepared above was used to detect PCV3 Cap protein of different concentrations, and the results showed that the detection limit of the test strip for PCV3 Cap protein was 10000-fold dilution, at this time the concentration of PCV3 Cap protein was 80 ng / mL, indicating that the test strip had good sensitivity.

[0129] ③Detection of clinical samples: 14 pig clinical blood samples were collected from a farm, and the porcine circovirus type 3 antigen test strip (latex method) developed above was used to detect according to the product instruction, and the detection results were as shown in Figure 20 The results showed that the 4th sample of the 14 clinical serum samples showed a positive band (T line), which was consistent with the real-time quantitative PCR results, indicating that it was positive for porcine circovirus type 3 antigen.

[0130] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A monoclonal antibody against porcine circovirus type 3 Cap protein, characterized in that: The anti-PCV3 Cap protein monoclonal antibody is monoclonal antibody 1D4-10-16, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1D4-10-16 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

2. A porcine circovirus type 3 antigen detection test strip, characterized in that: The test strip includes a test strip and a matching sample diluent, wherein the test strip includes a PVC base plate, on which a sample pad, a latex microsphere pad, a nitrocellulose membrane, and absorbent paper are fixed in sequence; the latex microsphere pad is a latex microsphere labeled with an effective amount of a polyclonal antibody against the PCV3 Cap protein; a test line and a quality control line are drawn on the surface of the nitrocellulose membrane, wherein the test line is an effective amount of the monoclonal antibody 1D4-10-16 according to claim 1, and the quality control line is an effective amount of the PCV3 Cap protein.

3. The test strip according to claim 2, characterized in that The concentration of the PCV3 Cap protein polyclonal antibody on the latex microsphere pad when labeling the latex microspheres is 1 mg / mL.

4. The test strip according to claim 2, characterized in that The PCV3 Cap protein polyclonal antibody is a rabbit anti-PCV3 Cap protein polyclonal antibody, and its ELISA titer is 1:80,000.

5. The test strip according to claim 2, characterized in that The concentration of monoclonal antibody 1D4-10-16 on the test line was 1 mg / mL.

6. The test strip according to claim 2, characterized in that The concentration of PCV3 Cap protein on the quality control line is 1 mg / mL.

7. The test strip according to claim 2, characterized in that The amino acid sequence of the PCV3 Cap protein is shown in SEQ ID NO.

1.

8. The test strip according to claim 2, characterized in that The codon-optimized nucleotide sequence of the PCV3 Cap protein is shown in SEQ ID NO.

2.

9. Use of the monoclonal antibody 1D4-10-16 according to claim 1 in preparing a detection reagent for PCV3 or PCV3 Cap protein.

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