CN116183913A - Antibody detection kit for sai card virus A and application thereof - Google Patents
Antibody detection kit for sai card virus A and application thereof Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention provides an antibody detection kit of a Sein card virus A and application thereof, and relates to the field of biotechnology detection. The antibody detection kit of the sai card virus A comprises an ELISA plate coated with a recombinant protein SVA-VP1-opti and a detection antibody; the amino acid sequence of the recombinant protein SVA-VP1-opti is shown as SEQ ID NO. 2; the detection antibody is a monoclonal antibody secreted by a hybridoma cell strain SVA-VP1-3 with the preservation number of CGMCC No. 45146. The kit is used for detecting the antibodies of the Sein card virus A, and has higher specificity and sensitivity.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to an antibody detection kit for a Sein card virus A and application thereof.
Background
The sai virus A (SenecavirusA, SVA) is a pathogenic pathogen causing swine primary vesicular disease (PIDV), and the clinical manifestations of swine infection are very similar to those of swine important vesicular diseases such as foot-and-mouth disease (FMD), swine Vesicular Disease (SVD), swine Vesicular Stomatitis (VS) and the like, and mainly comprise symptoms such as anorexia, lameness, fever, somnolence and the like. Pigs at all ages are susceptible, and the highest infection mortality rate of piglets at 1-3 days is 30-70%. Since 2018 SVA is transferred into Guangdong of China, a plurality of provinces are rapidly rolled, and the national spreading trend is shown. The disease brings difficulty to clinical workers in accurately reporting the swine vesicular disease, and huge waste of manpower and material resources is caused. In order to reduce the economic loss of pig industry in China, the prevention and control of SVA epidemic situation by means of laboratory means is a necessary path.
At present, the specificity and sensitivity of a kit for detecting SVA antibodies are not ideal.
Disclosure of Invention
The invention aims to provide a competition ELISA detection method for detecting an antibody of a Session initiation virus A, which aims to solve the problem that the specificity and sensitivity of the detection method of the antibody of the Session initiation virus A are not ideal.
The invention adopts the following technical scheme:
an antibody detection kit of a sai card virus A comprises an ELISA plate coated with a recombinant protein SVA-VP1-opti and a detection antibody; the amino acid sequence of the recombinant protein SVA-VP1-opti is shown as SEQ ID NO. 2; the detection antibody is a monoclonal antibody secreted by a hybridoma cell strain SVA-VP1-3 with the preservation number of CGMCC No. 45146.
In the invention, the recombinant protein SVA-VP1-opti is obtained by recombining the coding gene of the recombinant protein SVA-VP1-opti into an expression vector, transforming into a host cell and finally inducing expression.
In the invention, the coding gene of the recombinant protein SVA-VP1-opti is shown as SEQ ID NO.1, the expression vector is pGEX-6P-1, and the host cell is Escherichia coli.
In the invention, the concentration of the recombinant protein SVA-VP1-opti coating in the ELISA plate is 3-5 mug/mL.
In the present invention, the concentration of the detection antibody is 0.4 to 0.6. Mu.g/mL.
In the invention, the kit also comprises goat anti-mouse IgG antibody marked by horseradish peroxidase, a washing solution, positive control serum, negative control serum, a chromogenic solution and a stop solution.
In the invention, the positive control serum is obtained by immunizing rabbits with a recombinant protein SVA-VP1-opti; the washing liquid is PBST buffer solution; the stop solution is a 2M aqueous sulfuric acid solution.
The invention also provides application of the kit in detecting the antibody of the Sein card virus A for non-diagnosis.
The beneficial effects of the invention are as follows: the anti-SVA monoclonal antibody provided by the invention has high titer and high specificity. The kit is used for detecting antibodies of the Sein card virus A, has high specificity and high sensitivity, and can furthest reduce false positive rate, thereby accurately diagnosing whether animals are infected with SVA, further reducing erroneous judgment on other swine vesicular diseases, achieving accurate prevention and control on SVA epidemic situation, and reducing economic loss of pig industry. Compared with the existing SVA antibody detection method, the method has higher accuracy and lower detection cost.
Drawings
FIG. 1 is a schematic diagram of the structures of recombinant expression vectors pET-32a-SVA-VP1-opti and pGEX-6P-1-SVA-VP1-opti, wherein SVA-VP1 refers to SVA-VP1-opti fragment.
FIG. 2 shows the double restriction identification of recombinant expression vectors pET-32a-SVA-VP1-opti and pGEX-6P-1-SVA-VP1-opti by EcoR I and Not I, lane 1: vector pET-32a double cleavage product, lane 2: pET-32a-SVA-VP1-opti double cleavage product, lane 3: vector pGEX-6P-1 double cleavage, lane 4: pGEX-6P-1-SVA-VP1-opti double cleavage product.
FIG. 3 is an SDS-PAGE electrophoresis of supernatant and pellet of lysates of each recombinant strain after IPTG induction, M: protein molecular mass standard; lane 1, recombinant BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti lysate supernatant; 2, precipitation of recombinant BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti cleavage product; 3 recombinant BL21 (DE 3)/pGEX-6P-1 lysate supernatant; 4, precipitation of recombinant BL21 (DE 3)/pGEX-6P-1 lysate; recombinant BL21 (DE 3)/pET-32 a-SVA-VP1-opti lysate supernatant; precipitating recombinant BL21 (DE 3)/pET-32 a-SVA-VP1-opti lysate; recombinant BL21 (DE 3)/pET-32 a lysate supernatant; 8, precipitation of recombinant BL21 (DE 3)/pET-32 a lysate; arrows indicate the target protein.
FIG. 4 is a SDS-PAGE identification of the effect of induction expression time, IPTG concentration and induction expression temperature on GST-SVA-VP1-opti protein expression, respectively, A: SDS-PAGE identification of the effect of induction time on GST-SVA-VP1-opti protein expression, wherein M: protein molecular mass standard; lanes 0-8 are recombinant bacterium lysate supernatants after induced expression for 0, 1, 2, 3, 4, 5, 6, 7, 8h, respectively, and the arrow indicates the target protein; b: SDS-PAGE identification of the effect of inducer concentration on GST-SVA-VP1-opti protein expression, wherein M: protein molecular mass standard, lanes 1-6, are respectively 0, 0.2, 0.4, 0.6, 0.8, 1.0mmol/LIPTG concentration induced recombinant bacterium lysate supernatant, arrow indicates target protein; c: SDS-PAGE identification of the effect of induction temperature on GST-SVA-VP1-opti protein expression, wherein M: protein molecular mass standard; lanes 1-2 are the supernatants of recombinant bacterium lysates at 37℃and 28℃induction expression temperature, respectively; arrows indicate the target protein.
FIG. 5 is an electrophoretogram of the purified recombinant protein His-SVA-VP1-opti, lane 1: his-SVA-VP1-opti protein pre-purification stock (recombinant bacterium lysate supernatant); lane 2: liquid flowing out during protein loading; lane 3: washing impurity liquid in the purification process; lane 4: the purified recombinant protein His-SVA-VP1-opti; arrows indicate the target protein.
FIG. 6 is an electrophoretic pattern of purified recombinant protein GST-SVA-VP1-opti, lane 1: GST-SVA-VP1-opti protein purification pre-stock (recombinant bacterium lysate supernatant); lane 2: liquid flowing out during protein loading; lane 3: washing impurity liquid in the purification process; lane 4: purified recombinant protein GST-SVA-VP1-opti; arrows indicate the target protein.
FIG. 7 is a Western-blot specificity-identifying of the monoclonal antibodies SVA-VP1-3, lane 1: cell lysates collected from SVA infected cells; lane 2: cell lysates collected from PEDV infected cells; lane 3: cell lysates collected from PCV2 infected cells; lane 4: cell lysates collected from PDCoV infected cells; lane 5: and preparing a sample for the foot-and-mouth disease inactivated vaccine.
Detailed Description
The following examples are provided for a better understanding of the present invention and are in no way intended to limit the scope of the invention. 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 invention belongs. The following examples do not address the specific conditions, and generally employ conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or according to the methods recommended by the manufacturer.
EXAMPLE 1 construction of genetically engineered recombinant E.coli expressing SVA-VP1-opti protein
Acquisition of SVA-VP1-opti protein Gene: through analyzing and screening gene sequences of the sai virus, a plurality of antigen gene fragments are designed, and only SVA-VP1-opti protein (the sequence is shown as SEQ ID NO. 1) coded by the antigen gene fragments SVA-VP1-opti (the sequence is shown as SEQ ID NO. 2) and monoclonal antibodies prepared by the same are found, so that the effect of high sensitivity and high specificity can be achieved when the monoclonal antibodies are used for detecting the sai virus A antibodies.
EcoR I and Not I restriction enzyme recognition sites are respectively added at two ends of an antigen gene fragment SVA-VP1-opti, and the synthesis is carried out by the biotechnology company.
Construction of a prokaryotic expression vector of the SVA-VP1-opti gene: the SVA-VP1-opti and the prokaryotic expression vector pET-32a added with EcoR I and Not I restriction enzyme recognition sites at two ends are respectively subjected to double digestion by using restriction enzymes EcoR I and Not I, and the synthesized gene fragment SVA-VP1-opti and a vector framework are connected to obtain a recombinant expression vector pET-32a-SVA-VP1-opti (the structural schematic diagram is shown in figure 1). After cleavage of the recombinant expression vector pET-32a-SVA-VP1-opti by EcoR I and Not I, a vector band of 5.9kb and a target band of 291bp were observed by agarose gel electrophoresis, whereas only a vector band of 5.9kb was observed after cleavage of the pET-32a empty vector (see FIG. 2B). The recombinant expression vector pET-32a-SVA-VP1-opti is transformed into BL21 (DE 3) competent cells, and the obtained recombinant bacterium is named BL21 (DE 3)/pET-32 a-SVA-VP1-opti. The recombinant BL21 (DE 3)/pET-32 a-SVA-VP1-opti expressed protein consists of SVA-VP1-opti protein and His tag connected with the N end of the SVA-VP1-opti protein, and is marked as His-SVA-VP1-opti.
The SVA-VP1-opti and the vector pGEX-6P-1, to which EcoR I and Not I restriction enzyme recognition sites are added at both ends, are digested with restriction enzymes EcoR I and Not I, respectively, and the synthesized gene fragment and the vector backbone are ligated to obtain a recombinant expression vector pGEX-6P-1-SVA-VP1-opti (the schematic structure is shown in FIG. 1). After cleavage of the recombinant expression vector pGEX-6P-1-SVA-VP1-opti by EcoR I and Not I, a vector band of about 5.0kb and a target band of 291bp were observed by agarose gel electrophoresis, whereas only a vector band of 5.0kb was observed after cleavage of the pGEX-6P-1 empty vector (see FIG. 2). The recombinant expression vector pGEX-6P-1-SVA-VP1-opti is transformed into BL21 (DE 3) competent cells, and the obtained recombinant bacterium is named BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti. The BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti expressed protein consists of SVA-VP1-opti protein and GST tag connected with N end, and is marked as GST-SVA-VP1-opti protein.
In addition, empty vector pET-32a, pGEX-6P-1 were transformed into BL21 (DE 3) competent cells, respectively, to obtain recombinant bacteria BL21 (DE 3)/pET-32 a and BL21 (DE 3)/pGEX-6P-1.
Example 2: prokaryotic expression and purification of recombinant protein SVA-VP1-opti
Recombinant bacteria BL21 (DE 3)/pET-32 a-SVA-VP1-opti and BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti cultured to logarithmic phase are respectively inoculated into LB medium added with 100 mu g/mL ampicillin (Amp) according to the ratio (v/v) of 1:100 of inoculum size, and are subjected to shaking culture at 220r/min until bacterial liquid OD 600nm 0.5 was added with IPTG at a final concentration of 1mmol/L, cultured at 37℃for 5 hours for induction expression, bacteria were collected, and after washing 3 times with PBS buffer (pH=7.2), the bacterial pellet was resuspended, and then sonicated at 40Hz at 4s intervals, and the supernatant and pellet of the lysate were separated by centrifugation, and analyzed by SDS-PAGE, respectively.
In addition, recombinant bacteria BL21 (DE 3)/pET-32 a and BL21 (DE 3)/pGEX-6P-1-VP 1-opti were induced and expressed in the same manner as BL21 (DE 3)/pET-32 a-SVA-VP1-opti and BL21 (DE 3)/pGEX-6P-opti, and the expressed recombinant bacteria were sonicated, and the supernatant and pellet of the lysate were separated by centrifugation and analyzed by SDS-PAGE electrophoresis, respectively.
The results are shown in FIG. 3. A specific band appears in the supernatant of the recombinant BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti lysate at 35kDa, which indicates that GST-SVA-VP1-opti protein is expressed in a soluble way, and the supernatant and the sediment of the recombinant BL21 (DE 3)/pET-32 a-SVA-VP1-opti lysate both contain the specific band with the size of 30kDa, and the supernatant band is thicker than the sediment, which indicates that His-SVA-VP1-opti is mainly expressed in a soluble way.
Inoculating BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti bacterial liquid into LB liquid medium containing 100 μg/mL Amp, culturing to bacterial liquid OD 600nm The value was 0.5, and the induction time, the inducer concentration and the induction temperature were optimized, respectively, as follows:
(1) Optimization of induction time: when the bacterial liquid amounts are equal, the OD 600nm At a value of 0.5, IPTG with a final concentration of 1mmol/L was added, and after induction of expression at 37℃for 0, 1, 2, 3, 4, 5, 6, 7, 8 hours, equal amounts of bacterial liquid were taken, washed 3 times with PBS (pH=7.2) and then the bacterial pellet was resuspended, and then sonicated, centrifuged to obtain the supernatant of the lysate, which was analyzed by SDS-PAGE. As can be seen from FIG. 4A, the expression level of the target protein was highest at an induction time of 7 hours.
(2) Concentration of inducerIs optimized by: when the bacterial liquid amounts are equal, the bacterial liquid OD 600nm At a value of 0.5, IPTG inducer with final concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0mmol/L was added, respectively, and after induction of expression at 37 ℃ for 7 hours, equal amounts of bacterial liquid were taken, washed 3 times with PBS (ph=7.2), and then the bacterial pellet was resuspended, and then sonicated, centrifuged, and the supernatant of the lysate was taken for SDS-PAGE electrophoresis analysis. As is clear from FIG. 4B, the recombinant protein expression level was highest when the inducer concentration was 1.0 mmol/L.
(3) Optimization of induction temperature: when the bacterial liquid amounts are equal, the OD 600nm When the value is 0.5, adding IPTG inducer with the concentration of 1.0mmol/L, respectively placing the mixture in a shaking table at 28 ℃ and 37 ℃ to induce expression for 7 hours, taking an equivalent bacterial liquid, washing the bacterial liquid for 3 times by PBS (pH=7.2), re-suspending bacterial precipitation, then carrying out ultrasonic crushing and centrifugation, taking the supernatant of the lysate, and carrying out SDS-PAGE electrophoresis analysis. As can be seen from FIG. 4C, the protein expression level was high at 37 ℃.
Because GST tag is fused in recombinant protein GST-SVA-VP1-opti expressed by recombinant bacterium BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti, the supernatant of the cleavage product of recombinant bacterium BL21 (DE 3)/pGEX-6P-1-SVA-VP 1-opti is purified by utilizing GST tag protein purification pre-packed column; his tag is fused in recombinant protein His-SVA-VP1-opti expressed by recombinant bacterium BL21 (DE 3)/pET-32 a-SVA-VP1-opti, so that the supernatant of the cleavage product of the recombinant bacterium BL21 (DE 3)/pET-32 a-SVA-VP1-opti is purified by utilizing a nickel ion affinity chromatography column. Protein effluent (effluent liquid when protein is loaded), protein washing impurity liquid (impurity protein) and protein eluent (purified recombinant protein) in the purification process are collected, and then SDS-PAGE electrophoresis analysis is carried out together with a stock solution (recombinant bacterium lysate supernatant) before protein purification. His-SVA-VP1-opti protein purification results are shown in FIG. 5, and lane 4 shows that a single target protein band exists at 30kDa, which shows that the purification effect is better (the purity is more than 90 percent), and the purified recombinant protein His-SVA-VP1-opti is successfully obtained. The purification result of GST-SVA-VP1-opti protein is shown in FIG. 6, and lane 4 has a single target protein band at 35kDa, which shows that the purification effect is better (the purity is more than 90 percent), and the purified recombinant protein GST-SVA-VP1-opti is successfully obtained. Finally, 500mL of fermentation broth can obtain 34mg of purified recombinant protein GST-SVA-VP1-opti and 60mg of purified recombinant protein His-SVA-VP1-opti.
Example 3: preparation of monoclonal antibody by using recombinant protein His-SVA-VP1-opti as antigen
Mixing the purified recombinant protein His-SVA-VP1-opti solution (1 mg/mL) obtained in the example 2 and Freund's complete adjuvant according to a volume ratio of 1:1, and uniformly emulsifying, wherein the solution is used as an immunogen to immunize a 6-week-old BALB/c female mouse by adopting a subcutaneous multipoint injection mode, and the initial immunization dose is 70 mug (protein)/mouse; mixing the purified recombinant protein His-SVA-VP1-opti solution (1 mg/mL) and Freund's incomplete adjuvant according to a volume ratio of 1:1, emulsifying uniformly, and performing secondary immunization on the mice at intervals of 15 days after primary immunization, wherein the immunization dose is 70 mug (protein)/mouse; and carrying out third immunization 15 days after the second immunization, wherein the immunization dose and the method are the same as the second immunization. After 15d of the third immunization, serum is collected from blood of ocular venous plexus of the mice, antibody titer is detected by an indirect ELISA method, the mice with titer more than 1:10000 are selected for boosting, immunogen is recombinant protein His-SVA-VP1-opti solution (1 mg/mL), the intraperitoneal injection mode is adopted, the immune dose is 70 mug (protein)/animal, after 3 days of boosting, feeder cells (mouse peritoneal macrophages) are prepared in advance one day, and the feeder cells are spread in 96-well plates. The next day, SP2/0 cells (mouse myeloma cells) and spleen cells of mice with qualified antibody titers are subjected to cell fusion, the fused cells are paved in feeder cell holes, and then the fused cells are screened by an indirect ELISA method to obtain a hybridoma cell strain SVA-VP1-3 secreting monoclonal antibodies against SVAVP1 proteins.
Injecting the hybridoma cell strain SVA-VP1-3 into the abdominal cavity of a paraffin sensitized 8-week-old BALB/c female mouse to prepare ascites, and purifying the ascites by Protein G to obtain the monoclonal antibody SVA-VP1-3 secreted by the hybridoma cell strain.
Wherein the indirect ELISA method is used for detecting the serum antibody titer of mice, and comprises the following specific steps: by means of a coating solution (containing 0.03mol/LNaHCO 3 And 0.015mol/LNa 2 CO 3 Ph=9.6) the purified GST-SVA-VP1-opti protein (prepared in example 2) was diluted to 2 μg/mL, the elisa plate was coated at 100 μl/well and incubated overnight in a refrigerator at 4 ℃; discarding the bagAdding 200 mu LTBST (50 mL of Tris-HCL buffer solution with the concentration of 1M, pH 7.5.5, 8g of NaCl,0.2g of KCl and 0.5mL of Tween-20, mixing, adopting distilled water to fix the volume to 1L) into each hole, washing for 3 times, washing for 5min each time, beating on absorbent paper, adding 100 mu L of 5% skimmed milk powder solution prepared by taking TBST as a solvent into each hole, and sealing for 2h at 37 ℃; the blocking solution was discarded, washed 3 times with TBST and the serum to be tested was diluted in a gradient with TBST as follows: 1:200, 1:400 …, 1:25600, 151200, 100 μl per well, and incubating in 37 ℃ incubator for 1h; the plate was discarded, washed 3 times with TBST and patted dry, 100. Mu.L of HRP-goat anti-mouse IgG (purchased from Warbio, product number M21003S) diluted 1:5000 with TBST was added to each well and incubated for 1h at 37℃in an incubator; TBST was washed 3 times and patted dry, and a single-component TMB developing solution (purchased from Solarbio, cat. Number PR 1200) was added, 100. Mu.L/well, developed for 10min in the dark; mu.L of stop solution (2M H) was added to each well 2 SO 4 Aqueous solution), OD was measured by using an ELISA reader 450nm Values, titers were calculated according to conventional methods.
The specificity of the monoclonal antibody SVA-VP1-3 secreted by the hybridoma cell line SVA-VP1-3 was identified by Western-Blot. BHK-21 cells were inoculated with Seika Virus A (SVA), vero cells were inoculated with Porcine Epidemic Diarrhea Virus (PEDV), PK-15 cells were inoculated with porcine circovirus (PCV 2), and ST cells were inoculated with porcine delta coronavirus (PDCoV), respectively. When obvious lesions appear on cells but the cells do not fall off in a large area, removing culture medium supernatant, adding sterile PBS buffer solution for washing for 3 times, adding cell lysate, placing a cell plate on an oscillation instrument for 5min for lysing, collecting lysate, heating a metal bath at 95 ℃ for 10min to prepare a sample, loading the sample together with the sample prepared by the foot-and-mouth disease inactivated vaccine, performing SDS-PAGE electrophoresis, transferring proteins onto a polyvinylidene fluoride (PVDF) membrane, and sealing the sample for 1h at 37 ℃ by adopting 5% skimmed milk powder solution prepared by taking TBST as a solvent; adding a monoclonal antibody SVA-VP1-3 solution, and incubating overnight at 4 ℃; TBST was washed three times, and HRP-goat anti-mouse secondary antibody (purchased from Warbio, product number M21003S) diluted in a volume ratio of 1:5000 was added and incubated for 1h at room temperature; and (3) performing color development and imaging by using ECL luminous solution and a WB chemiluminescent imager, and identifying the reaction specificity of the monoclonal antibody and SVA. As shown in FIG. 7, monoclonal antibody SVA-VP1-3 reacts specifically with only lysates of SVA-vaccinated cells, indicating that monoclonal antibodies secreted by hybridoma cell lines SVA-VP1-3 can bind specifically to SVA.
The preservation information of hybridoma cell lines SVA-VP1-3 is as follows:
preservation unit: china general microbiological culture Collection center (CGMCC);
address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing;
reference biological materials (strain): SVA-VP1-3;
suggested class naming: a monoclonal antibody hybridoma cell strain secreting anti-sai virus AVP1 protein;
preservation number: CGMCC No.45146;
preservation date: 2022, 4 and 1.
Example 4: establishment of test kit and test method for competition ELISA of sai card virus A antibody
1. Composition of test kit for competition ELISA of sai card virus A antibody
The saint card virus a antibody competition ELISA detection kit (abbreviated as the kit of the present invention) comprises: washing solution, enzyme label plate coated with recombinant protein GST-SVA-VP1-opti, antibody, positive control, negative control, single-component TMB chromogenic solution and stop solution.
(1) Washing liquid
The washing liquid is PBST, and the preparation method is as follows: weighing 0.2g KH 2 PO 4 2.9g of Na 2 HPO 4 ·12H 2 O,8g of NaCl,0.2g of KCl and 0.5mL of Tween-20 were dissolved in distilled water and the volume was set to 1L.
(2) ELISA plate coated with recombinant protein GST-SVA-VP1-opti
Preparing a coating liquid: 0.795g of Na was weighed out 2 CO 3 And 1.465g NaHCO 3 Dissolve in ultrapure water and fix volume to 500mL, adjust ph=7.0.
The recombinant protein GST-SVA-VP1-opti solution with the concentration of 4 mug/mL is prepared by adopting coating liquid, 100 mug of each hole is added with an ELISA plate, and the mixture is coated for 3 hours at 37 ℃. Taking out the ELISA plate, washing 3 times with washing liquid, washing for 5min each time with 200 mu L of each hole, and drying on clean non-fiber absorbent paper; 100 mu L of a 5% (mass percent concentration) Bovine Serum Albumin (BSA) solution prepared by taking TBST (50 mL of Tris-HCl buffer solution with the concentration of 1M, pH 7.5.5, 8g of NaCl,0.2g of KCl and 0.5mL of Tween-20 as a solvent and adopting distilled water to fix the volume to 1L) as a solvent is added into each hole, the mixture is sealed for 2 hours at 37 ℃, the mixture is washed 3 times with a washing solution, 200 mu L of each hole is washed for 5 minutes each time, and the mixture is dried by beating on clean non-fibrous absorbent paper, so that the ELISA plate coated with the recombinant protein GST-SVA-VP1-opti is obtained.
(3) Antibodies to
Detection of antibody solution: the monoclonal antibody SVA-VP1-3 secreted by the hybridoma cell line SVA-VP1-3 of example 3 was present at a concentration of 0.5. Mu.g/mL.
And (2) secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG antibody (abbreviated HRP-goat anti-mouse IgG), available from warbrio under product number M21003S.
(4) Positive control serum and negative control serum
Positive control serum: the positive control serum is rabbit polyclonal antibody serum, and the preparation method is as follows: uniformly mixing the purified recombinant protein GST-SVA-VP1-opti solution (with the concentration of 1 mg/mL) obtained in the example 2 with Freund's complete adjuvant according to the volume ratio of 1:1, emulsifying, and immunizing a female New Zealand large ear rabbit with the age of 16 weeks by adopting a subcutaneous multipoint injection mode, wherein the immune dose is 0.6mg of recombinant protein per animal; after 2 weeks, uniformly mixing and emulsifying the purified recombinant protein GST-SVA-VP1-opti solution (the concentration is 1 mg/mL) and Freund's incomplete adjuvant according to the volume ratio of 1:1, and performing secondary immunization (0.6 mg recombinant protein/single); the third and fourth immunizations were performed in the same manner as the second immunization, with two weeks between adjacent immunizations. After one week of the fourth immunization, the rabbits were heart-collected, the serum of the rabbits was isolated, and the titer of the serum was 1:12800 by the indirect ELISA method established in example 3, and the serum was used as a positive control serum.
Negative control serum: in the preparation of positive control serum, a 16-week-old female New Zealand large-ear rabbit was subjected to collection of 5mL of blood from the vein at the front of immunization, and the serum was isolated as a negative control serum.
(5) Single-component TMB color development liquid
One-component TMB color development was purchased from Solarbio under the designation PR1200.
(6) Stop solution
The stop solution was an aqueous sulfuric acid solution having a concentration of 2M.
2. Establishment of method for detecting competition ELISA of antibodies of Senecio card virus A
The method for detecting the serum antibody of the Seecard virus A by adopting the kit comprises the following steps: and diluting the serum to be detected by adopting a washing solution according to the dilution of 1:20, and mixing the serum diluent with a detection antibody solution according to the volume ratio of 1:1 to obtain a mixed solution of the serum to be detected and the detection antibody. Adding 100 mu L of mixed solution of serum to be detected and detection antibody into each detection hole (marked as S) of an ELISA plate coated with recombinant protein GST-SVA-VP1-opti, carrying out shaking incubation at 37 ℃ for 5min, discarding liquid in the plate, washing 3 times with washing liquid for 5min each time, and drying on water-absorbing paper without fiber; adding 100 mu L of HRP-goat anti-mouse IgG diluted by adopting a washing solution according to a dilution ratio of 1:5000 into each hole, and placing the ELISA plate in a 37 ℃ incubator for incubation for 1h; discarding the liquid in the plate, washing 3 times by using the washing liquid, washing for 5min each time, and beating to dry on the water-absorbing paper without fiber; adding 100 mu L of single-component TMB color development liquid into each hole, and developing color for 10min in a 37 ℃ incubator in a dark place; adding stop solution in dark place to stop chromogenic reaction, adding enzyme-labeled instrument to determine OD at 50 μl each hole 450nm Is a value of (2). Simultaneously setting two positive control holes (marked as PC) and two negative control holes (marked as NC), wherein mixed liquid of positive control serum and detection antibody (obtained by diluting positive control serum with washing liquid according to the dilution of 1:20 and mixing the positive control serum with detection antibody solution according to the volume ratio of 1:1) is used for replacing mixed liquid of serum to be detected and detection antibody in the positive control holes, and other serum to be detected are used for detecting holes; the mixed solution of the negative control serum and the detection antibody (obtained by mixing the negative control serum and the detection antibody solution according to the volume ratio of 1:1) is used for replacing the mixed solution of the serum to be detected and the detection antibody in the negative control wells, and other serum to be detected are used for detecting the wells.
From the measurement results, the serum inhibition rate (I%) was calculated. Serum inhibitionThe formula for calculating the production rate is as follows: i% = (OD NC -OD S )/(OD NC -OD PC ) X 100%, where OD NC For negative control well detection value, OD S For the detection value of the serum detection hole to be detected, OD PC Positive control wells were tested.
20 parts of porcine serum samples that were tested as SVA negative using a commercially available SVA antibody test kit (purchased from Biovet under the trade designation TRM-567) were tested using the above-described kit of the invention and the serum inhibition was calculated. According to the statistical method, the formula is as follows:
analyzing the detection result, and (E) performing->
The average value of the negative serum inhibition rate was determined, and SD was the standard deviation of the negative serum inhibition rate. According to the results (Table 1),>
sd= 4.797%, so the negative-positive threshold is +.>
In order to judge the serum to be detected more clearly, when the inhibition rate (I%) of the serum to be detected is set to be more than or equal to 40%, the serum to be detected is judged to be positive, and the inhibition rate (I%) of the serum to be detected is set to be more than or equal to 40%<At 40%, the serum to be tested is judged as negative.
Table 120 results of detection of pig negative serum
Example 5: performance of the Seeca virus A antibody competitive ELISA detection kit
Pig serum samples were tested using a commercially available SVA antibody test kit (purchased from Biovet, cat# TRM-567) to find 20 SVA pig positive serum and 20 SVA pig negative serum.
The kit provided by the invention is used for detecting 20 SVA pig positive serum and 20 SVA pig negative serum with known backgrounds. When the inhibition rate (I%) of the serum to be detected is more than or equal to 40%, the serum to be detected is judged to be positive, and when the inhibition rate (I%) of the serum to be detected is less than 40%, the serum to be detected is judged to be negative. The detection results of the kit are shown in Table 2, and the yin-yang coincidence rate of the kit and a commercial SVA antibody detection kit (purchased from Biovet, product number: TRM-567) is 100%, so that the specificity and accuracy of the method are better, and the kit has wide application prospect.
TABLE 2 negative and positive serum test results
Example 6
The kit is used for respectively detecting PEDV positive porcine serum, PCV2 positive porcine serum and PDCoV positive porcine serum, SVA porcine positive serum is used as a positive control, SVA porcine negative serum is used as a negative control, and the serum inhibition rate (I%) is calculated according to the detection result. As shown in Table 3, the test results of the other serum except SVA pig positive serum are all negative, which indicates that the kit has good specificity.
TABLE 3 specificity test results
| PEDV-positive serum | PCV2 positive serum | PDCoV positive serum | Positive control | Negative control | |
| I(％) | 13.17 | 12.38 | 17.64 | 56.67 | 12.14 |
The kit of the invention and a commercial SVA antibody detection kit (purchased from Biovet, cat# TRM-567, abbreviated as commercial kit) were used to detect SVA positive porcine serum diluted by a factor of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, respectively. As shown in Table 4, the serum inhibition rate was 42.32% when the positive serum was diluted 1:160 and 33.81% when the positive serum was diluted 1:320, which was below the threshold for yin-yang (40%), indicating that the lowest assay dilution of the kit of the invention was 1:160. The commercial kit has a critical value of 50% for yin and yang and a serum inhibition of 51.3% when positive serum is diluted 1:40. The serum inhibition rate was 45.15% when diluted 1:80, at which point the value was below the threshold for yin-yang (50%), indicating a minimum assay dilution of 1:40 for the commercial kit.
TABLE 4 sensitivity test results
SEQ ID NO.1 sequence is as follows:
gctgagactggggttattgaggcaggtaacactgacaccgatttctctggtgaactggcggctcctggctctaaccatactaatgtcaaattcctgtttgaccgatctcgactactgaatgtaattaaggtactggagaaggacgccgtcttcccccgtcctttccccacagcaacaggtgcacagcaggacgatggttacttttgtcttctgacaccccgcccaacagtcgcttcccggcccgccactcgtttcggcctgtacgtcaacccatctgacagtggcgttctc
SEQ ID NO.2 sequence is as follows:
AETGVIEAGNTDTDFSGELAAPGSNHTNVKFLFDRSRLLNVIKVLEKUVFPRPFPTATGAQQDDGYFCLLTPRPTVASRPATRFGLYVNPSDSGVL
Claims (8)
1. an antibody detection kit of a sai card virus A is characterized by comprising an ELISA plate coated with a recombinant protein SVA-VP1-opti and a detection antibody; the amino acid sequence of the recombinant protein SVA-VP1-opti is shown as SEQ ID NO. 2; the detection antibody is a monoclonal antibody secreted by a hybridoma cell strain SVA-VP1-3 with the preservation number of CGMCC No. 45146.
2. The kit according to claim 1, wherein the recombinant protein SVA-VP1-opti is obtained by recombining the gene encoding the recombinant protein SVA-VP1-opti into an expression vector, transforming into a host cell, and finally inducing expression.
3. The kit according to claim 2, wherein the coding gene of the recombinant protein SVA-VP1-opti is shown as SEQ ID NO.1, the expression vector is pGEX-6P-1, and the host cell is Escherichia coli.
4. The kit according to claim 3, wherein the concentration of the recombinant protein SVA-VP1-opti coating in the ELISA plate is 3-5 μg/mL.
5. The kit according to claim 4, wherein the concentration of the detection antibody is 0.4-0.6. Mu.g/mL.
6. The kit of claim 4, further comprising horseradish peroxidase-labeled goat anti-mouse IgG antibodies, wash solutions, positive control serum, negative control serum, color development solutions, and stop solutions.
7. The kit according to claim 6, wherein the positive control serum is obtained by immunizing a rabbit with the recombinant protein SVA-VP1-opti; the washing liquid is PBST buffer solution; the stop solution is a 2M aqueous sulfuric acid solution.
8. Use of the kit of claim 1 for detecting antibodies to sai card virus a for non-diagnostic purposes.
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| CN117487006B (en) * | 2023-12-29 | 2024-04-12 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody for resisting A-type sai virus, epitope and application |
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