CN113336845A - Porcine single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit - Google Patents

Porcine single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit Download PDF

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CN113336845A
CN113336845A CN202110598030.7A CN202110598030A CN113336845A CN 113336845 A CN113336845 A CN 113336845A CN 202110598030 A CN202110598030 A CN 202110598030A CN 113336845 A CN113336845 A CN 113336845A
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张婧
卢曾军
李坤
曹轶梅
王健
魏德陇
张海霞
孙普
付元芳
白兴文
刘在新
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Lanzhou Veterinary Research Institute of CAAS
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Abstract

The invention provides a porcine single B cell antibody of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) nucleocapsid protein (N) and a competitive ELISA antibody detection kit, belonging to the technical field of virus detection. The invention provides porcine single B cell antibody C8 of prrsv protein. Tests show that the 3A epitope monoclonal antibody 3A24 of FMDV is used as a coating antibody, a 3AN fusion protein is captured to form a coated ELISA plate, the C8 monoclonal antibody is used as a detection antibody, a competitive ELISA reaction mode is adopted to detect the PRRSV antibody in the pig serum, and the prepared PRRSV antibody detection kit has good sensitivity and specificity and provides a new tool for PRRSV serum epidemiological investigation.

Description

Porcine single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a porcine single B cell antibody C8 of PRRSV nucleocapsid protein and a competitive ELISA kit.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and clinical symptoms are mainly characterized by reproductive disorders in sows, respiratory symptoms in piglets, and high mortality in suckling piglets. The disease causes great economic loss to the global pig industry and is one of the important epidemic diseases which affect the healthy development of the pig industry at present. The PRRSV is a single-stranded positive-strand RNA virus with an envelope, is very easy to mutate, has a plurality of epidemic strains, and can cause the recombination phenomenon of the epidemic virus and the vaccine virus when a commercial attenuated vaccine is used, thereby increasing the difficulty in preventing and controlling the PRRS.
At present, the prevention and control of PRRS in China mainly depend on attenuated vaccine immunity and strict biological safety measures. The disease is detected quickly and accurately in the early stage of infection, and the epidemic of PRRS is controlled to the maximum extent. The ELISA detection technology is a common method for detecting PRRSV antibodies, the detected target is mostly an N protein antibody, and the group infection or the immune condition can be judged by detecting the antibody. The N protein is nucleocapsid protein of PRRSV, has high conservation and strong immunogenicity, and can detect N protein antibody in serum 7 days after infection, so the N protein antibody is a main index for monitoring and evaluating PRRS infection condition. The monoclonal antibody can be developed by separating antigen-specific single B cells from a natural host and a non-natural host, and the monoclonal antibody produced by separating the antigen-specific single B cells from the natural host can more easily recognize natural antigen epitopes existing in virus antigens and has better application effects compared with the monoclonal antibody prepared by the non-natural host, but even the antigen-specific single B cell antibody separated from the natural host has difference in the specificity of recognizing PRRSV antigens, so that the provision of the antigen-specific single B cell antibody is crucial to the preparation of an ELISA detection kit.
Disclosure of Invention
In view of the above, the present invention aims to provide a porcine single B cell antibody of porcine reproductive and respiratory syndrome virus nucleocapsid protein, which has high binding specificity to porcine reproductive and respiratory syndrome virus.
The invention also aims to provide a porcine reproductive and respiratory syndrome virus competition ELISA antibody detection kit for monitoring PRRS infection or immune condition.
The invention provides a porcine single B cell antibody C8 of porcine reproductive and respiratory syndrome virus nucleocapsid protein, wherein a monoclonal antibody C8 generated by the porcine single B cell comprises a heavy chain variable region gene HV1 with an amino acid sequence shown as SEQ ID No.1 and a light chain variable region gene LV1 with an amino acid sequence shown as SEQ ID No. 2.
The invention provides an antibody composition of a group of swine single B cell antibodies of porcine reproductive and respiratory syndrome virus nucleocapsid protein and a monoclonal antibody of foot-and-mouth disease virus 3A protein, which comprises the swine single B cell antibody C8 and the monoclonal antibody of foot-and-mouth disease virus 3A protein 3A 24.
The invention provides application of the swine single B cell antibody C8 or the antibody composition in preparing a reagent or a kit for detecting an N protein antibody after infection of porcine reproductive and respiratory syndrome virus or immunization of animals.
Preferably, the immunoassay comprises colloidal gold immunoassay, immunoprecipitation assay, fluorescence immunoassay and/or enzyme-linked immunoassay.
The invention provides a competitive ELISA antibody detection kit for porcine reproductive and respiratory syndrome virus, which comprises the porcine single B cell antibody C8 or the antibody composition.
Preferably, the kit further comprises an antigen reaction plate, 100 times concentrated biotin labeled antibody, 100 times concentrated enzyme labeled avidin, 25 times concentrated washing solution, serum diluent, substrate solution, stop solution, positive control serum and negative control serum.
Preferably, the antigen reaction plate is prepared by pre-coating monoclonal antibody 3A24 and then combining 3A monoclonal antibody coating capture 3AN fusion protein;
the amino acid sequence of the 3A monoclonal antibody coating capture 3AN fusion protein is shown as SEQ ID No. 3.
Preferably, the 100 × concentrated biotin-labeled antibody is the swine-derived single B-cell antibody C8 labeled with biotin.
Preferably, the labeling concentration of the swine single B cell antibody C8 is 0.06-0.25 mu g/mL.
Preferably, the working concentration of the 100 multiplied concentrated biotin labeled antibody is 10000-30000.
The porcine single B cell antibody C8 of PRRSV nucleocapsid protein (N protein) provided by the invention comprises a heavy chain variable region HV1 and a light chain variable region LV1, wherein the amino acid sequence of the heavy chain variable region HV1 is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID No. 2. The swine single B cell antibody C8 is a swine PRRSV protein monoclonal antibody prepared by a single B cell antibody technology, and can recognize an epitope at the C end of PRRSV N protein, so that the specific recognition and detection of PRRSV can be realized.
The antibody composition provided by the invention comprises the porcine reproductive and respiratory syndrome virus nucleocapsid protein porcine single B cell antibody C8 and a foot-and-mouth disease virus 3A protein monoclonal antibody 3A 24. 3AN protein is captured by 3A monoclonal antibody 3A24 developed in the laboratory to prepare AN antigen reaction plate, a biotin-labeled swine-origin single B cell C8 monoclonal antibody is used as a detection antibody, and a competition ELISA detection mode is adopted, so that PRRSV antibody negative serum and PRRSV antibody positive serum can be distinguished, and therefore, the antibody composition can be used for antibody detection of porcine reproductive and respiratory syndrome virus and has better sensitivity and specificity.
The invention provides a competitive ELISA antibody detection kit for porcine reproductive and respiratory syndrome virus, which comprises the antibody composition. The detection sensitivity of the kit to 204 pigs from 21 days after infection is 98.5 percent, and the detection specificity to 65 healthy pig sera is 98.5 percent.
Drawings
FIG. 1 shows the result of indirect immunofluorescence assay for detecting the reactivity of 9 strain antibody with PRRSV after Marc-145 is inoculated with VR-2332 strain;
FIG. 2 shows the result of Western-blot verifying that the antibody of strain 9 recognizes N protein when N protein expression plasmids of GSWW/2015(A) and VR2332(B) strains are transfected in 293T cells;
FIG. 3 shows the result of the reaction between monoclonal antibody C8 and GST fusion protein truncation;
FIG. 4 shows the results of SDS-PAGE and Coomassie blue staining for 3AN protein detection;
FIG. 5 shows standard positive serum blocking rates for different concentrations of 3A24 antibody;
FIG. 6 shows the standard positive serum blocking rate at different concentrations of C8 antibody and 3AN protein antigen;
FIG. 7 shows the standard positive serum blocking rates at different dilutions;
FIG. 8 shows the mean blocking rate of three positive sera in reaction at 37 ℃ for different times;
FIG. 9 shows the blocking rate of detection of 65 pig negative sera and 204 positive sera;
FIG. 10 is a ROC curve plotted against the blocking rate for 65 swine negative sera and 204 positive sera;
FIG. 11 shows the stability test results of the kit prepared with 3A24 as the coating antibody and C8 as the detection antibody;
FIG. 12 shows the stability test results of the kit prepared with D11 as the coating antibody and C8 as the detection antibody.
Detailed Description
The invention provides a porcine single B cell antibody C8 of porcine reproductive and respiratory syndrome virus nucleocapsid protein, wherein the amino acid sequence of the porcine single B cell antibody C8 comprises a heavy chain variable region amino acid sequence HV1(SEQ ID No.1: GLVQPGGSLRLSCVASGFTFSSYIVTWVRQSPGKGLEWLAGTGVGEYALYYRNSVRGRFTLSRDNSQNTAYLQMNSLRVEETGRYFCRRGAAESVDLWGPGVEVVVSS) and a light chain variable region amino acid sequence LV1(SEQ ID No. 2: QEPAMSVSLGGTVTLTCAFSSGSVTRSHWPSWFQLTPGQPPRTLIVSTDSRPTGVPSRFSGAISGYKAALTITGAQPEDEADYVCGVYFTFTKRPFGGGTHLTVLG).
In the present invention, the preparation method of the monoclonal antibody C8 preferably comprises the following steps: the coding sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody C8 are respectively connected with the constant region sequences of the pig IgG heavy chain and the light chain, and then are respectively inserted into a pcDNA3.4 eukaryotic expression vector, the heavy chain recombinant expression plasmid and the light chain recombinant expression plasmid are mixed and transfected into CHO suspension culture cells according to the mol ratio of 2:3, and are expressed to obtain a complete antibody, and the antibody is obtained by affinity chromatography purification. The transfection method, the expression method and the affinity chromatography purification method of the recombinant expression plasmid are not particularly limited, and the technical scheme known in the field can be adopted.
The invention provides a porcine single B cell antibody composition of porcine reproductive and respiratory syndrome virus nucleocapsid protein, which comprises porcine single B cell antibody C8 and monoclonal antibody 3A24 produced by porcine single B cells; the epitope sequence recognized by the 3A24 monoclonal antibody is VDDAVNEYIEKANITTD DKTLDEAEKNPLETSGA ATVGKTLPGHKA (SEQ ID No. 4). 3A24 can be prepared by methods known in the art (FuY, Li P, Cao Y, et al. development of a blocking ELISA using a monoclonal antibody to a negative epitope in non-structural protein 3A of foot-and-mouth disease virus as a modifying test for a negative marker vaccine [ J ]. Plos One,2017,12(1): e 0170560.).
In the present invention, the ratio of the monoclonal antibody 3a24 and the monoclonal antibody C8 in the monoclonal antibody composition is not particularly limited, and the concentration of the two antibodies used in combination is determined according to the actual concentration of the reagents used in the preparation.
The invention provides application of the swine single B cell antibody C8 or the antibody composition in preparation of a reagent or a kit for detecting a porcine reproductive and respiratory syndrome virus N protein antibody. Preferably, the immunoassay comprises colloidal gold immunoassay, immunoprecipitation assay, fluorescence immunoassay and/or enzyme-linked immunoassay. The reagent or the kit comprises an in vitro diagnostic reagent or a kit prepared based on a competition method, such as a colloidal gold in vitro diagnostic reagent, a chemiluminescence in vitro diagnostic reagent or an ELISA detection reagent and the like.
The invention provides a competitive ELISA antibody detection kit for porcine reproductive and respiratory syndrome virus, which comprises the single B cell antibody composition. The kit preferably further comprises an antigen indirect coated ELISA plate, a 100 multiplied concentration biotin labeled antibody, a 100 multiplied concentration ELISA avidin, a 25 multiplied concentration washing liquid, a serum diluent, a substrate solution, a stop solution, positive control serum and negative control serum.
In the present invention, the antibody in the 100 × concentrated biotin-labeled antibody is the porcine single B-cell antibody C8. The working concentration of the 100 multiplied concentrated biotin labeled monoclonal antibody is preferably 0.06-0.25 mu g/mL, and more preferably 0.13 mu g/mL. The preparation method of the biotin-labeled monoclonal antibody is not particularly limited, and a labeling method known in the art can be used.
The preparation method of the antigen indirect coating ELISA plate comprises the steps of firstly coating the monoclonal antibody 3A24 in the monoclonal antibody composition on the ELISA plate, then capturing the fusion protein 3AN antigen by using the monoclonal antibody 3A24, adding a solid phase antigen stabilizing solution (PBS (phosphate buffered saline) containing 5-10% of sucrose and 1% of BSA (bovine serum albumin) by mass concentration), incubating, removing the supernatant, drying the ELISA plate by blowing, and sealing. The coating concentration of the monoclonal antibody 3A24 is preferably 0.12-3.9 mug/mL, and most preferably 0.49 mug/mL; the working concentration of the 3AN protein antigen is preferably 0.1 mu g/mL; the concentration of C8 is preferably 0.06-0.25. mu.g/mL, most preferably 0.13. mu.g/mL. The invention obtains the fusion protein 3AN by fusion expression of the N protein sequence of the porcine reproductive and respiratory syndrome virus and the identification epitope sequence of the foot-and-mouth disease virus 3A24 monoclonal antibody, adds the GPGPGP connecting peptide sequence in the middle, and has the N endAnd the C end is added with a6 × histidine tag, so that the purification of the fusion protein is convenient. The amino acid sequence of the fusion protein 3AN is' GHHHHHHVDDAVNEYIEKANITTDDKTLDEAEKNPLETSGAATVGKTLPGHKAGPGPGPMPNNNGKQQKKKKGNGQPVNQLCQMLGKIIAQQNQSRGKGPGKKNRKKNPEKPHFPLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCALSDSGRISYTVEFSLPTQHTVRLIRATASPSAHHHHHH”(SEQ ID NO:3)。
In the present invention, the kit comprises 100 × concentrated enzyme-labeled avidin. The 100 × concentrated enzyme-labeled avidin is preferably horseradish peroxidase (HRP) -labeled avidin. The source of HRP-labeled avidin is not particularly limited as long as HRP-labeled avidin known in the art is used.
In the present invention, the kit comprises 25 × concentrated washing solution. The 25 × concentrated washing solution is preferably prepared as follows: adding NaCl 200g, KCl 5g and Na into 500mL of ultrapure water2HPO4·12H2O 72.5g、KH2PO45g and 12.5mL of Tween 20, adding ultrapure water to a constant volume of 1000mL, adjusting the pH value to 7.4, autoclaving, and storing at room temperature for later use.
In the present invention, the kit comprises a serum dilution. The preparation method of the serum diluent comprises the following steps: preparing a phosphate buffer solution containing 2.0% of goat serum, 1.0% of bovine serum albumin (the purity is more than 98%) and 0.02% of thimerosal preservative, filtering and sterilizing, and storing at 2-8 ℃ for later use.
In the present invention, the kit comprises a substrate solution. The kind of the substrate solution is determined according to the kind of enzyme in the enzyme-labeled avidin. When HRP labels avidin, the substrate solution is 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate.
In the invention, the kit comprises a stop solution, and the stop solution is preferably 0.3mol/L H2SO4And (3) solution.
In the present invention, the kit comprises a positive control serum and a negative control serum. The positive control serum is collected from recovered pigs 120 days after PRRSV virus challenge, and is prepared by detecting that an N protein antibody is positive, performing aseptic treatment and adding a preservative agent. The negative control serum is collected from non-immune healthy pigs, is negative after being detected by PRRSV protein antibodies, and is preserved by adding a preservative after being subjected to aseptic treatment.
In the present invention, the method of using the kit preferably comprises the steps of:
A1. adding 30 mu L of serum diluent into each hole in an antigen coated plate, then sequentially adding a serum sample to be detected, a negative control serum sample and a positive control serum sample to be controlled with PBS (phosphate buffer solution) for controlling, wherein 20 mu L of each hole is added for 1 hole of the sample to be detected, and two holes are respectively added for the negative control serum sample, the positive control serum sample and the PBS blank control; then, diluting 100 times of biotin-labeled C8 monoclonal antibody (2.5 μ g/ml) with serum diluent at a ratio of 1: 100, 50 μ L per well, gently shaking and mixing, and acting at 37 deg.C for 1 h; the final dilution of the serum is preferably 1: 5;
A2. washing the plate 5 times with 1 XPBST washing solution, and drying the plate for the last time;
A3. diluting HRP-labeled avidin at a ratio of 1: 30000 into serum diluent, adding an enzyme label plate with 100 μ L per well, and reacting at 37 deg.C for 15 min; then washing with A2;
A4. adding 100 μ L substrate solution into each well, and reacting at 37 deg.C for 15 min;
A5. adding stop solution 100 μ L per well, mixing under gentle vibration, and reading light absorption value (OD) of 450nm with microplate reader within 10min450nm)；
A6. And (3) calculating the blocking rate: blocking Rate (PI) ═ 1-sample OD450nmValue/blank control OD450nmValue) × 100%;
A7. conditions for test establishment: blank control OD450nmvalue-Positive control OD450nmThe value is more than or equal to 1.0, and the positive control blocking rate is more than 50%; the negative control should block less than 25%.
A8. And (3) judging standard: the presence or absence of PRRSV antibodies was determined by calculating the PI value for each serum sample: the PI value is more than or equal to 40 percent, and the PRRSV antibody is judged to be positive; PI value is less than 40%, and the PRRSV antibody is judged to be negative.
In the invention, the detection sensitivity of the kit on 204 pigs from 21 days after infection is 98.5%, and the specificity on serum of healthy pigs is 98.5%.
The porcine single B cell antibody and competitive ELISA detection kit of porcine reproductive and respiratory syndrome virus nucleocapsid protein provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation and identification of PRRSV N protein porcine single B cell antibody
The pig is immunized by utilizing commercial PRRS attenuated vaccines and laboratory self-made epidemic strain vaccines. Simultaneously purifying whole virus antigen and marking virus particles by biotin; after the eighth immunization, venous blood of 0922# pigs was collected, mononuclear cells (PBMCs) in peripheral blood were isolated, biotin-labeled PRRSV was used as a capture antigen, and antigen-specific antibody secreting single B cells were sorted from PBMCs by flow cytometric sorting. Obtaining the gene sequences of the heavy chain and light chain variable regions (VH and VL) of the pig IgG antibody by a single B cell antibody gene amplification technology, and then inserting the gene sequences of the variable regions into a pcDNA3.4 eukaryotic vector containing the constant region of the pig IgG antibody to construct a complete antibody expression plasmid. The expression plasmid of IgG antibody is transfected with Chinese hamster ovary cells (CHO-S) adapted to suspension culture, and then the small expression of antibody is performed, and then the specificity of recognizing PRRSV by the small expression antibody is verified by using indirect immunofluorescence assay (IFA).
The result is shown in figure 1, after the PRRSV VR-2332 strain is inoculated to Marc-145 cells, IFA experiments show that 9 monoclonal antibodies can recognize PRRSV.
The 9-strain antibody is expressed and purified in large quantities. The purified antibodies are identified by Western-blot, and 9 strains of antibodies are found to be capable of specifically reacting with nucleocapsid protein (N protein) of PRRSV (figure 2). The N protein is truncated and then cloned into a pGEX-6P-1 vector, and is expressed in a fusion manner with a GST tag, and the result of FIG. 3 shows that the epitope of the C8 monoclonal antibody for recognizing the N protein is 81-110 sites at the C end.
Example 2
PRRSV nucleocapsid protein expression purification of fusion foot-and-mouth disease virus 3A protein epitope
Cloning the coding sequence of 3AN protein into pET-28a (+) vector by gene synthesis, transforming BL21 competent cell, selecting single clone, enlarging culture to 500ml, adding IPTG to the final concentration1mM, and inducing expression for 5 h. The cells were collected by centrifugation, resuspended and precipitated by adding 50ml of IB wash (EDTA 10mmoXXl/L, Tris-HCl 20mmol/L, pH 7.5, Triton X-1001%) in ice bath, sonicated until the cells were completely lysed, and centrifuged to collect the supernatant. According to the following steps of 4: 1, adding 50 percent of High Affinity Ni-Charged Resin FF, and incubating overnight at 4 ℃ at 200r/min to ensure that the target protein and Ni-NTA His Bind completely and specifically. With a washing solution (NaH) at pH8.02PO450mM/L NaCl 300mM, Urea 8M, imidazole 10mM) column 3 times, each time 2 times the volume, and 0.5 times the volume of pH8.0 eluent (NaH)2PO450mM, NaCl 300mM, Urea 8M, and imidazole 250mM) for 4 times, wherein the eluent is the purified N protein. The purified protein was dialyzed 3 times at 4 ℃ against a dialysis solution (containing Tris-HCl pH8.0 at a final concentration of 0.1M, L-Arg at 0.4M, EDTA at 0.2mM, L-glutaminone oxidized at 0.5mM and glutaminone at 2.5 mM) to obtain a solution of purified fusion protein 3 AN. FIG. 4 shows SDS-PAGE and Coomassie blue staining to detect fusion protein 3 AN.
Example 3
Preparation method of N protein competition ELISA antibody detection kit
1. Material
Carbonate buffer (Na)2CO3/NaHCO3) And BSA was purchased from SIGMA.
BCA protein concentration determination kit and biotin labeling kit were purchased from Thermo Fisher, Inc.
Horseradish peroxidase-labeled avidin was a commercial reagent (product of Genscript) purchased.
The PRRSV protein monoclonal antibody C8 was developed and stored in the laboratory by using single B cell antibody preparation technology at a concentration of 0.25 mg/mL. FMDV 3A monoclonal antibody 3A24 was developed by the laboratory using hybridoma technology and was found to be 1.95 mg/ml.
Negative and positive control serum, PRRSV N protein and 3A epitope fusion protein antigen of FMDV are prepared and stored in the laboratory.
2. Preparation of reagent and coating method of enzyme label plate
2.1. Preparation of antigen indirect coated enzyme label plate
Diluting the monoclonal antibody 3A24 in carbonate buffer solution according to the final concentration of 0.49 mu g/mL, coating an enzyme label plate at 100 mu L/hole overnight at 4 ℃; after PBST is used for washing for 3 times, 3AN fusion protein antigen with the final concentration of 0.1 mu g/mL is added, and the mixture is incubated for 2 hours at room temperature; washing with PBST for 3 times, adding a solid phase antigen stabilizing solution (PBS (phosphate buffered saline) containing 5-10% of sucrose and 1% of BSA), incubating at room temperature for 1h, sucking and removing supernatant, drying an ELISA plate, adding a drying agent, sealing, and refrigerating at 2-8 ℃. Each kit is packed with two enzyme label plates coated with antigen.
2.2. Preparation of biotin-labeled monoclonal antibody C8
The following procedure was followed:
a1, first, the monoclonal antibody C8 was replaced in PBS buffer using a cut-off 100kDa ultrafiltration tube and centrifuged at 4000r/min for 10 min.
A2, adding 180 mu L of ultrapure water into 1mg of biotin, and diluting to 10 mM; biotin is added in a ratio of 1: 10 and 1: 20, respectively, i.e., 50. mu.L, 25. mu.L to 500. mu.L of antibody are added, respectively.
A3, placing on ice at 4 ℃ for 4 h.
A4, displacement into antibody storage buffer with ultrafiltration tube.
A5, adding an equal volume of 100% glycerol, and storing at-70 ℃.
Each kit included 1 tube (300 μ L) of the biotin-labeled C8 antibody at 100-fold working concentration.
2.3.100 × concentrated horse radish peroxidase labeled avidin
The horseradish peroxidase (HRP) -labeled avidin is a purchased commercial reagent (BioFX), and the enzyme-labeled avidin is diluted in an antibody preservation solution (Sigma) according to a working concentration of 100 times and is preserved at 2-8 ℃ for later use. Each reagent cassette was packed in 1 tube (300. mu.L).
2.2.4. Concentrated washing solution (25X)
Adding 200g of NaCl, 5g of KCl and Na into 500mL of ultrapure water2HPO4·12H2O 72.5g、KH2PO45g and 12.5mL of Tween 20, adjusting the pH value to 7.4, adding ultrapure water to a constant volume of 1000mL, autoclaving (10Pa, 15min), and storing at room temperature for later use; each box is packed with 1 bottle (50)mL)。
2.2.5. Substrate solution
The single- component 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate solution was purchased as a commercial reagent (BioFX) and dispensed directly for use, 1 vial (25mL) per cassette, with a 4-year shelf life.
2.2.6. Stopping liquid
H with a concentration of 0.3mol/L2SO4The solution was prepared by diluting analytically pure concentrated sulfuric acid with ultrapure water, and each tank was filled with 1 bottle (25 mL).
2.2.7. Positive control serum
The protein N antibody is detected to be positive by recovered pigs 120 days after the PRRSV is detoxified, and is added with preservative after being sterilized and filtered for split charging and storage for later use.
2.2.8. Negative control serum
The PRRSV non-immune healthy pig is collected, is negative by detecting PRRSV protein antibody, is added with preservative after being filtered and sterilized, and is subpackaged and stored for later use.
2.2.9. Other articles: comprises 4 pieces of disposable closing plate film and an instruction for use.
2.2.10. Serum antibody detection procedure
And (3) detection procedures: the kit stored at 2-8 ℃ was equilibrated to room temperature and then operated according to the following procedure.
A1. Adding 30 mu L of serum diluent into each hole in an antigen coated plate, then sequentially adding a serum sample to be detected, a negative control serum sample and a positive control serum sample to be controlled with PBS (phosphate buffer solution) for controlling, wherein 20 mu L of each hole is added for 1 hole of the sample to be detected, and two holes are respectively added for the negative control serum sample, the positive control serum sample and the PBS blank control; then, diluting the biotin-labeled monoclonal antibody (2.5 mu g/ml) with a serum diluent of 1: 100 at a working concentration of 100 times, wherein each well is 50 mu L, gently shaking and uniformly mixing, and acting at 37 ℃ for 1 h; the final dilution of the serum is preferably 1: 5;
A2. washing the plate 5 times with 1 XPBST washing solution, and drying the plate for the last time;
A3. diluting HRP-labeled avidin at a ratio of 1: 30000 into serum diluent, adding an enzyme label plate with 100 μ L per well, and reacting at 37 deg.C for 15 min; then washing with A2;
A4. adding 100 μ L substrate solution into each well, and reacting at 37 deg.C for 15 min;
A5. adding stop solution 100 μ L per well, mixing under gentle vibration, and reading light absorption value (OD) of 450nm with microplate reader within 10min450nm)；
A6. And (3) calculating the blocking rate: blocking Rate (PI) ═ 1-sample OD450nmValue/blank control OD450nmValue) × 100%;
A7. conditions for test establishment: blank control OD450nmvalue-Positive control OD450nmThe value is more than or equal to 1.0, and the positive control blocking rate is not lower than 70%; the negative control should block less than 25%.
A8. And (3) judging standard: the presence or absence of PRRSV antibodies was determined by calculating the PI value for each serum sample: the PI value is more than or equal to 40 percent, and the PRRSV antibody is judged to be positive; PI value is less than 40%, and the PRRSV antibody is judged to be negative.
Example 4
Establishment of N protein competition ELISA antibody detection method
1 method
Determination of optimal working concentration of PRRSV 3AN fusion protein antigen and antibody
Monoclonal antibody 3A24 was coated with 3.9. mu.g/mL, 1.95. mu.g/mL, 0.98. mu.g/mL, 0.49. mu.g/mL, 0.24. mu.g/mL and 0.12. mu.g/mL, respectively, and then 3AN protein antigen was captured at different concentrations (0.1. mu.g/mL, 0.2. mu.g/mL, 0.4. mu.g/mL and 0.8. mu.g/mL), and a square matrix titration was performed by adding different dilutions (0.25. mu.g/mL, 0.13. mu.g/mL, 0.06. mu.g/mL and 0.03. mu.g/mL) of the biotinylated monoclonal antibody C8 and a fixed concentration of HRP-labeled avidin. Positive and negative control serum samples were tested against PBS blank according to the reaction procedure described in example 2. And calculating the blocking rate (PI) of the negative control serum and the positive control serum, and setting the optimal working concentration of the antigen and the antibody according to the reaction condition with the maximum difference between the positive control serum and the negative control serum.
PI ═ 1-positive control serum OD450nmValue/blank control serum OD450nmValue) × 100%
1.2. Determination of optimal serum dilution
After determining the optimal working concentration of antigen and antibody, the standard negative and positive sera were subjected to competitive ELISA at three dilutions of 1: 5, 1: 10 and 1: 20. Comparing the obtained blocking rates, and taking the dilution with the largest difference of the blocking rates of the negative serum and the positive serum as the optimal serum dilution.
1.3. Selection of reaction time after serum addition
The following reaction conditions were set: reaction at 37 deg.c for 30 min; ② reacting for 45min at 37 ℃; ③ reaction at 37 ℃ for 60 min. And (3) carrying out comparative analysis on the positive control serum blocking rate under each reaction condition, and taking the shortest action time when the blocking rate reaches the maximum as the optimal reaction condition.
1.4. Determination of decision criteria
Determining the judgment standard of the method according to the detection result of the blocking rate of the serum sample of the clinically healthy non-immune pig and the infected pig with clear background; detecting 65 parts of nonimmune healthy pig serum and 204 parts of infected pig serum, counting the distribution interval of the blocking rate of serum samples of infected animals and healthy animals, drawing an ROC curve, and determining the judgment standard of the competitive ELISA.
1.5. Specificity and sensitivity detection of the method
According to the determined judgment standard, the specificity and the sensitivity of the method are determined by analyzing the detection results of the serum samples of the non-immune healthy pigs and the infected pigs.
2. Results
2.1. Working concentration of antigen antibody
The blocking rate of the positive serum and the negative serum was determined by gradient dilution of the 3AN fusion protein antigen and the 3A24 monoclonal antibody, and as shown in FIG. 5, the difference between the positive serum and the negative serum was the greatest when the 3AN fusion protein antigen was 0.1. mu.g/ml and the C8 monoclonal antibody was 0.13. mu.g/ml, thus determining that the optimal concentration of the 3AN fusion protein antigen was 0.1. mu.g/ml and the optimal concentration of the C8 monoclonal antibody was 0.13. mu.g/ml.
And (3) coating the ELISA plate after the 3A protein monoclonal antibody 3A24 is subjected to gradient dilution, capturing 0.1 mu g/ml 3AN protein to indirectly coat the ELISA plate, and respectively detecting the blocking rate of positive serum and negative serum. As shown in FIG. 6, when the working concentration of 3A24 was 0.98. mu.g/ml, 0.49. mu.g/ml, 0.24. mu.g/ml, the difference between the blocking rates of the positive serum and the negative serum was large, and the optimal working concentration of 3A24 mAb was determined to be 0.24. mu.g/ml for cost savings.
2.2. Determination of optimal serum dilution
The blocking rates of the positive standard serum and the negative serum of each dilution are shown in FIG. 7, the blocking rate difference of the negative serum and the positive serum obtained by diluting the serum by 1: 5 is the largest, and the requirement on detection sensitivity can be met, so that the optimal dilution of the serum is determined to be 1: 5.
2.3 determination of reaction time after serum addition
After the positive serum is added, the standard positive serum blocking rate of different reaction time is compared and analyzed (see figure 8), and the result shows that the positive control serum blocking rate is highest after the reaction at 37 ℃ for 1h, so that the reaction time is determined to be 37 ℃ for 1 h.
2.4 determination of criteria for determination of results
A. Determination of Positive and negative decision criteria (thresholds)
According to the detection results of the infected swine serum and the healthy swine serum (FIG. 9), a ROC curve (FIG. 10) was plotted, and the optimal critical value was determined to be 40% according to the ROC curve. The decision criteria are therefore determined as: the blocking rate is not less than 40%, and the product is judged to be positive, and if the blocking rate is less than 40%, the product is judged to be negative. In a non-immune background, a positive result of the sample indicates that the animal has PRRSV infection, and corresponding prevention and control measures should be taken. The blocking rate (PI) of the test sample was calculated according to the following formula, PI ═ 1-test sample OD450nmValue/blank control mean OD450nmValue) × 100%.
B. Results of specificity and sensitivity determination
After the serum of the clinical healthy non-immune pig and the serum of the infected pig are detected, the specificity is calculated according to the ratio of the number of the detected negative serum to the total number of the healthy serum, and the sensitivity is calculated according to the ratio of the number of the detected positive serum to the total number of the serum of the infected animal. From the results (Table 1), it can be seen that the method has a detection sensitivity of 98.5% for 204 samples from infected pig sera and a specificity of 98.5% for healthy pig sera.
TABLE 1 detection results of specificity and sensitivity of N protein blocking ELISA antibody
Figure BDA0003091898440000121
Example 5
And 3A24 is used as a coating antibody to capture the 3AN fusion protein antigen to form a coated enzyme label plate, wherein the concentration of the C8 monoclonal antibody is 0.13 mu g/mL, the final concentration of the 3AN is 0.1 mu g/mL, and the coating concentration of the 3A24 monoclonal antibody is 0.24 mu g/mL. Specifically, the kit was prepared according to the method for preparing the N protein competition ELISA antibody detection kit described in example 3.
Comparative example 1
The C8 monoclonal antibody is used as a coating antibody to capture N protein antigen to form a coated enzyme label plate, the D11 monoclonal antibody is used as a detection antibody, the concentration of the D11 monoclonal antibody is 2 mu g/mL, the final concentration of the N protein antigen is 1 mu g/mL, and the working concentration of the D11 monoclonal antibody is 2 mu g/mL. Specifically, the kit was prepared according to the method for preparing the N protein competition ELISA antibody detection kit described in example 3.
Wherein monoclonal antibody D11 includes heavy chain variable region gene HV2 whose amino acid sequence is shown in SEQ ID No.5(GLVQPGGSLRLSCVASGFTLSNCAMTWVRQAPGKGLEWLAVINIRGDRSAYADSVRGRFTISKDDSQNTAYLEMKTLRVEDTARYYCKTADKAFPSESIQMDVWGPGVEVVASS) and light chain variable region gene LV2 whose amino acid sequence is shown in SEQ ID No.6 (WVPGARCAIQVTQSPASLAASLGDTVSITCRASLSMSNYLAWYQQKPGKTPRRLIYYASTLESGVPSRFKGRGVGTEFTLTISGLQAEDVATYYCLQYWSAPWTFGQGSKVELKR). The monoclonal antibody D11 was prepared by in vitro recombinant production by referring to the method for producing a monoclonal antibody in example 1. The amino acid sequence of the N protein antigen is shown as SEQ ID NO: shown at 7 (MPNNNGKQQKKKKGNGQPVNQLCQMLGKIIAQQNQSRGKGPGKKNRKKNPEKPHFPLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCALSDSGRISYTVEFSLPTQHTVRLIRATASPSAHHHHHH).
Example 6
The kits prepared in example 5 and comparative example 1 were stored at 37 ℃ for 7 days respectively for aging experiments, after the experiments were completed, the treated kits and the freshly prepared kits were taken out to measure N protein positive serum and negative samples respectively, a blank control group was set, the OD values detected in each group were counted, and a histogram was drawn.
The results are shown in FIGS. 11 and 12. FIG. 11 shows that 3A24 is used as coating antibody to capture 3AN fusion protein antigen and coat the ELISA plate. As can be seen from FIG. 11, the OD values of the positive serum, the negative serum and the blank control detected by comparing the fresh coated ELISA plate with the ELISA plate stored at 37 ℃ for 7 days with C8 as the detection antibody are not significantly different, which indicates that the combination of the 3A24 antibody capture antigen 3AN fusion protein and C8 as the detection antibody is stable. FIG. 12 uses C8 as coating antibody to capture N protein antigen and coat the ELISA plate. D11 is a detection antibody, and the differences of the OD values of the positive serum, the negative serum and the blank control are detected by comparing a fresh coated ELISA plate with an ELISA plate stored for 7 days at 37 ℃. The decrease in OD values of the negative serum and blank control detection after 7 days of storage at 37 ℃ was found to be significant, indicating that the combination of the antigen N protein captured by the C8 antibody and the detection antibody D11 was unstable.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> porcine single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit
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Asn Ser Gln Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Val Glu
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Leu Trp Gly Pro Gly Val Glu Val Val Val Ser Ser
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His

Claims (10)

1. A porcine single B cell antibody C8 of porcine reproductive and respiratory syndrome virus nucleocapsid protein, characterized in that the monoclonal antibody C8 produced by the porcine single B cell comprises a heavy chain variable region gene HV1 with an amino acid sequence shown as SEQ ID No.1 and a light chain variable region gene LV1 with an amino acid sequence shown as SEQ ID No. 2.
2. A combination of swine single B cell antibody of porcine reproductive and respiratory syndrome virus nucleocapsid protein and foot-and-mouth disease virus 3A protein monoclonal antibody, which is characterized in that the combination comprises the swine single B cell antibody C8 of claim 1 and the foot-and-mouth disease virus 3A protein monoclonal antibody 3A 24.
3. Use of the porcine single B-cell antibody C8 of claim 1 or the antibody composition of claim 2 for the preparation of a reagent or kit for detecting N protein antibodies following infection with porcine reproductive and respiratory syndrome virus or immunization of an animal.
4. The use of claim 3, wherein the immunoassay comprises a colloidal gold immunoassay, an immunoprecipitation assay, a fluorescent immunoassay and/or an enzyme-linked immunoassay.
5. A competitive ELISA antibody detection kit for porcine reproductive and respiratory syndrome virus, comprising the porcine single B cell antibody C8 of claim 1 or the antibody composition of claim 2.
6. The competitive ELISA antibody assay kit of claim 5 further comprising an antigen-reactive plate, 100 x concentrated biotin-labeled antibody, 100 x concentrated enzyme-labeled avidin, 25-fold concentrated washing solution, serum diluent, substrate solution, stop solution, positive control serum and negative control serum.
7. The competitive ELISA antibody assay kit of claim 6 wherein the antigen-reactive plate is prepared by pre-coating monoclonal antibody 3A24 and then combining 3A monoclonal antibody coated capture 3AN fusion protein;
the amino acid sequence of the 3A monoclonal antibody coating capture 3AN fusion protein is shown as SEQ ID No. 3.
8. The competitive ELISA antibody assay kit of claim 6 wherein the 100 x concentrated biotin labeled antibody is biotin labeled with the porcine single B cell antibody C8.
9. The competitive ELISA antibody assay kit of claim 8 wherein the concentration of the porcine single B cell antibody C8 label is 0.06-0.25 μ g/mL.
10. The competitive ELISA antibody detection kit of claim 6, wherein the working concentration of the 100 x concentrated biotin labeled antibody is 1: 10000-30000.
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