CN117025545B - Hybridoma cell strain secreting highly pathogenic porcine reproductive and respiratory syndrome virus specific monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting highly pathogenic porcine reproductive and respiratory syndrome virus specific monoclonal antibody and application thereof Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention discloses a hybridoma cell strain secreting a highly pathogenic porcine reproductive and respiratory syndrome virus specific monoclonal antibody and application thereof, and belongs to the field of biological medicine. The hybridoma cell strain XX10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45495 and the preservation time of 2023, 03 and 03 days. The hybridoma cell strain XX10 can stably secrete a monoclonal antibody specifically recognizing HP-PRRSV, and can be used for establishing a competition ELISA method specifically aiming at HP-PRRSV. The antibody differential diagnosis kit established based on XX10 can distinguish infection of HP-PRRSV and other types of PRRSV, has strong specificity and high sensitivity, and fills up the blank of the HP-PRRSV specific detection method.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a hybridoma cell strain secreting a highly pathogenic porcine reproductive and respiratory syndrome virus specific monoclonal antibody and application thereof.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an acute infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) that is primarily characterized by sow abortion and piglet respiratory symptoms. PRRSV contains two genotypes, PRRSV-1 and PRRSV-2, respectively, PRRSV-1 is further classified into subtype 1 (subtype I (Global)), subtype 2 (subtype I (Russa) and subtype III) and subtype 3 (subtype III), PRRSV-2 is further classified into linear 1 to 9, linear 1 is further classified into linear 1.1 to 1.9 or L1A to 1H, linear 3 is further classified into L3.1 to 3.5, linear 5 is further classified into L5.1 to 5.2, and linear 8 is further classified into L8.1 to L8.9. At present, PRRSV in China has the coexistence of double genotypes and multiple genotypes. In order to control the harm of PRRSV to the pig industry in China, the agricultural sector approves a plurality of vaccines including classical inactivated vaccine CH-1a strain, classical live vaccine CH-1R strain, respPRRS MLV strain, R98 strain, highly pathogenic live vaccine JXA1-R strain, huN-F112 strain, TJM strain and GDr180 strain, and chimeric vaccine PC strain.
At present, a plurality of PRRSV antibody detection kits mainly aim at broad-spectrum PRRSV antibodies, and HP-PRRSV live vaccines are widely applied in China, however, a method for specifically detecting the HP-PRRSV antibodies is not available, so that the infection of HP-PRRSV and other types of PRRSV cannot be accurately distinguished.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain secreting a monoclonal antibody specific to highly pathogenic porcine reproductive and respiratory syndrome virus and application thereof, so as to solve the problems in the prior art. The XX10 hybridoma cell strain can stably secrete the monoclonal antibody capable of specifically recognizing the HP-PRRSV, and can be used for establishing a competition ELISA method for specifically detecting the HP-PRRSV antibody.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a hybridoma cell strain secreting a high pathogenicity porcine reproductive and respiratory syndrome virus specific monoclonal antibody, named as mouse monoclonal antibody hybridoma cell strain XX10, which is preserved in China general microbiological culture collection center (CGMCC) No.45495, and the preservation time is 2023 and 03 months 03.
The invention also provides an antibody secreted by the hybridoma cell line XX10 and resisting the highly pathogenic porcine reproductive and respiratory syndrome virus.
The invention also provides an application of the hybridoma cell strain XX10 or the antibody in preparation of a product for detecting the porcine reproductive and respiratory syndrome virus.
Further, the highly pathogenic porcine reproductive and respiratory syndrome virus is an HP-PRRSV strain.
The invention also provides an antibody competition ELISA kit for detecting the highly pathogenic porcine reproductive and respiratory syndrome virus, which comprises the antibody.
Further, an ELISA plate coated with inactivated highly pathogenic porcine reproductive and respiratory syndrome virus is included; in ELISA plates, the highly pathogenic porcine reproductive and respiratory syndrome virus is HuN-F112 strain with the preservation number of CGMCC NO.2484.
Further, the inactivated highly pathogenic porcine reproductive and respiratory syndrome virus has a coating concentration of 300 ng/. Mu.L.
Further, the kit also comprises goat anti-mouse IgG marked by horseradish peroxidase, a blocking solution, a washing solution, a diluting solution, a color development solution and a stop solution.
Further, the horseradish peroxidase-labeled goat anti-mouse IgG is diluted by a PBST solution with a dilution multiple of 10000 times.
Further the blocking solution was a 1% w/v BSA solution.
The invention discloses the following technical effects:
the invention discloses a hybridoma cell strain XX10 secreting a highly pathogenic porcine reproductive and respiratory syndrome virus specific monoclonal antibody and establishment of a competition ELISA method thereof. The XX10 hybridoma cell strain can stably secrete the monoclonal antibody specifically recognizing the HP-PRRSV, and can be used for establishing a competition ELISA method specifically aiming at the HP-PRRSV.
The XX10 hybridoma cell strain and the monoclonal antibody secreted by the same can be used for preparing a competition method antibody diagnosis kit, and the antibody differential diagnosis kit established based on XX10 can distinguish infection of HP-PRRSV and other types of PRRSV, has strong specificity and high sensitivity, and fills up the blank of a HP-PRRSV specific detection method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the identification of immune effects in mice;
FIG. 2 shows IFA results after subcloning of monoclonal antibodies;
FIG. 3 shows the result of the IFA reaction of monoclonal antibody XX10 with each type of PRRSV;
FIG. 4 shows the purification results of monoclonal antibody XX 10;
FIG. 5 is a HP-C-ELISA negative and positive sample threshold analysis, wherein A: critical value ROC curve analysis, B: analyzing interaction points of the critical values;
FIG. 6 shows the results of HP-C-ELISA specificity;
FIG. 7 shows the specificity of the competition ELISA method (A) established by the HP-C-ELISA monoclonal antibody compared with the commercially available kit (B);
FIG. 8 shows the sensitivity of the competition ELISA method (A-B) established by the HP-C-ELISA monoclonal antibody compared with the sensitivity of the commercial kit (C-D).
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 preparation of monoclonal antibodies specifically recognizing HP-PRRSV
1 Material
1.1 viruses, cells and laboratory animals
The viral antigens used for immunization were strain HuN-F112, strain JXA1-R and strain TJM, strain JXA1-R purchased from Guangdong Yongshun biopharmaceutical Co., ltd; TJM strain was purchased from the company of bio-pharmaceuticals, inc. Strains for immunofluorescence for monoclonal antibody specific analysis include: huN4 strain F112 (HP-PRRSV, linear 8.7), from the China general microbiological culture Collection center, accession number: CGMCC No.2484.
CH-1R strain (classical strain, linear 8.7), SD-R strain (NADC 30-like PRRSV, linear 1.8), LNTZJ1341 (NADC 34-like PRRSV, linear 1.5), HNTZJ1714 (QYZ-like PRRSV, linear 3.5), ZD1 strain (PRRSV-1, subtype 1) were isolated and identified by Harbin veterinary institute and have been disclosed in Chinese patent No. CN113801854B in hybridoma cell lines secreting European porcine reproductive and respiratory syndrome virus specific monoclonal antibodies and applications thereof.
RespPRRS MLV strain (classical American strain, linear 5.1) live vaccine was purchased from Boringer John biopharmaceutical (China).
Haemophilus Parasuis (HPS), streptococcus (SS), infectious pleuropneumonia (APP), transmissible gastroenteritis virus (TGEV), porcine Epidemic Diarrhea Virus (PEDV), porcine pseudorabies virus (PRV), swine fever virus (CSFV), foot and Mouth Disease Virus (FMDV), european PRRSV ZD-1 strain, NADC30-like PRRSV SD-R strain, WK108 strain, VR2332-like PRRSV SD192 strain, NADC34-like PRRSV ZD32-1901 strain, classical strain CH-1R strain, HP-PRRSV HuN4-F112 strain positive serum is provided by the haerbin veterinary research and has been disclosed in the chinese patent "CN113801854B hybridoma cell line secreting monoclonal antibodies specific for european porcine reproductive and respiratory syndrome virus" and uses thereof. The JXA1-R strain and TJM strain positive serum is prepared from the JXA1-R strain and TJM strain immune double-negative piglets.
Marc-145, SP2/0 myeloma cells of African green monkey kidney cells are provided by the pig disease comprehensive prevention and control laboratory of the Harbin veterinary institute, and PRRSV-2 monoclonal antibody 1C8 is disclosed in the "Characterisation ofnovel linear antigen epitopes on North American-type porcine reproductive and respiratory syndrome virus M protein" literature; PRRSV-1 monoclonal antibody EU1 has been disclosed in the literature "establishment and use of European PRRSV competition ELISA antibody detection methods", clean-grade female BALB/c mice were purchased from Vetong LiHua Corp.
1.2 major reagents
Fusion agents PEG/DMSO (Mw, 1450), HAT salt (50 x), HT salt (50 x), FITC-labeled goat anti-mouse IgG, freund's complete adjuvant, and freund's incomplete adjuvant, all purchased from Sigma; HRP-labeled goat anti-mouse IgG was purchased from soribao corporation; DMSO was purchased from Ameresco company; DMEM is available from Gibco company; inlet quality fetal bovine serum (PAA) was purchased from Nalgene, spain; 96 well cell culture plates were purchased from JET biochemicals, canada; monoclonal antibody subclass identification kits were purchased from Southern Biotech company.
2 method
2.1 preparation of monoclonal antibodies
2.1.1 immunization of mice
(1) Preparation and purification of immunogens
The HP-PRRSVHuN4-F112 strain, the JXA1-R strain and the TJM strain are respectively inoculated with Marc-145 cells which grow into a monolayer, after more than 80% of the cells are diseased, the supernatant of the cultured cells is taken, the supernatant is centrifuged at 10000rpm at 4 ℃ for 50min, the supernatant is collected, and then the supernatant is subjected to super-isolation at 35000rpm at 4 ℃ for 4h. Centrifuging, discarding supernatant, dissolving and re-suspending the precipitate with PBS, re-centrifuging at low speed after re-suspending the precipitate with PBS, collecting supernatant, purifying virus by molecular sieve chromatography, ultrafiltering and concentrating the collected liquid respectively, measuring protein content by BCA protein quantitative kit, and storing at-80deg.C for use.
(2) Immunization
Mixing the purified HP-PRRSV HuN4-F112 strain, JXA1-R strain and TJM strain as immunogens, adding equal volume Freund complete adjuvant into 100 mu g/100 mu L, fully emulsifying, and injecting into 6-week-old female BALB/c mice subcutaneously through the abdomen; immunization was performed 2 weeks later, changing the adjuvant to Freund's incomplete adjuvant; after 2 weeks, immunization was performed 3 rd with the same amount of antigen without adjuvant. After 3 days of 1 week, tail venous blood was collected with an EP tube and serum isolated. The sterile PBS is utilized to perform double dilution on the mouse serum, the IFA fluorescence method is used for detecting the immune condition, the negative mouse serum is used as a negative control, the PRRSV-2 monoclonal antibody 1C8 is stored in a laboratory and used as a positive control, and the dilution degree reaches 1: obvious fluorescence was seen after 5000 as an immune success, and boost could be continued. To stimulate the mice to rapidly develop a strong immune response, boost was performed 3d prior to fusion and double doses of antigen were injected.
2.1.2 cell fusion
(1) Preparation of myeloma cells: and (3) culturing the myeloma cells in an enlarged mode 36-48 hours before fusion, so that the cells are in a logarithmic growth phase. On the day of fusion, prepared SP2/0 cells from 3 75T cell flasks were blown down with 20 mM MEM and placed in 50mL centrifuge tubes for further use.
(2) Preparation of immune spleen cells: mice were removed from their eyeballs prior to fusion to collect blood, and positive serum was collected and frozen. And (5) sterilizing in 75% alcohol for 5min after neck removal and sacrifice. The mice were lying down on the dissecting plate with the abdominal cavity facing upwards, fixing the limbs. The abdominal cavity is gently cut off by the sterilized scissors, the spleen of the mouse is found, peripheral tissues are slowly peeled off by the sterilized forceps, the spleen is taken out and placed in a cell culture dish, and then the peripheral connective tissues are completely peeled off.
A1 mL syringe needle was used to puncture a few small holes in the mouse spleen, and 20mL DMEM medium was then aspirated by the syringe and injected gently into the mouse spleen, where the spleen cells were allowed to flow out with the DMEM. The effluent DMEM was collected in a 50mL centrifuge tube for later use.
(3) Spleen cells fused with myeloma cells: SP2/0 cells and spleen cells prepared in the previous step were centrifuged at 1500rpm for 4min. After centrifugation, the supernatants were discarded, and the two cells were resuspended and mixed well with 10 mM MEM and centrifuged at 1500rpm for 4min. The supernatant was removed, the tube was shaken, the bottom cells were shaken down, and the two cells were thoroughly mixed.
The centrifuge tube is placed into a beaker filled with distilled water at 37 ℃, 1mL of PEG preheated in advance is sucked and slowly dripped into the centrifuge tube (dripping is completed in about 1 min), liquid in a mixing tube of the centrifuge tube is shaken at any time in the whole dripping process, and then the centrifuge tube is kept stand for 1.5min. 1mL of preheated DMEM is sucked and slowly dripped into the pipe at 37 ℃ for about 1min, and the pipe is continuously shaken and uniformly mixed. Then 1mL of preheated DMEM is sucked and slowly dripped into the pipe, and the dripping is completed for about 30 seconds, and the pipe is continuously shaken and uniformly mixed. The preheated DMEM was added continuously for the next 2min, mixed by shaking, the tube was filled with liquid to 10mL and centrifuged at 1500rpm for 4min. Cells were resuspended in 80mL of DMEM containing 10% clone Easy and 20% FBS and plated into 4 96 well cell culture plates 200. Mu.L per well at 37℃with 5% CO 2 Culturing in an incubator.
2.1.3 screening and subcloning of Positive hybridoma cells
And observing cells in the 96-well plate by using a microscope 7-10 d after fusion, and performing indirect immunofluorescence detection when the cells to be fused grow into cell clusters at the bottom and the size of the cell clusters reaches about 1/4 area of each well. And observing the incubated glass slide by using a fluorescence microscope, wherein the visible green fluorescence of cells in the visual field is positive, recording the position of a positive hole, and performing hybridoma cell subcloning by using a limiting dilution method and flow sorting. Subcloning the indirect immunofluorescence positive hole for 7-10 d, subcloning the indirect immunofluorescence positive hole again, repeating the whole process for 3-4 times, when the detection positive rate of the 96-well plate with the hybridoma cells reaches 100%, picking 1 hole for gradual expansion culture, and expanding to 75T cell bottles for mass freezing.
2.2 monoclonal antibody identification
2.2.1 subclass identification of monoclonal antibodies
The screened hybridoma cell lines were identified for Ig subclasses using SBA ClonotypingTM System/HRP according to the kit instructions.
2.2.2 specific identification of monoclonal antibodies
HuN4-F112 strain (HP-PRRSV, linear 8.7), JXA1-R strain (HP-PRRSV, linear 8.7), TJM strain (HP-PRRSV, linear 8.7), CH-1R strain (classical strain, linear 8.7), SD-R strain (NADC 30PRRSV, linear 1.8), LNTZJ1341 (NADC 34 SV, linear 1.5), HNTZJ1714 (QYYZ PRRSV, linear 3.5), respPRRS MLV strain (classical American strain, linear 5.1), ZD1 strain (PRRSV-1, subtype 1) were inoculated into two wells of Marc-145 cells, when 10% lesion effect (CPE) appears on the cells, washed with PBS for two times, fixed with absolute ethanol for half an hour, and washed with PBS for two times after the absolute ethanol. mu.L of monoclonal antibody 1C8/EU1 mixed supernatant and monoclonal antibody supernatant secreted by the hybridoma cell line obtained by 2.1.3 were added respectively, incubated at 37℃for 1 hour, washed 3 times with PBS, 100. Mu.L of FITC-labeled goat anti-mouse IgG antibody diluted 1:200 was added, incubated at 37℃for 30 minutes, and fluorescence intensity was observed under an inverted fluorescence microscope after washing.
2.2.3 identification of sensitivity of monoclonal antibodies
Taking the culture supernatant of hybridoma cell strain cells, diluting the cell culture supernatant by a ratio of 2 times with DMEM for later use.
The inactivated and purified HuN-F112 virus was diluted with carbonate buffer (0.05 mol/L, pH 9.6), added to a 96-well ELISA plate, 100. Mu.L/well, and coated overnight at 4 ℃. The wells were discarded, and 300. Mu.L of PBST was added to each well and washed 3 times repeatedly, and the wells were dried by pipetting. mu.L of 1% BSA blocking solution was added to each well, and the wells were blocked at 37℃for 2 hours. The wells were discarded, and 300. Mu.L of PBST was added to each well and washed 3 times repeatedly, and the wells were dried by pipetting.
100 mu L of diluted hybridoma cell strain culture supernatant is added into each hole, DMEM is used as negative control, and the mixture is incubated for 30min at 37 ℃. The wells were discarded, and 300. Mu.L of PBST was added to each well and washed 3 times repeatedly, and the wells were dried by pipetting. 100 mu L of goat anti-mouse IgG enzyme-labeled antibody (diluted 1:10000 times) was added to each well, and incubated at 37℃for 30min. The wells were discarded, 300 μl of PBST was added to each well, washing was repeated 3 times, and the wells were dried. 100. Mu.L of TMB substrate solution was added to each well, and the wells were incubated at 37℃for 15min. Adding 50 mu L of stop solution into each hole, slightly vibrating and uniformly mixing, and reading OD (optical density) at the wavelength of 450nm by using an enzyme-labeled instrument 450nm Values (readings should be completed within 15min after addition of stop solution) and the results recorded. The P/N ratio is greater than 2.1 (P/N=sample OD to be tested) 450nm Value/negative control OD 450nm Value), positive.
2.2.4 purification of monoclonal antibodies specifically recognizing HP-PRRSV
The method adopts octanoic acid-saturated ammonium sulfate to purify the prepared ascites, and the operation steps are briefly described as follows:
(1) Adding 6mL of 0.06mol/LPH4.8 acetic acid buffer solution into 3mL of pretreated ascites; adding 99 mu L of octanoic acid into ascites, stirring for 30 minutes at room temperature, and standing at 4 ℃ for more than 2 hours; taking out 11000rpm, centrifuging for 30 minutes, and discarding the precipitate; the supernatant was adjusted to pH7.4 with 2 mol/LNaOH;
(2) Adding saturated ammonium sulfate to 50% saturation at 4deg.C, stirring in ice bath for 30min, and standing at 4deg.C for more than 2 hr; centrifuging at 11000rpm for 30min, and discarding supernatant; the pellet was dissolved in 5mL of 0.1M PBS at pH 7.4;
(3) Adding a proper amount of saturated ammonium sulfate solution into the precipitate suspension while stirring to ensure that the concentration of the ammonium sulfate solution is 30% -40%, stirring in an ice bath for 30 minutes, and standing at 4 ℃ for more than 2 hours; centrifugation at 11000rpm for 30min, taking the pellet and dissolving the pellet in 2ml of 0.1m PBS ph 7.4;
(4) The precipitate suspension was loaded into a dialysis belt, dialyzed against 0.1M PBS pH7.4 at 4℃and exchanged for 2 hours for one time, and dialyzed for 2 days. After the dialysis is completed, the purified ascites in the dialysis bag is recovered, the protein concentration is measured by an ultraviolet spectrophotometer, and the purity is detected by SDS-PAGE.
Further purifying the monoclonal antibody by DEAE-Sephadex A-50 (GE company) column chromatography, recovering the purified monoclonal antibody, measuring the protein concentration by ultraviolet spectrophotometer, and detecting the purity by SDS-PAGE.
3 results
3.1 monoclonal antibody screening against HP-PRRSV
3.1.1 identification of immune Effect
Tail blood collection was performed on mice immunized three times, 1: after 5000 dilution, the mice were tested by IFA method, and the results showed that three randomly selected mice were successfully immunized and the mice were boosted and cell fused (fig. 1).
3.1.2 selection of hybridoma cells capable of secreting specific monoclonal antibodies
After 10d of cell fusion, detecting the supernatant of a hybridoma cell culture solution by using an IFA method, subcloning positive holes for 3 times, carrying out IFA detection on the supernatant of the cell culture solution in each subcloning process until 96-well plates are positive, successfully preparing a hybridoma cell line capable of secreting PRRSV specific antibodies (figure 2), and preparing a cell strain of a monoclonal antibody, namely a mouse monoclonal antibody hybridoma cell strain XX10, and preserving the cell strain in China general microbiological culture Collection center with the following microorganism preservation number: CGMCC No.45495, and the preservation date is 2023 and 03.
3.2 identification of monoclonal antibody XX10 specifically recognizing HP-PRRSV
3.2.1 subclass identification of monoclonal antibodies
Antibody subclass identification is carried out on the antibody secreted by XX10 by using an antibody subclass identification kit, and the identification result shows that the XX10 antibody heavy chain is IgG 2a The light chain is the K chain.
3.2.2 specific detection of monoclonal antibody XX10
The results of the specific detection of monoclonal antibody XX10 show that monoclonal antibody XX10 does not react with American type PRRSV strain CH-1R (classical strain, linear 8.7), SD-R strain (NADC 30-like PRRSV, linear 1.8), respPRRS MLV strain (classical North American type strain, linear 5.1), LNTZJ1341 strain (NADC 34-like PRRSV, linear 1.5), HNTZJ1714 strain (QYYZ-like PRRSV, linear 3.5) and European type strain ZD-1 strain, and reacts well with HP-PRRSV HuN4-F112 strain (HP-PRRSV, linear 8.7), JXA1-R strain (HP-PRRSV, linear 8.7), TJM strain (HP-PRRSV, linear 8.7) (FIG. 3). Monoclonal antibody XX10 was shown to react only with HP-PRRS strains, but not with other types of PRRS strains.
3.2.3 detection of sensitivity to monoclonal antibody XX10
The harvested XX10 cell culture supernatant was diluted 2-fold with DMEM and XX10 was finally identified at 1: the P/N is still higher than 2.1 at 256 times, so XX10 has higher sensitivity.
3.2.4HP-PRRSV monoclonal antibody XX10
The purified antibody is detected by an ultraviolet spectrophotometer to obtain the result of 2.16mg/mL, and SDS-PAGE electrophoresis detection shows that the monoclonal antibody XX10 is better purified, the impurity protein in ascites is basically removed, and the heavy chain and the light chain of the monoclonal antibody are clear (figure 4).
EXAMPLE 2 establishment of a competitive ELISA method for specifically identifying HP-PRRSV antibodies
1. Establishment of HP-PRRSV antibody competition ELISA method
(1) Antigen coating: ELISA plates were coated with 300ng of purified inactivated HuN-F112 virus per well at 4℃overnight.
(2) Closing: taking out the coated ELISA plate, washing the PBST for 3 times, and beating to dryness; 300. Mu.L/well of blocking solution was added and incubated in a constant temperature incubator at 37℃for 2h. The blocked ELISA plates were removed from the incubator, washed 3 times with PBST and patted dry.
(3) Serum to be tested and primary antibody incubation: serum to be tested was combined with XX10 monoclonal antibody (prepared in example 1) 1:1, adding 100 mu L of ELISA plate into each hole, simultaneously setting positive serum and negative serum as control, incubating for 30min in a constant temperature incubator at 37 ℃, washing with PBST for 3 times, and drying.
(4) Secondary antibody incubation: HRP-goat anti-mouse IgG was 10000-fold diluted with PBST, 100. Mu.L of each well was added, incubated in a 37℃incubator for 30min, washed 3 times with PBST, and patted dry.
(5) Developing a substrate: adding substrate TMB for color development, adding 100 mu L of TMB into each hole, and placing in a 37 ℃ constant temperature incubator for shading for 15min.
(6) Terminating the reaction: 50 mu L of 2M H are added to each well 2 SO 4 Termination was performed, and absorbance A (OD) was measured at 450nm with a microplate reader 450nm Values).
(7) Serum inhibition was calculated: serum inhibition (%) = (negative control OD 450nm Value-serum OD to be examined 450nm Value)/negative control OD 450nm The value was 100%.
2. Comparison of HP-C-ELISA with commercially available kit
(1) Specificity comparison
The specificity of HP-C-ELISA was determined by detecting positive serum of Haemophilus Parasuis (HPS), streptococcus (SS), infectious pleuropneumonia (APP), transmissible gastroenteritis virus (TGEV), porcine Epidemic Diarrhea Virus (PEDV), porcine pseudorabies virus (PRV), classical Swine Fever Virus (CSFV), foot and Mouth Disease Virus (FMDV), european PRRSV ZD-1 strain, NADC30-like PRRSV SD-R strain, WK108 strain, VR2332-like PRRSV SD192 strain, NADC34-like PRRSVDZD32-1901 strain, classical strain CH-1R strain, HP-PRRSVHuN4-F112 strain, JXA1-R strain and TJM strain.
(2) Sensitivity comparison
2 parts of PRRSV-2HuN4-F112 strain immunized serum are diluted by 2 times ratio respectively, and are detected by HP-C-ELISA and IDEXX PRRS X3 antibody detection kit respectively, and the sensitivity of the HP-C-ELISA is compared with that of the commercial kit.
3. Results
3.1 preparation of coating antigen
The concentration of HuN4-F112 whole virus (inactivated) after molecular sieve purification is 300 ng/. Mu.L by a BCA protein concentration determination kit.
3.2 establishment of HP-C-ELISA method and determination of reaction conditions
The optimal reaction conditions for the HP-C-ELISA method established by matrix titration are shown in Table 1.
TABLE 1 optimization of reaction conditions for HP-C-ELISA detection methods
3.3 determination of the threshold value for HP-C-ELISA
156 parts of negative serum and 130 parts of HP-PRRSV positive serum were tested by the optimized HP-C-ELISA method, and the analysis result of the MedCalcv15.8 software showed a threshold value of 45%, a sensitivity of 96.9% and a specificity of 97.4% (FIG. 5). Therefore, the result of the determination is positive when PI of the serum sample is not less than 45%, and negative when PI of the serum sample is less than 45%.
3.4HP-C-ELISA specificity test results
The HP-C-ELISA method is used for detecting positive serum of Haemophilus Parasuis (HPS), streptococcus (SS), infectious pleuropneumonia (APP), transmissible gastroenteritis virus (TGEV), porcine Epidemic Diarrhea Virus (PEDV), porcine pseudorabies virus (PRV), classical Swine Fever Virus (CSFV), foot-and-mouth disease virus (FMDV), european PRRSV ZD-1 strain, NADC30-like PRRSV SD-R strain and WK108 strain, VR2332-like PRRSV SD192 strain, NADC34-like PRRSV DZD32-1901 strain, classical strain CH-1R strain, HP-PRRSV HuN4-F112 strain, JXA1-R strain and TJM strain, and the result shows that the method can only compete with HP-PRRSV positive serum specifically for coating antigen, other American type various lineages strains and common porcine virus and bacterial positive serum PI values are far below a critical value (figure 6).
3.5HP-C-ELISA repeatability test results
The established HP-C-ELISA method is used for carrying out repeatability tests, and the results show that the variation coefficient of the repeated tests in the batch and between the batches is less than 5 percent (Table 2), thus indicating that the method has better repeatability.
TABLE 2 within-batch and inter-batch reproducibility experiments (n=5) of HP-C-ELISA methods
3.6 comparison of HP-C-ELISA with commercially available kits
Specific comparison of 3.6.1HP-C-ELISA and commercially available kits
Compared with IDEXX PRRSV ELISA antibody kit, the HP-C-ELISA method established by the invention is used for detecting 5 parts of animal experiment positive serum of American PRRSV SD-R strain (NADC 30PRRSV, linear 1.8), VR2332-like PRRSV SD192 strain (L5.1), NADC34-like PRRSV DZD32-1901 strain (L1.5), classical strain CH-1R strain (L8.7) and HP-PRRSV HuN4-F112 strain (L8.7), and the result shows that the HP-C-ELISA established by the invention can specifically detect HP-PRRSV antibody (figure 7).
Sensitivity of 3.6.2HP-C-ELISA to commercially available kit
2 parts of PRRSV-2 HuN-F112 strain immunized serum was diluted 2-fold, and the serum was detected by HP-C-ELISA and IDEXX commercial kits, respectively, to compare the sensitivities of the HP-C-ELISA and the commercial kits. The results show that the inhibition rate is still greater than a critical value when the dilution ratio of 2 parts of PRRSVHuN4-F112 positive serum is 1:8 and 1:16; the IDEXX PRRS X3 antibody detection kit can also detect 2 parts of PRRSV HuN4-F112 positive serum (figure 8) diluted by 8 times and 16 times at most respectively, and the results show that the HP-C-ELISA method established by the invention has sensitivity similar to that of a commercially available indirect ELISA kit.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The hybridoma cell strain XX10 secreting the monoclonal antibody specific to the highly pathogenic porcine reproductive and respiratory syndrome virus is characterized in that the hybridoma cell strain XX10 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.45495 and the preservation time of 2023 and 03 months 03.
2. A monoclonal antibody against a highly pathogenic porcine reproductive and respiratory syndrome virus secreted by the hybridoma cell line XX10 of claim 1.
3. Use of the hybridoma cell line XX10 according to claim 1 or the monoclonal antibody according to claim 2 for the preparation of a product for detecting pathogenic porcine reproductive and respiratory syndrome virus.
4. The use according to claim 3, wherein the highly pathogenic porcine reproductive and respiratory syndrome virus is an HP-PRRSV strain.
5. An antibody competition ELISA kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus comprising the monoclonal antibody of claim 2.
6. The antibody competition ELISA kit of claim 5 further comprising an inactivated highly pathogenic porcine reproductive and respiratory syndrome virus coated ELISA plate; in ELISA plates, the highly pathogenic porcine reproductive and respiratory syndrome virus is HuN-F112 strain with the preservation number of CGMCC NO.2484.
7. The antibody competition ELISA kit of claim 6 wherein the inactivated highly pathogenic porcine reproductive and respiratory syndrome virus has a coating concentration of 300ng/μl.
8. The antibody competition ELISA kit of claim 6 further comprising horseradish peroxidase-labeled goat anti-mouse IgG, blocking solution, wash solution, diluent, chromogenic solution, and stop solution.
9. The antibody competition ELISA kit of claim 7 wherein the horseradish peroxidase-labeled goat anti-mouse IgG is diluted 10000-fold with PBST solution.
10. The antibody competition ELISA kit according to claim 7 characterized in that the blocking solution is a 1% w/v BSA solution.
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