CN114164179A - Hybridoma cell secreting anti-SVA antibody, antibody secreted by hybridoma cell and application of antibody - Google Patents

Abstract

The invention relates to the technical field of immunodetection, and particularly discloses a hybridoma cell secreting an anti-SVA antibody, an antibody secreted by the hybridoma cell and application of the hybridoma cell. The hybridoma cell comprises a hybridoma cell strain SVA-VP2-6D5 and/or a hybridoma cell strain SVA-VP3-7A 9; the hybridoma cell strain SVA-VP2-6D5 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021277, and the hybridoma cell strain SVA-VP3-7A9 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021276. The monoclonal antibody secreted by the hybridoma cell strain SVA-VP2-6D5 and SVA-VP3-7A9 is jointly used as competitive CLEIA constructed by competitive antibodies, and can be used for quantitative detection of the A-type Seneca virus antibody. The detection method is sensitive, rapid and accurate, and provides a reliable serological detection method for SVA antibody detection.

Description

Hybridoma cell secreting anti-SVA antibody, antibody secreted by hybridoma cell and application of antibody

Technical Field

The invention relates to the technical field of immunodetection, in particular to a hybridoma cell secreting an anti-SVA antibody, an antibody secreted by the hybridoma cell and application of the hybridoma cell.

Background

Porcine seneca virus disease is a swine vesicular disease caused by Senecavirus a (SVA). SVA was first called Seneca Valley virus SVV (Seneca Valley virus SVV) and was listed in the genus Senecavirus of the picornaviridae family by the International Committee for Virus Classification (ICTV) in 2015, where SVA shares closest homology with members of the family myocarditis virus genus. SVA can cause diarrhea of piglets, and vesicles and ulcers appear around noses, palate, oral cavity, lips and hoof crowns of multiparous sows and boars, and occasionally cause diarrhea symptoms, and the death rate of newborn piglets can reach 30-70% in 1-3 days. The occurrence of this disease has been reported in various countries so far. However, at present, the recognition and research on SVA are less at home and abroad, and once large-scale outbreak happens, serious economic loss is brought to the breeding industry.

SVA is a single-stranded positive-stranded RNA virus without envelope, with an icosahedron structure, a diameter of about 30nm, a genome of about 7.2-7.3 kb in overall length, a viral gene having 1 Open Reading Frame (ORF), encoding a polyprotein precursor of 2181 amino acids, which is then cleaved into 1 leader protein and 3 major polyproteins (P1, P2 and P3). The P1 gene region encodes mainly 4 structural proteins (VP4, VP2, VP3 and VP1), forming the nucleocapsid of the virus, while the P2 and P3 gene regions encode 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). Studies have shown that the VP2 and VP3 proteins can induce the body to produce main neutralizing antibodies, which are used for eliminating viruses in vivo and play a crucial role in resisting SVA infection and relieving clinical symptoms. Meanwhile, a serological method is established by taking the antigen as a detection antigen, and the method has high consistency with a serum neutralization test.

Because clinical symptoms caused after SVA infects pigs are difficult to distinguish from foot-and-mouth disease, swine vesicular disease and the like, SVA is difficult to diagnose only by clinical symptoms and pathological dissection. Therefore, virus separation and identification are the most common pathogenic detection method, but the method is complex in operation and long in detection period; the PCR and fluorescent quantitative PCR detection method has high cost, high price of instruments and equipment and difficult popularization and application in the basic level; the ELISA method has the advantages of simple operation, high sensitivity and suitability for large-scale sample detection, and is a detection method commonly used in laboratories. At present, a blocking ELISA reagent for detecting SVA antibody based on a monoclonal antibody is available, but the process is relatively complicated and the operation is long; there are also other studies on SVA antibody detection blocking and competitive ELISA methods, but they are all based on polyclonal antibodies with low specificity and sensitivity.

The chemiluminescence enzyme immunoassay (CLEIA) is a novel immunoassay technology which combines a chemiluminescence system with an enzyme-linked immunoassay technology and detects trace antigens or antibodies according to the proportional relation between chemiluminescence intensity and reactant concentration. Compared with the common indirect ELISA detection method, the technology has simple operation and short reaction time; quantitative analysis can be realized by drawing a standard curve; the sensitivity and the accuracy are obviously superior to other methods, and the detection rate of standard positive samples can reach 100 percent; the detection range is wide and can reach 6 orders of magnitude. At present, the method is widely applied to the items of human body external diagnostic reagents such as tumor marker detection, hepatitis C and AIDS antibody detection, food safety detection and the like, but the method is not widely applied in the field of animal epidemic disease detection.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to establish a sensitive, quick and accurate SVA competition CLEIA detection method.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the invention provides a hybridoma cell strain which is named as SVA-VP2-6D5 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021277; or is named as SVA-VP3-7A9 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021276.

The hybridoma cell strain SVA-VP2-6D5 is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, Wuhan, China center for type culture Collection, zip code 430072) at 2021, 9 months and 30 days, and the preservation number is CCTCC NO: C2021277.

The hybridoma cell strain SVA-VP3-7A9 is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, Wuhan, China center for type culture Collection, zip code 430072) at 2021, 9 months and 30 days, and the preservation number is CCTCC NO: C2021276.

The invention researches and obtains two hybridoma cells SVA-VP2-6D5 and SVA-VP3-7A9 which can secrete monoclonal antibodies against SVA r-VP2 and SVAr-VP3 respectively, and the antibodies can be used as competitive antibodies in SVA competitive CLEIA detection after being subjected to enzyme labeling, so that an ideal SVA detection effect is realized.

In a second aspect, the invention provides a hybridoma cell comprising a hybridoma cell strain SVA-VP2-6D5 and a hybridoma cell strain SVA-VP3-7A 9; the hybridoma cell strain SVA-VP2-6D5 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021277, and the hybridoma cell strain SVA-VP3-7A9 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021276.

In a third aspect, the invention provides a hybridoma cell line or a monoclonal antibody produced by the hybridoma cell line.

In a fourth aspect, the invention provides an application of the hybridoma cell strain or the hybridoma cell or the monoclonal antibody in preparation of a reagent or a kit for detecting the A-type seneca virus.

In a fifth aspect, the present invention provides a reagent or a kit for detecting Seneca virus type A, which comprises the monoclonal antibody described above.

Preferably, the reagent or the kit contains two enzyme-labeled monoclonal antibodies, wherein the two monoclonal antibodies are respectively an rVP2 monoclonal antibody and an rVP3 monoclonal antibody, the rVP2 monoclonal antibody is generated by a hybridoma cell strain SVA-VP2-6D5, and the rVP3 monoclonal antibody is generated by a hybridoma cell strain SVA-VP3-7A 9;

more preferably, the mass ratio of the rVP2 monoclonal antibody to the rVP3 monoclonal antibody is 1: (1.1-1.8), and further preferably 1:1.2, to obtain better detection sensitivity;

when the kit is used for detection, a competitive chemiluminescence enzyme-linked immunoassay method is utilized, specifically, purified SVA inactivated concentrated virus protein is used as an antigen, and two enzyme-labeled monoclonal antibodies are used as competitive enzyme markers.

The invention discovers that when monoclonal antibodies secreted by two hybridoma cell strains SVA-VP2-6D5 and SVA-VP3-7A9 are labeled by enzyme and then are jointly used as competitive antibodies in a detection kit, the detection effect is better.

And/or the kit also contains an enzyme-labeled monoclonal antibody diluent; the enzyme-labeled monoclonal antibody diluent is a Tris-cl solution containing 1.4-1.6%, preferably 1.5% casein, and can be used for improving the detection sensitivity.

In a sixth aspect, the invention provides an antibody competitive chemiluminescent enzyme-linked immunosorbent assay for detecting the A-type Seneca virus, which uses the monoclonal antibody as a component of a competitive antibody.

In the method, the competitive antibodies are an enzyme-labeled rVP2 monoclonal antibody and an enzyme-labeled rVP3 monoclonal antibody, the rVP2 monoclonal antibody and the rVP3 monoclonal antibody are as described above, and the mass ratio of the rVP2 monoclonal antibody to the rVP3 monoclonal antibody is 1: (1.1-1.8), preferably 1: 1.2.

In the method, when the coating antigen is combined, the concentration of the rVP2 monoclonal antibody in the enzyme-labeled rVP2 monoclonal antibody is 0.75-1 mug/mL, preferably 0.86 mug/mL; the concentration of the rVP3 monoclonal antibody in the enzyme-labeled rVP3 monoclonal antibody is 1.7-2.65 mug/mL, and preferably 2.1 mug/mL, so that the sensitivity is ensured, and meanwhile, raw materials are saved.

In the method, the concentration used for coating the antigen is 0.9-1.1 mug/mL, preferably 1 mug/mL, so as to ensure that better sensitivity and specificity for detecting positive and negative samples are obtained while the minimum raw materials are used;

and/or the action time of the sample to be detected, the competitive antibody and the coating antigen is 28-32min, preferably 30 min;

and/or the action time of the luminescent substrate is 4.5-5.5min, preferably 5 min.

Specifically, the method takes inactivated A-type seneca virus as a coating antigen, takes horse radish peroxidase-labeled anti-A-type seneca virus VP2 and VP3 protein monoclonal antibodies as enzyme-labeled antibodies, and draws a standard curve with a calibrator prepared by diluting positive serum to realize quantitative detection.

The invention has the beneficial effects that:

the invention prepares the enzyme label of the anti-SVA r-VP2 and SVA r-VP3 monoclonal antibodies, takes the purified SVA inactivated concentrated virus protein as antigen and two enzyme-labeled monoclonal antibodies as competitive enzyme labels, and successfully establishes a sensitive, rapid and accurate SVA competitive CLEIA detection method. The detection can be completed within 45min, and the calibrator traced serum with the maximum dilution multiple of 1: 2048 can be detected, and has no cross reaction with the standard positive serum of other 5 virus antigens such as foot-and-mouth disease and the like; the variation coefficient in and among analysis in batches is not more than 15%, and the repeatability and the stability are better; 480 clinical serums are detected by a neutralization test respectively, the positive coincidence rate is 95.33 percent, the negative coincidence rate is 97.58 percent, the total coincidence rate is 96.88 percent, and the high consistency is achieved. The invention fills the blank of the SVA antibody quantitative detection kit and provides a reliable serological detection method for clinical SVA antibody detection.

Drawings

FIG. 1 is an electrophoretic image of the purified monoclonal antibody analyzed by SDS-PAGE in example 1, wherein M: a protein Marker; 1: SVA-VP2-6D5 monoclonal antibody; 2: SVA-VP3-7A9 monoclonal antibody.

FIG. 2 is the electrophoresis chart of the result of using SVA-VP2-6D5 monoclonal antibody as primary antibody Western blot in example 2, wherein, M: a protein Marker; 1: rVP2 protein; 2: rVP3 protein; 3: pET32a/BL21 empty vector.

FIG. 3 is the electrophoresis chart of the result of using SVA-VP3-7A9 monoclonal antibody as primary antibody Western blot in example 2, wherein, M: a protein Marker; 1: rVP2 protein; 2: rVP3 protein; 3: pET32a/BL21 empty vector.

FIG. 4 is a graph obtained by plotting a calibrator (antibody dose value of 0 to 100NCU/mL) in example 3, wherein the abscissa represents the antibody dose value, the unit is NCU/mL, and the ordinate represents the luminescence value.

FIG. 5 is a graph plotting ROC curves in example 3, wherein the abscissa represents 1-specificity and the ordinate represents sensitivity.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Some materials and instrument information used in the embodiment of the present invention are as follows:

1. viruses, bacteria, cells, serum and experimental animals:

SVV-HeXX/Swine/2017 is preserved in China center for type culture collection, the preservation number is CCTCC NO: V201767 (disclosed in China patent CN109182278A, the preservation information of SVV-HeXX/Swine/2017 is as follows: the preservation number of microorganism is CCTCC NO: V201767; the preservation address is university of Wuhan, China; the preservation time is 11 and 22 days in 2017; the preservation organization is China center for type culture collection), the virus culture, the recombinant pET32a (+) -rVP2, pET32a (+) -rVP3 protein (the construction method is as follows: extracting SVV-HeXX/Swine/2017 virus strain RNA, respectively amplifying VP2 and VP3 genes after reverse transcription by PCR, respectively cloning to pGEM-T Easy vector, respectively connecting to prokaryotic expression vector pET32 Escherichia coli a (+), transforming to DE Rosetta (3) sensitive cell, recombinant bacteria pET32a (+) -rVP2 and pET32a (+) -rVP3 are constructed. Inducible expression was performed using 0.5mmol/L IPTG. Under the non-denaturing condition, recombinant SVA VP2 and SVA VP3 proteins and Escherichia coli BL21pLysS mycoprotein are purified from the supernatant of the thallus lysate by using a nickel column and are obtained and supplied by expression of a prevention and control center for epidemic diseases of animals in Henan province. Myeloma cells (SP2/0) are preserved by laboratories of animal epidemic prevention and control centers in Henan province; the positive sera of Foot-and-mouth disease virus (FMDV) type O and A are provided by Lanzhou biological pharmaceutical factory of Zhongmu practical corporation, and the titer is not lower than 1: 256; the Swine Vesicular Disease Virus (SVDV) positive serum is derived from PrioCHECK SVDV enzyme-linked immune antibody detection kit of PrioCHECK of Prionics company of Switzerland, the Swine Pseudorabies virus (PRV) positive serum is PRV gB antibody positive serum national reference (Z89), the Swine reproduction and respiratory syndrome virus (PRRSV) positive serum is provided by Harbin nationality organisms, and the titer is not lower than 1: 27; escherichia coli-positive serum with titer not less than 1: 128 was purchased from Beijing Solaibao Tech Co. SVA clinical immune antibody positive serum and SVA negative serum which are collected from porcine epikavirus inactivated vaccine (SVV-HeXX/swine/2017 strain, suspension culture) clinical tests are detected by a Serum Neutralization Test (SNT); female BALB/c mice 8-10 weeks old are provided by the Experimental animal center in Henan province.

2. Main apparatus and reagents:

bam HI, Hind III, pre-staining protein Marker, BCA protein quantification kit and the like are purchased from TaKaRa company; the His Bind protein purification kit is purchased from exert oneself Ri Biotech limited of Shanghai; goat anti-mouse IgG (HRP-labeled) secondary antibody and anti-His-labeled mouse monoclonal antibody were purchased from Kyoto China fir Jinqiao biotechnology, Inc.; PEG2000, X-gal, IPTG, NaSCN, BSA, Casein, etc. were purchased from Solambio; fetal bovine serum was purchased from seror corporation; DMEM and DMSO are purchased from Strobilanthes Wuhan, Dr bioengineering, Inc.; HT, HAT, 8-Azaguanine and monoclonal antibody subclass identification kit is purchased from Sigma company in the United states; high-speed liquid protein chromatography system instrument (NGC)^TM Chromatography Systems)、SUPrA^TMCartidge affinity chromatography columns were purchased from Bio-RAD, usa; the luminescent substrate liquid is provided by Luoyang Laipeng information technology limited; LUMO chemiluminescence apparatus was purchased from Zhengzhou Antu bioengineering, Inc.; FMDV O-type antibody detection kit and FMDV A-type antibody detection kit are purchased from Korea Jinnuo (MEDIAN) diagnostics; the PRV gB antibody detection kit and the PRRSV antibody detection kit are purchased from IDEXX Edison biotechnology company in the United states; the swine vesicular disease virus SVDV antibody detection kit is purchased from Prionics, Switzerland.

Example 1 preparation of monoclonal antibodies against SVA rVP2 and SVA rVP3 proteins

According to the known monoclonal antibody preparation method, Balb/c mice are immunized after emulsifying the inactivated and concentrated culture solution of SVV-HeXX/sine/2017 Strain (SVA) according to the established immunization program (see Chinese patent application No. 201710671265.8). An indirect ELISA method for screening the antibody is established by taking purified rVP2 and rVP3 proteins as coating antigens and goat anti-mouse IgG (HRP marker) as an enzyme-labeled secondary antibody, and the specific antibody titer of the mouse after three times of immunization is detected. Selecting an immune mouse with the highest titer for enhancing immunity, taking the spleen of the immune mouse for cell fusion, screening specific positive holes by using an established indirect ELISA method, carrying out cloning culture on positive hybridoma cells by using a limiting dilution method, and carrying out amplification culture when the positive rate reaches 100% after 5 times of subcloning. Preparing monoclonal antibody from hybridoma cell strain capable of being stably passaged by in vivo induced ascites method, performing coarse purification on aseptically collected ascites identified as IgG subtype by monoclonal antibody subclass by caprylic acid-saturated ammonium sulfate precipitation method, and combining high speed liquid phase protein chromatography system with SUPrA^TMThe cartidge affinity column was further purified, desalted and the final mab concentration determined by BCA method. The indirect ELISA method is used for measuring the titer of the monoclonal antibody, and ascites of a mouse induced by SP2/0 cells is used as a negative control.

The concrete result is as follows: and (3) respectively measuring the serum antibody titer of the mice completing three times of immunization by using an indirect ELISA method, wherein the SVA rVP2 and the SVA rVP3 protein antibody titer are higher than 1: 16000. Selecting mice with highest antibody titer of rVP2 and rVP3, and taking spleens of the mice for fusion, wherein the fusion rate reaches 100%. Respectively obtaining 1 hybridoma cell strain SVA-VP2-6D5 capable of specifically secreting rVP2 monoclonal antibody by 5 times of subclone positive screening, nonspecific reaction determination and subtype determination screening, wherein the subtype is IgG1, and the titer of the supernatant is 1: 16000; 1 hybridoma cell strain SVA-VP3-7A9 capable of specifically secreting rVP3 monoclonal antibody, subtype is IgG2a, titer of supernatant is 1:16000, SVA-VP2-6D5 and SVA-VP3-7A9 hybridoma cell strains can stably secrete antibody after repeated freeze thawing for 15 times and continuous passage for more than 15 generations, and titer is still as high as 1: 16000; the purity of the monoclonal antibody obtained by preparing ascites is up to more than 90 percent after purification, and an electrophoretogram is shown in figure 1.

After desalination and concentration, the concentrations of the SVA-VP2-6D5 and SVA-VP3-7A9 monoclonal antibodies are 6.2mg/mL and 5.7mg/mL respectively, and the titers are 1: 10 ten thousand and 1: 4 ten thousand.

Example 2 identification of monoclonal antibodies against SVA rVP2, rVP3 proteins

1. Western blot analysis

The rVP2, rVP3 protein and pET32a (+)/BL21 empty carrier protein are used as antigens, the rVP2(SVA-VP2-6D5) and rVP3(SVA-VP3-7A9) monoclonal antibodies prepared in example 1 are used as primary antibodies, the HRP-labeled goat anti-mouse IgG is used as an enzyme-labeled secondary antibody, and Western blot is used for identifying the reactivity of the monoclonal antibodies, and the specific steps are as follows:

1.1 sample preparation: sucking a certain amount of sample, adding 4 Xloading buffer solution, mixing, boiling for 10 min, and centrifuging at 10000r/min for 1 min.

1.212% separation gel formulation (15.08 mL): 5.1mL of double distilled water, 6.0mL of 30% acrylamide stock solution, 3.8mL of Tris-HCl (1.5mol/L, pH 8.8), 0.15mL of 10% SDS, 0.1mL of 10% ammonium persulfate, and 0.005mL of TEMED.

1.35% concentrated gum formulation (6.09 mL): 4.2mL of double distilled water, 1.0mL of 30% acrylamide stock solution, 0.82mL of Tris-HCl (1.0mol/L, pH 6.8), 0.06mL of 10% SDS, 0.06mL of 10% ammonium persulfate, and 0.01mL of TEMED.

1.4 glue filling: slowly loading 12% of separation gel into an electrophoresis plate, slowly adding 5% of concentrated gel after solidification, and quickly inserting into a comb; after the concentrated gel is solidified, the electrophoresis plate is placed into an electrophoresis tank, a proper amount of electrophoresis buffer solution is added, a comb is taken out, and the sample is loaded at 10 mu l/hole.

1.5 switching on the power supply, carrying out electrophoresis under the condition of constant current of 15mA until bromophenol blue moves to the bottom of the electrophoresis plate, terminating electrophoresis, unloading the electrophoresis tank, and lightly taking out the gel plate.

1.6 Polyacrylamide gel-film "sandwiches" were prepared in the following order: filter paper-gel-PVDF membrane-filter paper.

A clean glass rod was gently rolled through the gel-film "sandwich" to eliminate air bubbles between layers. The sponge pad and filter paper, PVDF membrane were equilibrated in transfer buffer for 10 minutes in advance. Transferring the fixed gel-membrane sandwich into an electrotransformation instrument, wherein the gel side faces to a negative electrode, the PVDF membrane side faces to a positive electrode, connecting a cooling device, and transferring at a constant current of 200mA for 1 hour.

1.7 taking down the PVDF membrane after the transfer is finished, and sealing: putting the PVDF membrane into a glass plate with a proper size, and sealing the PVDF membrane for 12 hours at the temperature of 2-8 ℃ by using 5% skimmed milk powder-TBST;

1.8 washing membrane I: removing the confining liquid, and slowly shaking and washing the membrane with TBST for three times, 5 minutes each time; adding a primary antibody: the rVP2 and rVP3 monoclonal antibodies (rVP2 at an initial concentration of 6.2mg/mL and rVP3 at an initial concentration of 5.7mg/mL, both diluted 1: 2000-fold) obtained in example 1 were each used as primary antibodies, and were slowly shaken for 2 hours; and (3) washing a membrane II: discarding the primary antibody, and slowly shaking and washing the membrane with TBST for 3 times, 5 minutes each time; adding a secondary antibody: adding horse radish peroxidase labeled goat anti-mouse IgG (initial concentration 1mg/ml, 1:2000 times dilution) into TBST, and slowly shaking for 0.5 h; washing a membrane III: discarding the secondary antibody, and slowly shaking and washing the membrane with TBST for 3 times, 5 minutes each time;

1.9 substrate: 12mg of DAB was weighed out and dissolved in 20mL of TBST, filtered and then 60. mu.l of 30% H was added₂O₂And placing the PVDF membrane in a developing solution, and immediately immersing the PVDF membrane into double distilled water to wash the PVDF membrane after a specific reaction strip appears to terminate the reaction. And (5) absorbing water on the dry film by using absorbent paper, keeping dry in the dark, observing and recording the result.

Western blot results show that the SVA-VP2-6D5 monoclonal antibody can specifically react with rVP2 protein, the SVA-VP3-7A9 monoclonal antibody can specifically react with rVP3 protein, and the non-specific cross reaction does not exist with pET32a/BL21 empty vector, and the results are shown in FIG. 2 and FIG. 3.

2. Identification of specificity

The specificity of the monoclonal antibody is identified by using a detection method established by using an escherichia coli BL21pLysS mycoprotein envelope plate and a porcine pseudorabies gB, foot-and-mouth disease O/A type, porcine reproductive and respiratory syndrome virus and SVDV antibody detection kit envelope plate, goat anti-mouse IgG (HRP mark) is used as an enzyme-labeled secondary antibody, and a negative control is arranged for analysis. The negative control was negative mouse serum.

The results show that the SVA-VP2-6D5 and SVA-VP3-7A9 monoclonal antibodies can respectively react with rVP2 and rVP3 specifically, and do not react with Escherichia coli mycoprotein, inactivated SVDV, porcine pseudorabies gB, foot-and-mouth disease O/A type and porcine reproductive and respiratory syndrome virus pathogens in a non-specific way, which is shown in Table 1.

TABLE 1 identification of the specificity (OD) of the monoclonal antibodies₄₅₀Value)

Note: the S/N value (the ratio of the OD value of the sample to the negative control) is less than 2.1, and the sample is judged to be negative to the antibody; the S/N value is more than or equal to 2.1, and the sample is judged to be positive by the antibody.

3. Application assay

Taking the inactivated and concentrated SVA as a coating antigen, respectively adding SPF pig serum, 50 parts of SVA positive pig serum and 50 parts of VA negative pig serum, adding the monoclonal antibody to be detected, finally adding goat anti-mouse IgG (HRP mark) as an enzyme-labeled secondary antibody, and reading OD (optical density) after color development termination₄₅₀. OD when added into serum well of SVA positive pig serum₄₅₀The value is lower than the OD of the porcine serum well added with SPF₄₅₀When the value is about half, the SVA monoclonal antibody can be specifically blocked by immune antibodies in pig serum, namely the SVA monoclonal antibody is a monoclonal antibody aiming at a specific SVA antigenic site and can be applied to a subsequent establishment method.

Detecting, adding 50 parts of OD of SVA immune pig serum hole₄₅₀The values are all lower than the OD of the pig serum added with SPF and 50 SVA negative pig serum₄₅₀Half the value, see table 2. The monoclonal antibodies SVA-VP2-6D5 and SVA-VP3-7A9 can be specifically combined with the antigenic sites of SVA, and can be used for application of subsequent method establishment.

TABLE 2 determination of the applicability of monoclonal antibodies (OD)₄₅₀Value)

Example 3 establishment of SVA antibody competitive chemiluminescence enzyme-linked immunoassay method (CLEIA)

1. Determination of optimum concentration

Diluting the purified SVA inactivated concentrated virus protein by taking a carbonate buffer solution with the pH of 9.6 as a coating buffer solution according to the concentrations of 2 mug/mL, 1 mug/mL, 0.5 mug/mL and 0.25 mug/mL respectively, coating a chemiluminescence plate with the SVA inactivated concentrated virus protein, mixing 5 parts of SVA positive pig serum, 5 parts of SVA negative pig serum and an enzyme-labeled monoclonal antibody 1:1 containing an HRP-labeled monoclonal antibody respectively, adding 100 mug L of the mixture into the fluorescence plate, placing the mixture in the fluorescence plate, reacting at 37 ℃ for 30min, adding a luminol chemiluminescence substrate after PBST washing, determining the result by a chemiluminescence immunoassay analyzer, and determining the concentration corresponding to the maximum N/P chemiluminescence value ratio as the optimal coating concentration.

After the detection of horseradish peroxidase-labeled anti-SVA rVP2 protein monoclonal antibody (SVA-VP2-6D5), the enzyme amount is 1.387mg/mL, the IgG amount is 3.42mg/mL, and the gram-molecule ratio (E/P) is 1.62; after the monoclonal antibody (SVA-VP3-7A9) of the anti-SVA rVP3 protein is labeled by horseradish peroxidase, the enzyme amount is 0.913mg/mL, the IgG amount is 2.087mg/mL, and the gram-molecule ratio (E/P) is 1.75. And (3) diluting the horseradish peroxidase-labeled anti-SVA rVP2 and SVA rVP3 protein monoclonal antibodies according to the ratio of 1: 500, 1: 1000, 1:2000 and 1: 4000 (the diluent is a Tris-cl solution containing 3% Bovine Serum Albumin (BSA)), and determining the optimal working concentration of the enzyme-labeled monoclonal antibody according to the maximum ratio of the chemiluminescence value of N/P.

The rVP2 and rVP3 monoclonal antibodies are labeled by horseradish peroxidase and then the detected titers are 1:12000 and 1:8000 respectively, so that the subsequent research is carried out by adopting a coordination mode that the concentration of rVP2 is lower than that of rVP 3.

As a result: the value of the yin-yang ratio N/P is more than 1 along with the dilution of the rVP2 enzyme-labeled monoclonal antibody: after 4000, the dilution degree is reduced and decreased, and the dilution degree of the enzyme-labeled monoclonal antibody of rVP3 is more than 1: the concentration of the SVA whole virus protein coating is 1 mug/mL, and the N/P value is the maximum and is 94.3 when the dilution of the rVP2 and the rVP3 enzyme-labeled monoclonal antibodies are 1: 4000 and 1:2000 respectively, and the result is shown in Table 3. Therefore, the optimal coating concentration is determined to be 1 mug/mL, the dilution of the rVP2 enzyme-labeled monoclonal antibody is 1: 4000, and the dilution of the rVP3 enzyme-labeled monoclonal antibody is 1: 2000.

TABLE 3 determination of optimal concentration of SVA antibody to compete for CLEIA

2. Determination of optimal enzyme-labeled monoclonal antibody diluent

Tris-cl containing 15% fetal calf serum, 1.5% casein, 3% Bovine Serum Albumin (BSA) and 1% tryptose peptone is respectively used as enzyme-labeled monoclonal antibody diluent, and the enzyme-labeled monoclonal antibody diluent corresponding to the maximum N/P chemiluminescence value ratio is used as the optimal enzyme-labeled monoclonal antibody diluent by the method adopted when the optimal concentration is determined.

The detection result shows that the enzyme-labeled monoclonal antibody diluent containing 1.5% casein effectively improves the sensitivity, the N/P value is the largest, and the result is shown in table 4, so that a Tris-cl solution containing 1.5% casein is used as the enzyme-labeled monoclonal antibody diluent.

TABLE 4 determination of enzyme-labeled monoclonal antibody dilutions

3. Determination of optimal reaction time

Mixing the serum to be detected and the enzyme-labeled monoclonal antibody at a ratio of 1:1, adding the mixture into a luminescent plate coated with SVA protein, reacting at 37 ℃ for 15min, 30min and 60min respectively, performing 2 sets of repeated tests, adding a luminescent substrate, and then performing light-shielding action at 15-25 ℃ for 3 min, 5min and 10 min respectively, wherein each time point is repeated for 2 sets, and the time corresponding to the maximum chemiluminescence value N/P ratio is the optimal reaction time and the optimal substrate action time of the serum to be detected and the enzyme-labeled monoclonal antibody.

The detection result shows that when the serum to be detected, the enzyme-labeled monoclonal antibody and the antigen act for 30min at 37 ℃, the luminescent substrate is added to act for 5min, the N/P value is the maximum, and the N/P value is shown in tables 5 and 6, so that the optimal acting time is 30min +5 min.

TABLE 5 optimal reaction time of enzyme-labeled monoclonal antibody, serum to be detected and antigen

TABLE 6 optimal action time of the substrate

4. Preparation of calibrator

The neutralization titer was determined by a menses serum neutralization test to be 1:2⁹The serum positive to the Seneca virus antibody is used as the traceable serum of a calibrator, the value is assigned to 5000NCU/mL, the serum is diluted by the determined enzyme-labeled monoclonal antibody diluent in a multiplying ratio, the luminous value of the diluted serum is determined, the dosage value of the antibody is used as the abscissa, the luminous value is used as the ordinate, ELISACalc software is used for analysis, and a calibration curve is fitted by a four-parameter curve fitting method. Selecting 5 characteristic points from the curve, diluting the standard positive serum according to the dosage value by using an enzyme-labeled monoclonal antibody diluent according to the proportion respectively to be used as a calibrator 2-6, and diluting the SPF pig serum by 1:20 times by using the enzyme-labeled monoclonal antibody diluent to prepare a calibrator 1. And (4) according to the traceable serum detection value of the calibrator and a calibration curve drawn by the calibrator, calculating the relationship of the dosage value of the antibody, quantitatively subpackaging the calibrator after the calibration is qualified, and storing the calibrator below-20 ℃ for later use.

The concrete result is as follows: and (3) performing 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 and 2048-fold dilution on the traceable serum of the calibrator to draw a four-parameter fitting curve of the calibrator. According to the curve, the luminescence values are most obviously distinguished when the antibody dosage value is less than or equal to 100NCU/mL, therefore, serum samples with different antibody dosage values are sequentially prepared by taking 100NCU/mL as a starting point (the retrospective serum of the calibrator is diluted by 50 times), four-parameter fitting curves are repeatedly drawn, when the antibody dosage values are 5NCU/mL, 12NCU/mL, 25NCU/mL, 50NCU/mL and 100NCU/mL, the corresponding coordinate points have the best repeatability, and r of the curves has the best repeatability²All > 0.99, see FIG. 4. Three batches of calibrators were prepared, and the deviation between the antibody dose value of the prepared calibrators and the theoretical antibody dose value was within 5.0%, and the results are shown in table 7.

TABLE 7 comparison of theoretical antibody dosage value and measured dosage value of prepared calibrator

5. Determination of a threshold value

Detecting 243 pig serum with clear background in SVA vaccine clinical test by determined optimal reaction conditions and reagents, and identifying the pig serum with clear background to be positive SVA antibody by using a serum neutralization test; detecting and collecting 181 parts of pig serum without SVA vaccine immunization, and identifying the pig serum to be SVA antibody negative by using a serum neutralization test; the serum neutralization test is used for detecting that the SVA antibody titer is less than 2⁵30 positive sera were assayed and the antibody dose value, expressed in NCU/ml, was calculated from the standard curve obtained above. Antibody dose values for all serum samples were analyzed using SPSS 16.0 software. An ROC curve is constructed by a nonparametric method, and a point with the highest Youden index (Youden index ═ sensitivity- (1-specificity)) is used as a critical point for positive and negative judgment, and the corresponding antibody dose value is the critical value.

The detection results of all the samples were subjected to data analysis using SPSS 16.0 software, and an ROC curve was plotted with sensitivity as ordinate and 1-specificity as abscissa, as shown in fig. 5. The Youden index (Youden index ═ sensitivity- (1-specificity)) was calculated for each cut-off value, with a maximum Youden index of 0.974 and a corresponding antibody dose value of 10.2NCU/mL, see table 8 (only data near the critical cut-off values are shown). Therefore, the critical value of the method is determined to be 10, and finally the determination criterion of the method is determined to be: when the dosage value of the sample antibody is more than or equal to 10NCU/mL, the sample antibody is positive for SVA antibody; when the sample antibody dosage value is less than 10NCU/mL, the sample is SVA antibody negative. Under this criterion, the cut-off corresponds to a sensitivity of 98.0% and a specificity of 99.4%.

TABLE 8 sensitivity, specificity and Youden index for different dosage values

6. Finally established detection method

According to the steps, the invention establishes a complete SVA antibody competition CLEIA detection method, which comprises the following steps:

6.1 Balancing

Before use, the coated plate, enzyme-labeled monoclonal antibody suspension (the mixed solution of two enzyme-labeled rVP2 and rVP3 monoclonal antibodies with specific concentration/dilution prepared by Tris-cl solution of 1.5% casein determined by the method of the invention) and luminescent substrate are taken out from a refrigerated cabinet, balanced to room temperature, and the calibrators 1 to 6 are gently rotated or shaken and uniformly mixed before use.

6.2 preparation of washing working solution

Before use, 25-fold dilution of the concentrated PBST washings with purified or distilled water was carried out 25-fold. 1 part of 25-fold concentrated washing solution to 24 parts of distilled water (e.g., 40ml of 25-fold concentrated washing solution to 960ml of distilled water).

6.3 working procedure

6.3.1 sample application: and (4) taking out the antigen coated plate. Each experiment required a series of 6 wells of calibrator. Firstly, adding 60 mu l of sample into each hole of a serum dilution plate sample, adding 60 mu l of calibrator into each hole of the calibrator, adding 60 mu l of enzyme-labeled monoclonal antibody suspension into each hole, and uniformly mixing by oscillation; each well was pipetted 100. mu.l to the corresponding well of the coated plate.

6.3.2 incubation: incubate at 37 ℃ for 30 minutes (+ -1 minute).

6.3.3 plate washing: the reaction plate was removed, the reaction solution was discarded, and 300. mu.l of washing solution was added to each well to wash the plate well for a total of 5 times. The liquid in the wells should be discarded after each wash. After the last wash was discarded, the residual wash in the wells was patted dry on absorbent paper.

6.3.4 addition of substrate: add 50. mu.l luminescent substrate A and 50. mu.l luminescent substrate B to each well, shake and mix (or add 100. mu.l luminescent substrate A, B after mixing in equal proportion). The luminescent substrate A is luminol solution (24.23 g of trihydroxymethyl aminomethane is weighed and dissolved in 800ml of pure water to be fully stirred uniformly, the pH value is adjusted to 8.0, 0.026g of luminol dry powder is added, purified water is added to the mixture to be constant volume to 1000ml after the luminol dry powder is completely dissolved), and the luminescent substrate B is amino acid oxidase solution (15.42 g of ammonium acetate is weighed and dissolved in 800ml of pure water to be adjusted to pH value to 5.2, 0.066g of amino acid oxidase is added, and then purified water is added to be constant volume to 1000 ml).

6.3.5 standing: the mixture is placed at room temperature (15-25 ℃) in a dark place for 5 minutes.

6.3.6 reading: the luminescence values were read with a chemiluminescence apparatus.

6.4 judging the result: the four-parameter fitting curve of the calibrator was plotted by ELISA Calc software using the luminescence values of the calibrators 1 to 6 as ordinate and the corresponding antibody dose values (0NCU/ml, 5NCU/ml, 12NCU/ml, 25NCU/ml, 50NCU/ml, 100NCU/ml) as abscissa.

6.5 conditions are satisfied: the antibody dosage values and the luminous values corresponding to the calibrators 1 to 6 are fitted by a calibration curve, and when the correlation coefficient r of the calibration curve is²The experiment was established when the value was 0.99 or more. Otherwise, it should be re-detected.

6.6 decision criteria: when the antibody dosage value of the sample is less than 10NCU/ml, the sample is judged to be SVA antibody negative; if the antibody dose value of the sample is more than or equal to 10NCU/ml, the sample is determined to be SVA antibody positive.

Example 4

1. Specificity test

The SVA antibody competition CLEIA established in the embodiment 3 is used for respectively detecting 10 parts of FMDV O type, FMDV A type, SVDV, PRV and PRRSV positive serum and 2 parts of escherichia coli positive serum, and simultaneously SVA serum neutralization test is used for parallel detection to verify the specificity of the establishment method.

The results show that, as shown in Table 9, the established SVA antibody competes with CLEIA to detect FMDV O type, FMDV A type, SVDV, PRV, PRRSV positive serum and Escherichia coli positive serum respectively, which are all negative, have no cross reaction and are consistent with the result of the serum neutralization test. In Table 9, the contents of the calibrators 1 to 6 are specifically described in "4" and preparation of calibrators "in example 3.

TABLE 9 results of specificity test

Note: the result of the serum neutralization test is judged asSerum antibody titer is less than 1:2⁴Is negative, and the serum antibody titer is more than or equal to 1:2⁴It is positive.

2. Sensitivity test

Diluting the traceable serum of the calibrator with an enzyme-labeled monoclonal antibody diluent according to the ratio of 1:16 to 1: 32 to 1: 64 to 1: 128 to 1: 256 to 1: 512 to 1: 1024 to 1: 2048 to 1: 4096, respectively using the established SVA antibody to compete for CLEIA for detection, and simultaneously using a serum antibody neutralization test for identification to verify the sensitivity of the established method.

The detection results of the established SVA competitive CLEIA and the neutralization test are consistent, and the calibrator retrograded serum with the maximum dilution multiple of 1: 2048 can be detected, and the results are shown in Table 10.

TABLE 10 results of sensitivity test

Note: the determination result of the serum neutralization test is that the serum antibody titer is less than 1:2⁴Is negative, and the serum antibody titer is more than or equal to 1:2⁴It is positive. In table 10, the contents of the calibrators 1 to 6 are described in example 3 "4" and preparation of calibrators ".

3. Repeatability test

3 batches of SVA antibody competition CLEIA reagent are used for respectively detecting SVA antibody negative, strong positive and weak positive samples determined by serum antibody neutralization test (SVA antibody titer is less than 2 determined by serum neutralization test)⁵Positive sera) were tested in 5 replicates, the results were statistically analyzed, and the Coefficient of Variation (CV) between batches was calculated (standard deviation/average) × 100%.

The detection results are shown in tables 11 to 14, and the coefficient of variation between the inner analysis and the analysis of the established SVA competition CLEIA is between 2.11 percent and 8.43 percent and is less than 10 percent; the inter-batch variation coefficient is between 3.69 and 12.16 percent and is less than 15 percent.

TABLE 1120200601 batch repeatability test results

TABLE 1220200602 batch repeatability test results

TABLE 1320200603 batch repeatability test results

TABLE 143 kit for detecting the coefficient of variation of the same 13 serum dishes

4. Study of shelf life

Assembling and matching the sample diluent, the packaged coated luminescent plate, the calibrator, the enzyme-labeled monoclonal antibody solution and the luminescent substrate, randomly extracting 3 sets of reagents from 3 batches of SVA antibody competitive CLEIA reagents, respectively storing at 2-8 ℃, respectively inspecting for 1, 2, 3, 6, 9, 12 and 15 months, and determining the storage period of the reagent according to the sensitivity and specificity test results.

3 sets of SVA competition CLEIA reagents of different batches are placed and stored at 2-8 ℃ for 15 months, positive traceable serum with the maximum dilution multiple of 1: 2048 can be detected, FMDV O type, FMDV A type, SVDV, PRV, PRRSV positive serum and Escherichia coli positive serum are detected, the dosage values are all less than 10NCU/ml, and no cross reaction exists. Thus, the shelf life of the method can be determined to be 12 months.

Example 5 comparative experiment

And respectively adopting the established SVA antibody competitive CLEIA and serum neutralization tests to respectively detect 480 pig sera collected from different pig farms, and determining the coincidence rate between the established method and the neutralization tests.

The detection results show that, as shown in table 15, the positive coincidence rate of the established SVA competitive CLEIA detection result and the neutralization test is 95.33%, the negative coincidence rate is 97.58%, and the total coincidence rate is 96.88%.

TABLE 15 comparison of SVA Competition CLEIA detection methods with neutralization test results

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A hybridoma cell strain is characterized in that the hybridoma cell strain is named as SVA-VP2-6D5 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021277; or is named as SVA-VP3-7A9 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021276.

2. The hybridoma cell is characterized by comprising a hybridoma cell strain SVA-VP2-6D5 and a hybridoma cell strain SVA-VP3-7A 9; the hybridoma cell strain SVA-VP2-6D5 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021277, and the hybridoma cell strain SVA-VP3-7A9 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021276.

3. A monoclonal antibody produced by the hybridoma cell line of claim 1 or the hybridoma cell of claim 2.

4. Use of the hybridoma cell strain of claim 1, the hybridoma cell of claim 2 or the monoclonal antibody of claim 3 in the preparation of a reagent or a kit for detecting Seneca virus type A.

5. A reagent or a kit for detecting Selenecar virus type A, comprising the monoclonal antibody according to claim 3.

6. The reagent or the kit according to claim 5, which comprises two enzyme-labeled monoclonal antibodies, wherein the two monoclonal antibodies are respectively rVP2 monoclonal antibody and rVP3 monoclonal antibody, the rVP2 monoclonal antibody is produced by hybridoma cell strain SVA-VP2-6D5, and the rVP3 monoclonal antibody is produced by hybridoma cell strain SVA-VP3-7A 9;

preferably, the mass ratio of the rVP2 monoclonal antibody to the rVP3 monoclonal antibody is 1: (1.1-1.8), more preferably 1: 1.2;

and/or the kit also contains an enzyme-labeled monoclonal antibody diluent; the enzyme-labeled monoclonal antibody diluent is a Tris-cl solution containing 1.4-1.6%, preferably 1.5% casein.

7. An antibody competitive chemiluminescent enzyme-linked immunosorbent assay for the detection of Selenecar virus type A, characterized in that the monoclonal antibody of claim 3 is used as a component of a competitive antibody.

8. The method of claim 7, wherein the competing antibodies are an enzyme-labeled rVP2 monoclonal antibody and an enzyme-labeled rVP3 monoclonal antibody, wherein the rVP2 monoclonal antibody and the rVP3 monoclonal antibody are as set forth in claim 6, and wherein the mass ratio of the rVP2 monoclonal antibody to the rVP3 monoclonal antibody is 1: (1.1-1.8), preferably 1: 1.2.

9. The method according to claim 7 or 8, characterized in that the concentration of the rVP2 monoclonal antibody in the enzymatically labelled rVP2 monoclonal antibody is 0.75-1 μ g/mL, preferably 0.86 μ g/mL, when binding to the coating antigen; the concentration of rVP3 monoclonal antibody in the enzyme-labeled rVP3 monoclonal antibody is 1.7-2.65. mu.g/mL, preferably 2.1. mu.g/mL.

10. The method according to any one of claims 7 to 9, wherein the antigen is coated at a concentration of 0.9 to 1.1 μ g/mL, preferably 1 μ g/mL;

and/or the action time of the sample to be detected, the competitive antibody and the coating antigen is 28-32min, preferably 30 min;

and/or the action time of the luminescent substrate is 4.5-5.5min, preferably 5 min.

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