CN115094043B

CN115094043B - Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application

Info

Publication number: CN115094043B
Application number: CN202210924308.XA
Authority: CN
Inventors: 夏振强; 刘伟; 石晶; 赵雪; 张蕊
Original assignee: Changchun Sr Biological Technology Co ltd
Current assignee: Changchun Sr Biological Technology Co ltd
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2023-08-25
Anticipated expiration: 2042-08-02
Also published as: CN115094043A

Abstract

The invention provides a hybridoma cell strain for canine coronavirus and canine parvovirus, a monoclonal antibody and application thereof, and belongs to the technical field of biological detection. In order to provide hybridoma cell lines and monoclonal antibodies produced thereby for the detection of canine coronaviruses and canine parvoviruses. The invention provides a hybridoma cell strain, a hybridoma cell strain 3G4 and a hybridoma cell strain 5H8, wherein the preservation number of the hybridoma cell strain 3G4 is CGMCC No.45197, and the preservation number of the hybridoma cell strain 5H8 is CGMCC No.45198. The triple colloidal gold detection test strip prepared by the monoclonal antibody has high sensitivity and good specificity, thereby more accurately and rapidly detecting the canine coronavirus and the canine parvovirus.

Description

Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a hybridoma cell strain for canine coronavirus and canine parvovirus, a monoclonal antibody and application thereof.

Background

Canine parvovirus (Canine Parvovirus, CPV) and canine coronavirus (Canine coronavirus) are major diseases which are harmful to the health of dogs, and clinical symptoms caused by the two diseases in the onset period are similar and often mixed infection, and it is difficult to identify which virus infection is specific by clinical symptoms alone, and laboratory detection means are necessary. The three common detection methods specified in the current national standard are an immunoenzymatic method, an immunohistochemical method and an RT-PCR method respectively. There is also only one colloidal gold technology that can detect only one virus in one clinical test.

The method in national standard is used for detecting the canine parvovirus and the canine coronavirus, which requires special equipment and professional technicians, has long detection time and higher cost, and is not beneficial to popularization and use in animal hospitals and the like. The colloidal gold detection method has the advantages of simple operation, sensitive detection, stable storage, no need of special equipment and skilled professional technicians, convenient clinical or field use of veterinary personnel, low cost and the like.

At present, colloidal gold test strips for separately detecting canine coronaviruses and canine parvoviruses exist on the market, but the detection sensitivity is low, viruses with high concentration are needed to be detected, and detection omission and false negative results appear in the detection process; and only a single colloidal gold detection method is adopted, two viruses cannot be detected at the same time, and the use cost is high.

Disclosure of Invention

The present invention provides hybridoma cell lines for detecting canine coronavirus and canine parvovirus and monoclonal antibodies produced thereby.

It is another object of the present invention to provide a colloidal gold triple test strip for canine coronavirus and canine parvovirus, which can be used for detecting canine coronavirus and canine parvovirus simultaneously.

The invention provides a hybridoma cell strain for producing a monoclonal antibody of canine parvovirus, which is 3G4 or 5H8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No.45197; or the preservation number of the hybridoma cell strain 5H8 is CGMCC No.45198, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

The invention provides a pair of hybridoma cell strains for generating monoclonal antibodies of canine parvovirus, wherein the hybridoma cell strains are 3G4 and 5H8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No.45197; the preservation number of the hybridoma cell strain 5H8 is CGMCC No.45198, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

The invention provides a monoclonal antibody of canine parvovirus, which is canine parvovirus antibodies 3G4 and 5H8, wherein the antibody 3G4 is an antibody generated by a hybridoma cell strain 3G 4; the antibody 5H8 is an antibody produced by hybridoma cell line 5H 8.

The invention provides application of the hybridoma cell strain, the pair of hybridoma cell strains or the monoclonal antibody in preparation of a triple detection kit for detecting canine coronavirus and canine parvovirus or a canine parvovirus detection kit.

The invention provides a triple colloidal gold detection test strip for canine coronavirus and canine parvovirus, which consists of the following components: sample pad, colloidal gold pad, water-absorbing filter paper, nitrocellulose membrane; the nitrocellulose membrane is provided with a quality control line and a detection line, the detection line 1 is coated with an antibody 3C2, the detection line 2 is coated with an antibody 5H8, and the quality control line is coated with goat anti-mouse IgG; the colloidal gold pad is coated with colloidal gold-labeled antibodies 1B2 and 3G4.

Further defined, the preparation method of the colloidal gold-labeled antibodies 1B2 and 3G 4:

step 1: the colloidal gold solution is stirred, the pH value is regulated to 8.5, the average is divided into 2 bottles, the canine coronavirus monoclonal antibody 1B2 is slowly added dropwise into one bottle to ensure that the final concentration of the canine coronavirus monoclonal antibody 1B2 is 10 mug/ml, the canine parvovirus monoclonal antibody 3G4 is slowly added dropwise into the other bottle to ensure that the final concentration of the canine parvovirus monoclonal antibody 3G4 is 10 mug/ml, the mixture is kept stand at room temperature for 30 minutes, and 0.01ml of 10% NaCl solution is added into the two bottles, and the mixture is kept stand for 1 hour after uniform mixing.

Step 2: adding 10% BSA solution into two bottles of colloidal gold solution of monoclonal antibody respectively to make the final concentration of the colloidal gold solution be 1%, uniformly mixing, sealing for 30 minutes, centrifuging for 20 minutes at 8000r/min, discarding the supernatant, and re-suspending the centrifuged sediment by using gold-labeled buffer solution to make the volume of the gold-labeled heavy suspension be 1/10,1B2 of the original volume and the gold-labeled heavy suspension of 3G4 be 1:1, and obtaining the monoclonal antibodies 1B2 and 3G4 marked by colloidal gold.

Further defined is the preparation method of the colloidal gold solution in step 1: adding 0.01% chloroauric acid solution into a siliconized glass container, placing the container on a magnetic heating stirrer, boiling for 3 minutes, rapidly adding 1.2ml of 1% trisodium citrate aqueous solution at one time under stirring, continuously heating for 6-8 minutes, cooling to room temperature, recovering to 100ml with purified water, filtering with a 0.22 μm filter membrane, and filling into a clean glass bottle; the colloidal gold solution in the step 1 is placed on a magnetic stirrer and stirred at the rotating speed of 300 g/min; the pH was adjusted to 8.5 with a 0.2mol/L potassium carbonate solution.

Further defined, the gold-labeled buffer in step 2 is composed of the following components: 0.02mol/L PBS pH7.2, bovine serum albumin final concentration 1% (m/V), sucrose final concentration 0.5% (m/V) and sodium azide final concentration 0.01% (m/V).

Further defined, the antibodies 1B2 and 3G4 labeled with colloidal gold were sprayed onto the glass fiber film in an amount of 60. Mu.l/100 mm per 1ml ² 。

Further limiting, diluting the canine coronavirus monoclonal antibody 3C2 with a membrane coating liquid, and spraying the diluted solution on a nitrocellulose membrane according to the concentration of 1.0 mug/cm to serve as a detection line 1; diluting canine parvovirus antibody 5H8 with a film coating liquid, and spraying on a nitrocellulose film according to the concentration of 1.0 mug/cm to serve as a detection line 2; spraying goat anti-mouse IgG on a nitrocellulose membrane according to the concentration of 1.0 mug/cm to serve as a quality control line; the concentration of the canine coronavirus monoclonal antibody 3C2 is 20 mug/ml, and the concentration of the canine parvovirus monoclonal antibody 5H8 is 75 mug/ml.

The beneficial effects are that:

after canine parvovirus WQ8401 cell culture fluid is purified, 6-8 week old female BALB/c mice are immunized, 6 indirect ELISA detection cell strains of more than 3 positive monoclonal holes are screened out, ascites is prepared, and monoclonal antibody identification is carried out after purification. 6 strains of monoclonal antibodies are subjected to affinity measurement, namely 1A4>3G4>5H8>4H2>2G8>1A4 is carried out from top to bottom, the monoclonal antibodies are identified as 3G4 and 5H8 belonging to the subclass IgG2a kappa, 1A4 and 2G3 belonging to the subclass IgG2b kappa, and 2G8 and 4H2 belonging to the subclass IgG1 kappa. Indirect immunofluorescence showed that canine parvovirus monoclonal antibodies 1A4, 3G4, 4H2 and 5H8, all with a fold no less than 6400 times the fluorescence signal could be detected.

Monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 were confirmed by additive ELISA assays, and the AI after addition of 4 mAbs was greater than 50%, indicating that they acted on different antigenic sites.

And respectively carrying out pairing combination tests on monoclonal antibodies 1A4, 3G4, 4H2 and 5H8, wherein the 3G4 and the 5H8 coated are marked as optimal combinations, detection line signals are stronger than other combinations, and the detection line signals are respectively used as capture monoclonal antibodies and detection monoclonal antibodies and are used for colloidal gold marking and detection lines coated on a nitrocellulose membrane.

Canine parvovirus hybridoma cell lines 3G4 and 5H8, which are 3G4 or hybridoma cell line 5H8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No.45197, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day; or the preservation number of the hybridoma cell strain 5H8 is CGMCC No.45198, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

The canine coronavirus hybridoma cell strain 1B2 and 3C2, wherein the preservation number of the hybridoma cell strain 1B2 is CGMCC No.45200, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day; the preservation number of the hybridoma cell strain 3C2 is CGMCC No.45199, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

Deposit unit address: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.

Drawings

FIG. 1 is a standard curve of BCA protein assay.

FIG. 2 is an OD coating different CCV antigen concentrations _450nm And (5) detecting a result.

FIG. 3 shows indirect immunofluorescence results, wherein A is the result of indirect immunofluorescence of monoclonal antibody 1B 2; b is a blank cell control; c is the result of indirect immunofluorescence of monoclonal antibody 3C 2; d is a blank cell control; e is the indirect immunofluorescence result of monoclonal antibody 2B 5; f is a blank cell control; g is the result of indirect immunofluorescence of monoclonal antibody 2D 3; h is a blank cell control.

FIG. 4 shows the electrophoretic purity detection of purified ascites 1B2 and 3C 2.

FIG. 5 shows indirect immunofluorescence results, A is the monoclonal antibody 1A4 indirect immunofluorescence results; b is a blank cell control; c is the result of indirect immunofluorescence of monoclonal antibody 3G 4; d is a blank cell control; e is the indirect immunofluorescence result of monoclonal antibody 4H 2; f is a blank cell control; g is the indirect immunofluorescence result of monoclonal antibody 5H 8; h is a blank cell control.

FIG. 6 is a schematic diagram of a 4-strain monoclonal antibody pairing assay; 1-12 are respectively 1. The 3G4 label coats 1A4;2. marking the 4H2 coating 1A4;3. tag 5H8 coating 1A4;4. tag 1A4 coated 3G4;5. marking 4H2 coating 3G4;6. marking 5H8 coated 3G4;7. tag 1A4 coated with 4H2;8. marking 3G4 coating 4H2;9. labeling 5H8 coated 4H2;10. tag 1A4 coated 5H8;11. marking 3G4 coating 5H8;12. the label 4H2 coats 5H8.

Fig. 7 is a schematic diagram of a test strip structure.

Detailed Description

EXAMPLE 1 preparation of canine coronavirus hybridoma cells

The purified canine coronavirus CCV2015 strain (which is obtained by autonomous isolation and identification of vincristoceno biological limited company) is adopted as an immunogen, a BALB/c mouse with the age of 6-8 weeks is immunized, the tail is cut and blood is taken simultaneously after three immunization for 7 days to measure the serum antibody titer in the mouse body, the mouse myeloma cell SP2/0 is fused with splenic lymphocyte, and positive fusion holes are screened and subjected to individual treatment.

1. Purification and detection of the immune source: the purified canine coronavirus CCV2015 strain was used as an immune source, and the specific procedures were as follows: centrifuging at a low speed of 2000-3000 r/min for 20-30 min to remove larger host cell fragments and other larger impurities; and (3) centrifuging the virus supernatant at 35000r/min for 2 hours by adopting a sucrose density gradient centrifugation method, and dissolving the precipitation zone by using PBS with the pH of 7.2 to obtain the purified immune source. The detection was performed according to the BCA kit detection procedure, and OD values were measured at 562nm wavelength. At the same time, standard 2.0mg/ml, 1.0mg/ml, 0.5mg/ml, 0.25mg/ml and 0.125mg/ml protein solution were prepared to prepare a standard curve. Deducing the protein content of the protein to be detected according to the standard curve.

And (3) carrying out protein quantification on the purified virus by using a Pierce BCA protein Assay Kit protein content detection kit. y=2.4553x+0.185 (Y represents absorbance, X represents protein content, R) ² = 0.9828). The absorbance of the purified virus protein after 50-fold dilution was 1.232, and the protein content thereof was 0.385mg/ml, the original concentration was 19.25mg/ml, and the results are shown in FIG. 1.

2. Animal immunization: the purified canine coronavirus CCV2015 strain was used as an immune source at 200 μg protein per mouse. First, exempt from: mixing antigen and Freund's complete adjuvant at a volume of 1:1, and making into emulsion for subcutaneous multipoint injection in abdomen of mice. And (2) avoiding: after 14 days of priming, mixing the antigen and Freund's incomplete adjuvant at a volume of 1:1 to obtain emulsion, and immunizing according to priming immunization method. Three-way: immunization was performed 14 days after the second immunization, by the same procedure as the second immunization. Mice were bled by tail-off after 7 days, serum neutralization antibody titers were determined by indirect ELISA, and when the antibody titers reached the requirements, mice were given one intraperitoneal injection of antigen with 50 μg protein of non-emulsified antigen per mouse 3 days prior to fusion.

3. Mouse serum potency assay

Establishment of indirect ELISA detection method

The purified canine coronavirus antigen was used to dilute it with coating solution to a final concentration of 4. Mu.g/. Mu.l, 2. Mu.g/. Mu.l, 1. Mu.g/. Mu.l, 0.5. Mu.g/. Mu.l, 0.25. Mu.g/. Mu.l, 0.125. Mu.g/. Mu.l and 0.0625. Mu.g/. Mu.l, 100. Mu.l/well, respectively, and coated overnight at 4℃and then washed 3 times with washing solution, 5% skim milk blocking solution, 200. Mu.l/well, incubated at 37℃for 2 hours, washed 3 times with washing solution, mouse positive serum as positive control, blank control added PBS buffer, mouse negative serum as negative control, 100. Mu.l per well incubated at 37℃for 1 hour; after washing 3 times with wash solution, adding PBS to dilute 4000 times secondary antibody (HRP-labeled goat anti-mouse IgG), incubating for 1 hour at 37 ℃, taking out, washing 3 times with wash solution, adding 100. Mu.l of color development solution per hole, developing for 5 minutes, adding 50. Mu.l of stop solution per hole, measuring OD value at 450nm, recording storage data, and determining that the optimal coating concentration of the purified canine coronavirus is 0.25. Mu.g/. Mu.l as can be seen from FIG. 2.

4. Mouse serum potency assay

The purified canine coronavirus antigen was diluted with coating solution to a final concentration of 0.25. Mu.g/. Mu.l, 100. Mu.l/well, coated overnight at 4℃and then washed 3 times with washing solution, blocked with 5% skim milk blocking solution, 200. Mu.l/well, incubated for 2 hours at 37℃and washed 3 times with washing solution, 2-fold gradient dilution of mouse positive serum from 200-fold, addition of PBS buffer to blank control, and 200-fold dilution of negative serum to negative control. All 100. Mu.l/well, incubated at 37℃for 1 hour; after 3 washes, 4000-fold dilution of secondary antibody (HRP-labeled goat anti-mouse IgG) with PBS was added, incubated at 37℃for 1 hour, taken out and washed 3 times with wash, 100. Mu.l of color development solution was added per well, developed for 5 minutes, and each well was terminated with 50. Mu.l of stop solution, OD was measured at 450nm, and the data were recorded. The titer of 4-immunized mice was checked using canine coronavirus ELISA method, wherein the dilution corresponding to the minimum OD reading with titer greater than the maximum OD/2 was determined as the final canine coronavirus serum titer, and the mice with the highest canine coronavirus serum ELISA titers were number 4, and the results are shown in Table 1.

TABLE 1 ELISA titers results after mice immunization

5. Preparation of myeloma cells, spleen lymphocytes and thymocytes: the method comprises the steps of (1) carrying out culture passage by using an IMDM complete medium (containing 20% serum) without generating immunoglobulin and through mycoplasma detection, selecting a mouse eyeball with the highest canine coronavirus titer, taking blood, killing, immersing in 75% alcohol for 5 minutes, pouring a small amount of serum-free IMDM into a plate, placing a cell sieve and an inner core of a syringe into the plate, taking down spleen of the mouse with scissors and tweezers, placing the spleen on the cell sieve, lightly grinding the spleen with the inner core of the syringe, sucking the ground cells into a centrifuge tube, washing for 3 times with PBS for later use, taking down thymus of the mouse with scissors and tweezers, grinding, and taking the ground thymus cells to 15ml for later use.

6. Fusion assay: blowing off the SP2/0 cells in good state from the wall of a culture flask gently, sucking the cells into a 50ml centrifuge tube, sucking the prepared spleen lymphocyte cells into the centrifuge tube filled with the SP2/0, and centrifuging at 1500r/min for 5 minutes; the centrifuged cells were decanted, gently and gently blown with serum-free IMDM, centrifuged at 1500r/min for 5 min, and the centrifuged cell supernatant was decanted as much as possible. The bottom of the centrifuge tube is beaten to fully suspend the cells, the centrifuge tube is placed in warm water at 37 ℃, 1ml of PEG is slowly added within 1 minute, after the addition is completed, the centrifuge tube is placed in warm water for 1 minute, then 2ml of serum-free IMDM is slowly added within 2 minutes, then 8ml of serum-free IMDM is slowly added within 2 minutes, and the centrifuge tube is centrifuged at 1000r/min for 5 minutes. The supernatant was removed, 45ml of HAT complete medium was added, the cells were carefully homogenized, and the previously prepared thymocytes were poured and thoroughly mixed. The cells were added to 3 96-well cell culture plates with a dispenser, 150. Mu.l per well, placed in a wet box, and then placed in an incubator for culture.

7. Screening of positive fusion wells: after 7 days of HAT complete medium, half of the original medium was replaced with IMDM medium containing HAT,20% FBS, and after 16 days of fusion, 100. Mu.l of cell supernatant was aspirated into ELISA plates previously coated with purified canine coronavirus CCV2015 strain antigen, while 100. Mu.l of HT complete medium was supplemented into 96 well plates. And if the ELISA test result is positive, performing subcloning.

8. Hybridoma cell titer assay: ELISA screening is carried out on the fused hybridoma cells to determine positive cell strains, and the steps are as follows: diluting the canine coronavirus CCV2015 purified virus with the coating solution to a final concentration of 0.25 μg/μl,100 μl/well, and overnight at 4deg.C; washing with washing liquid for 3 times; blocking with 5% skim milk blocking solution, incubating at 37deg.C for 2 hr, washing with washing solution for 3 times, adding primary antibody (cell culture supernatant or ascites), negative control (SP 2/0 culture supernatant), blank control (PBS), positive control (positive serum PBS 1000-fold dilution), all 100 μl/hr, incubating at 37deg.C for 1 hr, washing with washing solution for 3 times, adding PBS to dilute 4000-fold secondary antibody (HRP-labeled goat anti-mouse IgG), incubating at 100deg.C for 1 hr, taking out, washing with washing solution for 3 times, adding developing solution for 100 μl/hr, developing for 5 min, adding 50 μl of stop solution for each well, measuring OD value at 450nm, recording preservation data, and titer is the corresponding degree of minimum OD reading greater than maximum OD/2.

The canine coronavirus CCV2015 strain is coated, fusion cell holes are detected by ELISA, xx positive hybridoma cell holes are screened, namely 1B2, 1E8, 1F3, 2B5, 2C10, 2D3, 3C1, 3C2 and 3H11 are obtained, H12 is positive control, G12 is blank control, F12 is negative control, and the results are shown in tables 2, 3 and 4.

Table 2 canine coronavirus antibody positive cell screen 1 (OD _450nm Value of

TABLE 3 canine coronavirus antibody positive cell screen 2 (OD _450nm Value of

TABLE 4 canine coronavirus antibody positive cell screen 3 (OD _450nm Value of

9. Hybridoma cell singulation: to achieve homogeneity of the genetic background of hybridoma cells, positive cloning wells in 96-well plates were subjected to limiting dilution to 2 cells/well. The total cell number was counted by a hemocytometer, diluted to a cell number of about 200 cells in 10ml of IMDM medium containing 20% serum, and added to 100 μl/well of a 96-well plate, 2 cells per well. After 7-10 days, 1 colony hole is selected, after 14 days, the hole is transferred to 24 holes, after 18 days, the hole is transferred to 25cm ² In the cell culture flask, the antibody hybridoma and the antibody secreted therefrom were designated as 1B2, 1E8, 1F10, 2B5, 2C10, 2D3, 3C1, 3C2 and 3H11, as confirmed by microscopic observation as individual clones.

EXAMPLE 2 preparation of canine coronavirus antibodies

1. Identification of monoclonal antibodies

Hybridoma cell and ascites titer test the screened monoclonal hybridoma cells are used for preparing mouse ascites according to a 2.10 method, cell supernatant and mouse ascites are used as primary antibodies respectively, purified CCV2015 strain virus is used as an antigen coating ELISA plate, and an indirect ELISA method is used for titer test, wherein the titer is the dilution corresponding to the minimum OD reading which is larger than the maximum OD/2.

The 9 monoclonal hybridoma cell supernatants 1B2, 1E8, 1F10, 2B5, 2C10, 2D3, 3C1, 3C2 and 3H11, and the corresponding prepared mouse ascites were re-coated with purified CCV2015 strain virus, and the results of the titer test were shown in Table 5 by ELISA.

TABLE 5 ELISA titers of monoclonal hybridoma cell canine coronavirus results

2. Identification of monoclonal antibody subtypes: coating 96-well ELISA plate (100 μl/well) with optimized antigen concentration, washing with PBST for 2 hr at 37deg.C, beating, adding 1:5000 diluted monoclonal antibody ascites to be detected (50 μl/well), incubating at 37deg.C for 1 hr, washing, beating, adding 1:1000 diluted goat anti-mouse IgG1, igG2a, igG2b, igG3, igM, igA, ig k, ig-specific subclass serum (50 μl/well), beating at 37deg.C for 1 hr, washing, beating, adding 1:5000 diluted HRP-rabbit anti-sheep IgG (100 μl/well), beating for 15 min at room temperature (15-25deg.C), adding TMB substrate in the well, developing color at 37deg.C for 5-10 min (100 μl/well), adding 2M H ₂ SO ₄ Termination color development (100. Mu.l/well), reading OD _450nm The antibody type with a significantly higher read than the other wells was chosen as the mab subclass.

And carrying out subclass identification on the 9 positive cell strains selected, and finally obtaining 9 positive hybridoma cell strains with IgG types. The 1E8, 2B5, 2C10, 2D3 and 3C1 cell lines are IgG1, kappa chain, and the 1F10 and 3H11 cell lines are IgG1, lambda chain; 1B2 cells are IgG2B, kappa chain; the 3C2 cells were IgG2b, lambda chain and the results are shown in Table 6.

TABLE 6 monoclonal cell subclass identification (OD _450nm Value of

3. Monoclonal antibody neutralization activity assay: the neutralizing activity of each monoclonal antibody was determined by the immobilized virus-diluted serum method: will be 100TCID ₅₀ Mixing diluted canine coronavirus virus solution with culture supernatant (2 times serial dilution) or ascites (10 times serial dilution) of equal volume hybridoma cells, standing in 37 deg.C incubator for 1 hr, inoculating the virus-antibody mixture into 96 well plate (100 μl/hole), spreading into F81 cells, standing in 37 deg.C 5% CO ₂ Culturing in incubator for several days, observing cytopathy day by day, and synchronously setting positive serum against canine coronavirus, canine coronavirus and normal F81 cells as controls.

mAb-1B2 culture supernatant was assayedNeutralizing titer of neutralizing antibody of 2 ⁶ The neutralization titer of ascites is 1.28X10 ³ None of the other 8 monoclonal antibodies had neutralizing activity.

4. Determination of affinity constant of the cloned antibody: the affinity of the antibodies was determined using a non-competitive ELISA. The coated antigen is purified canine coronavirus, and the ELISA plate is coated with 1, 0.5, 0.25 and 0.125 mug/ul, 100 mug/hole is coated overnight at 4 ℃; 1% BSA was added and each well was blocked at 37℃for 2 hours in 150. Mu.l; after PBST washing, the monoclonal antibody was diluted at a specific concentration of 5. Mu.g/. Mu.l by a double ratio, and the logarithmic value of the antibody concentration (moL/L) was taken as the abscissa and the corresponding OD was taken as the corresponding OD ₄₅₀ The values are on the ordinate, and 4S-curves are made in one coordinate system. Find the top of the S-curve, set to OD _max . Finding out 50% OD of each of the 4 curves _max Corresponding antibody concentration. The affinity constants of the monoclonal antibodies were calculated according to the formula, with 4 concentrations in pairs.

Ka＝(n-1)/2(n[Ab']t-[Ab]t)

n is the multiple of the concentration of two coating antigens in each group, [ Ab ]']t and [ Ab]t is two 50% OD in each group _max Corresponding antibody concentration (moL/L).

The average value of 6 Ka's of the canine coronavirus monoclonal antibody 1B2 was 2.67×10 ⁷ M ^-1 6 Ka averages of 3C2 are 2.6X10 ⁷ M ^-1 6 Ka averages of 2B5 are 2.3X10 ⁷ M ^-1 6 Ka averages of 2D3 are 1.6X10 ⁷ M ^-1 6 Ka averages of 1E8 are 0.8X10 ⁶ M ^-1 6 Ka averages 1.6X10 for 1F10 ⁶ M ^-1 6 Ka averages of 2C10 are 3.0X10 ⁶ M ^-1 6 Ka averages of 3C1 are 1.2X10 ⁶ M ^-1 And 6 Ka averages of 3H11 are 2.0X10 ⁶ M ^-1 . Nine monoclonal antibodies with affinity of 1B2 from high to low in sequence>3C2>2B5>2D3>2C10>3H11> 1F10>3C1>1E8。

It is generally considered that the affinity constant is 10 ⁷ ～10 ¹² M ^-1 When belonging to high affinity antibodies, the affinity constant is 10 ⁵ ～10 ⁷ M ^-1 In the time-course of which the first and second contact surfaces,belonging to the class of low affinity antibodies. Four strains of mab 1B2, 3C2, 2B5 and 2D3 were chosen as markers and coating studies.

5. Indirect immunofluorescence assay: f81 cells were grown at 2X 10 ⁵ Mu.l to 3X 10 ⁵ Mu.l of the cell suspension was added to a 96-well plate at 100. Mu.l/well. Dilution of canine coronavirus to 100TCID ₅₀ 100 μl/well, and 100 μl/well of canine distemper virus, canine parvovirus, canine adenovirus and canine parainfluenza virus, each of which has a dilution, are added to the cells of the following viruses. Blank cell control was also established at 37℃with 5% CO ₂ The cells were cultured in an incubator for 48 hours. After 48 hours of virus infection of cells, the virus solution was discarded, and the cells were fixed with 80% cold acetone at 200. Mu.l/well for 30 minutes at room temperature (15 to 25 ℃). PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, followed by pipetting the wells. Hybridoma cell culture supernatants were diluted 2-fold, 50-fold, 100-fold, 200-fold, 400-fold, 819200-fold, and 15 dilution gradients were added sequentially to 96-well plates, 50. Mu.l/well, and incubated for 1 hour at 37 ℃. PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, followed by pipetting the wells. The secondary antibody is FITC-labeled goat anti-mouse IgG, and secondary antibody diluent (FITC-labeled goat anti-mouse IgG 1:200-fold dilution, evans blue 1:100-fold dilution) is added into 96-well plate cells, and incubated for 1 hour at 37 ℃. PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, followed by pipetting the wells.

After hybridoma cell lines 1B2, 3C2, 2B5 and 2D3 are full, taking the supernatant for antibody property identification, and using an indirect immunofluorescence method, the result shows that 1B2, 3C2, 2B5 and 2D3 can be specifically combined with CCV strains and are not combined with canine distemper virus, canine parvovirus, canine adenovirus and canine parainfluenza virus. The indirect immunofluorescence results by fold dilution showed that the fluorescence signal could still be detected when the antibody was diluted 6400 fold, as shown in fig. 3.

6. Monoclonal antibody antigen recognition site differential analysis: the differences in the action antigen sites of the monoclonal antibodies of each strain are analyzed by adopting additive ELISA test: respectively diluting the ascites of each monoclonal antibody strain toSaturation working concentration (OD) _450nm The value did not increase with increasing antibody concentration), one monoclonal antibody was added, incubated for 1.5 hours at 37 ℃, after 4 washes, the other monoclonal antibody was added, incubated for 1.5 hours at 37 ℃, after 4 washes, enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG) was subsequently added and tested according to the procedure of the indirect ELISA method. Determination of the total OD after mixing and addition, respectively _450nm Value and OD of each independent reaction _450nm Values, each set of experiments were repeated 3 times, averaged. The AI value of 2 strains of monoclonal antibodies superimposed on each other was calculated according to the following formula: ai=ai (%) = [ (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100，A ₁ And A ₂ Is the absorption value of each of two monoclonal antibodies, A ₁₊₂ Is the absorbance measured after the addition of the two monoclonal antibodies. If AI<50, then both monoclonal antibodies bind to the same antigenic site, if AI>50, the two monoclonal antibodies bind to different antigenic sites.

According to the OD value result of the detected monoclonal hybridoma cell strain and the calculation formula AI (%) = [2A ₁₊₂ /(A ₁ +A ₂ )-1]X100 to analyze epitope, if AI is more than or equal to 50%, two monoclonal antibodies are different epitopes, if AI<50% of the two monoclonal antibodies are the same epitope. The addition ELISA test shows that the AI of the 4 mAbs added is greater than 50%, which indicates that the mAbs act on different antigen sites, and the result is shown in Table 7.

Table 7 4 strain McAb addition test for AI value

Note that: AI (%) = [ (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100

Pairing test of coating and detection antibody 4 purified mAbs were labeled with colloidal gold at the same concentration (10. Mu.g/. Mu.l), NC membrane was coated at the same concentration, dried at 37℃for 5 hours, paired in cross, positive control was added dropwise, and the best combination of capture mab and coating mab was selected as the one with the highest signal intensity.

Pairing experiments are carried out on the monoclonal antibodies 1B2, 3C2, 2B5 and 2D3 and corresponding labeled antibodies respectively, so that the effect of the coated monoclonal antibody 3C2 and the labeled monoclonal antibody 1B2 on detecting canine coronavirus sensitive reference substances is best, and the results are shown in Table 8.

TABLE 8 test results for different combinations of monoclonal antibodies

7. Hybridoma cell ascites preparation

Recovering hybridoma cell strains 3C2 and 1B2 for production in frozen storage in a water bath kettle at 37 ℃ for 2-5 minutes, and adding the recovered hybridoma cell strains into the kettle at 75cm ² Cell culture flask in 5% CO ₂ CO at 37 DEG C ₂ Growing for 3-5 days in an incubator. Taking hybridoma cells which are full of a monolayer, discarding the supernatant, sucking 10ml of IMDM into a 10ml suction tube, repeatedly blowing a cell bottle, collecting cell suspension, adding the cell suspension into a 50ml centrifuge tube, centrifuging at 1000rpm for 10 minutes, discarding the supernatant, and re-dissolving the precipitate with 2ml of IMDM. Sucking the re-dissolved hybridoma cell suspension by using a maximum range 1ml injector, and inoculating 8-10 week old female BALB/c mice, each 1X 10 ⁶ Individual cells. After 10 days of inoculation, the ascites of the mice are sucked by a syringe, each mouse is collected 1-3 times, 3-10 ml each time, until the mice die. The collected ascites (12000 g) was centrifuged for 5 minutes, and the supernatant was collected and repeated once. Adding merthiolate with final concentration of 0.01% into ascites, subpackaging, and preserving at-70deg.C.

8. Antibody purification: the mouse ascites antibody is affinity purified by using G protein, the filling material is loaded into a chromatographic column, the ascites is diluted by about 5 times by buffer (20mM PBS,300mM NaCl,pH7.4-8.5), and the column is put on the column for purification. After loading, the unbound protein fraction is removed with equilibration buffer (20mM PBS,300mM NaCl,pH7.4-8.5); then, an elution buffer (0.1M sodium citrate, pH 4.0) was added to elute the binding protein, and finally, a neutralization buffer (1M Tris-HCl, pH 9.0) was used to adjust the pH.

9. Purity test (SDS-PAGE method): mixing 5 times volume of purified antibody and 1 volume of Buffer protein, boiling for 5 minutes, centrifuging, and loading the supernatant: sample 10. Mu.l, protein Marker 5. Mu.l. Electrophoresis conditions: the voltage was 95V and the current was about 75mA, and the electrophoresis was performed for 2 hours. And after electrophoresis, taking out the electrophoresis gel, putting the electrophoresis gel into a staining solution for 1 hour, slowly shaking the shaking table, taking out the electrophoresis gel, putting the electrophoresis gel into a dehydration solution after the staining is completed, slowly shaking the shaking table for 1-2 hours, and taking out an observation result after the decoloring is completed.

10. Potency assay: purified antibodies were titered using viral ELISA, coating: after dilution and purification of the coating solution, the final concentration of the canine coronavirus CCV2015 strain is 0.25 mug/μl,100 μl/hole, and the temperature is kept at 37 ℃ for 1 hour; closing: sealing 200/hole with 5% skimmed milk, sealing overnight, washing the plate 3 times, diluting the purified antibody 100 times, diluting with PBS (phosphate buffered saline) times, and washing the plate 3 times at 37deg.C for 1 hr; HRP-labeled goat anti-mouse IgG (secondary antibody) diluted at 1:4000 PBS, 100 μl of ELISA plate added per well at 37deg.C for 0.5 hr; washing the plate for 3 times; color development: TMB 100PBS for 3 minutes at room temperature; 2mol/L H ₂ SO ₄ And (5) stopping liquid.

Monoclonal antibodies 1B2 and 3C2 mouse ascites were prepared and affinity purified using G protein. The purified sample was subjected to purity detection by SDS-PAGE, BCA protein content detection and canine coronavirus ELISA, and the results are shown in FIG. 4 and Table 9.

TABLE 9 purity, concentration and potency results for 1B2 and 3C2 after purification

Hybridoma cell lines 3C2 and 1B2 expanded and frozen: after the CCV hybridoma cells 3C2 and 1B2 grow to be full of monolayers, the culture medium is discarded, 10ml of IMDM complete culture medium (containing 20% serum) is used for blowing the cells out of the bottom of the cell bottles, the process is carried out according to the ratio of 1:3, the hybridoma cells in 1 cell bottle are evenly distributed into 3 cell bottles, 30-40 ml of IMDM complete culture medium (containing 20% serum) is injected into each cell bottle, the culture is continuously observed, and the growth condition of the cells is recorded every day. Cells were passaged every 4 days, serially passed for 3 passages, and the results were recorded. Continuously transferring 1 st generation CCV hybridoma cells 3C2 and 1B2 for 5 generations in the presence of5％CO ₂ In an incubator at 37℃for 5 days, the growth of the cells was recorded daily.

Adding small amount of serum-free IMDM after cell digestion, centrifuging at 1000rpm for 5 min, collecting the bottom cells, dispersing with 10% bovine serum IMDM, continuously washing for 3 times, collecting the bottom cells, and adjusting cell concentration to 10 ⁶ Mu.l of a freezing medium (IMDM 95% containing 20% foetal calf serum, dimethyl sulfoxide 5% and antibiotics) was added, frozen at 20℃for 2 hours and at 70℃for 4 hours, and stored in liquid nitrogen. According to the method, 3 rd-10 th generation hybridoma cells are preserved, the cell generation times and the preparation date are noted, and the hybridoma cells are preserved in liquid nitrogen.

The CCV hybridoma cells 3C2 and 1B2 proliferated 5-10 times, the cell morphology was that of mouse myeloma cells, and the results of good proliferation characteristics of the cells are shown in Table 10.

TABLE 10 proliferation results of CCV hybridoma cells 1B2 and 3C2 passages

Results of hybridoma cell line secretion antibody assay hybridoma cell line 1B2,3C2 was grown to the bottom of the cell flask, and the supernatant was collected for secretion antibody assay, and the results are shown in table 11.

TABLE 11 determination of antibody secretion by hybridoma cell lines

Hybridoma cell passage stability P6 generation cells of hybridoma cell lines 1B2 and 3C2 are taken, the cells can be stably proliferated after 15 passages are continuously produced, supernatant fluid is collected for measuring secreted antibody, the properties of the secreted antibody are stable, and the results are shown in Table 12.

TABLE 12 determination of hybridoma cell passage stability

Cell cryopreservation assay results

3.17.1 cell viability assay cell viability assays were performed on 4 batches of liquid nitrogen-preserved cells with cell viability rates of 88.6%,89.5%,92.5% and 93.5% for the P3, P5, P8 and P10 passages, respectively.

Growth characterization of 3.17.2 resuscitated cells P5 and P10 passages were resuscitated and then assayed for growth characteristics, cells were cultured for 5 days, and counted to give P5 passages 5.5X10 cells ⁵ 6.1X10 th generation cell per ml, 10 th generation cell ⁵ And each ml.

The detection results of exogenous factors show that the bacteria and mould of hybridoma cell strains 1B2 and 3C2 are negative, and the results of the detection of mycoplasma are negative by adopting a liquid culture medium. The detection is carried out by using fluorescent antibodies for resisting canine parvovirus, rabies virus and bovine viral diarrhea virus, the results are negative, and the specificity is good.

EXAMPLE 3 preparation of canine parvovirus hybridoma cells

1. Obtaining canine parvovirus antigen: the canine parvovirus in the feces of the suspected canine parvovirus diseased dogs collected clinically is separated and identified, and the separated canine parvovirus is identified by means of an electron microscope observation method, a physical and chemical property identification method, a red blood cell hemagglutination spectrometry method, a specificity test detection method, a nucleic acid detection method and the like.

The results show that: the separated pathogen is canine parvovirus, named canine parvovirus WQ8401 strain (obtained by autonomous separation and identification of vincristoceno biological Co., ltd.), has obvious trabecular lesions after F81 cells are synchronously inoculated, virus particles of the virus are visible under an electron microscope, the virus has the most obvious agglutination phenomenon on porcine erythrocytes at pH 7.2, specific detection proves that the virus can be specifically combined with canine parvovirus fluorescent monoclonal antibody, and a nucleic acid detection method proves that the virus is a canine parvovirus positive result. Thus, the WQ8401 strain is a canine parvovirus strain.

2. After purifying canine parvovirus WQ8401 virus liquid, immunizing female BALB/c mice with 6-8 weeks of age, and simultaneously cutting tail to collect blood and measure the serum antibody titer in the mice after two weeks of secondary immunization, and simultaneously establishing an indirect ELISA detection method. Screening out cell strains of a monoclonal hole with more than 3 times of positive detection by indirect ELISA, preparing ascites, purifying anti-canine parvovirus monoclonal antibody, carrying out identification such as monoclonal antibody subtype identification, affinity constant measurement and the like, and selecting the mark and the coating of the monoclonal antibody by combining the preparation process of a test strip. The result is that 4 anti-canine parvovirus monoclonal hybridoma cell strains with higher affinity, namely 1A4, 3G4, 5H8 and 4H2 are screened out, an indirect ELISA test proves that 4 monoclonal antibodies are aimed at different epitopes of antigen, a pairing test finds that the mark 3G4 and the coating 5H8 are the optimal combination, and the detection line signals are stronger than other combinations and are respectively used as capture monoclonal antibodies and detection monoclonal antibodies, and are used for colloidal gold marks and detection lines on the coating nitrocellulose membrane.

3. Concentrating and purifying antigen: the third generation virus culture solution of canine parvovirus WQ8401 strain is repeatedly frozen and thawed for 3 times at 37 ℃ minus 20 ℃. After centrifugation of the freeze-thaw liquid at 3500r/min for 35 min at 4℃the supernatant was collected. PEG8000 was added to the collected supernatant to a final concentration of 12%, and NaCl was added to a final concentration of 0.5mol/L. The mixing was repeated four times every 15 minutes, and the virus solution was placed in a refrigerator at 4℃overnight. After centrifugation of overnight virus solution at 4℃and 15000r/min for 50 min, taking out carefully, discarding supernatant, adding an appropriate amount of PBS according to the precipitation amount for resuspension, and freezing at-80℃for standby.

4. Immunization of animals: 5 female BALB/c mice of 6-8 weeks of age were selected, and injected with canine parvovirus purified virus and equivalent Freund's complete adjuvant emulsion, 200. Mu.l each subcutaneously. Two weeks later, the mice were given a second immunization, and canine parvovirus concentrated purified virus and equivalent amount of Freund's incomplete adjuvant emulsion, in the same amount and method as the first immunization. The second immunization was followed by a third immunization, and the immunization method and virus injection were identical to the second immunization. And after three or two weeks, selecting mice with high serum titers after tail cutting and blood sampling for intraperitoneal injection to purify the virus liquid.

5. Indirect ELISA assay for in vivo serum titers in mice: the optimal coating concentration of CPV-VLPs and optimal dilution of serum were determined by the method of matrix. The purified CPV-VLPs were diluted with coating solution to different concentrations of 10, 8, 7, 6, 5, 4, 3, 1ug/ml, each coated with 12 wells, 100u L/well. CPV positive serum and CPV negative serum diluted by blocking solution are used as primary antibodies, the dilution ratio is 1:10, 1:20, 1:40, 1:80 and 1:160 respectively, and the positive serum and the negative serum with the same dilution are added into two adjacent rows of enzyme-labeled plate holes. Other conditions were performed according to conventional indirect ELISA detection methods.

The antibody titers of the culture supernatants (2-fold dilution) and ascites (10-fold dilution) of the hybridoma cells were determined by indirect ELISA assay, and the results are shown in Table 1.

TABLE 1 monoclonal antibody Indirect ELISA titers

6. Screening of mouse myeloma cells (SP 2/0)

(1) Two weeks prior to cell fusion, SP2/0 cells were resuscitated: 20. Mu.g/ml of 8-azaguanine (8-AG) was prepared and filtered through a 0.22 μm filter.

SP2/0 cells (round and transparent cells) with good states are selected and added into RPMI-1640 cell culture solution containing 8-AG for screening. After one week, the cells were changed to normal cell culture.

(2) Cell fusion: preparation of feeder cells

BALB/c mice around 8 weeks old were sacrificed at cervical scission and immersed in 75% alcohol for 10 minutes. The sacrificed mice were fixed with their abdomen facing upwards in an ultra clean bench. The left hand lifts the mouse belly skin with forceps, the right hand shears a small opening at the lifted position with surgical scissors, and the mouse skin is torn along the sheared small opening to expose the mouse belly. After the exposed abdomen of the mice was sterilized with an alcohol cotton ball, the incomplete culture solution of RPMI-1640 was aspirated with a 10ml syringe, gently injected into the abdomen of the mice, and gently rubbed with an alcohol cotton ball. The syringe was pushed gently into the abdominal cavity, then gently withdrawn, and aspirated back and forth twice. The injected liquid was gently aspirated and transferred to a sterile centrifuge tube. After centrifugation of the feeder cells-containing liquid at 1500r/min for 8 min, the supernatant was discarded, the RPMI-1640 incomplete culture solution was added and washed once with gentle pipetting, and the supernatant was discarded again by centrifugation under the same centrifugation conditions. Cell pellet was suspended and counted in RPMI-1640 complete medium containing 12% FBS of HAT, and then added to a 96-well cell culture plate at 100. Mu.l/well. Feeder cells prepared from one mouse can be used in 3-4 96-well plates. The 96-well plates were placed in a cell incubator for 24 hours.

(3) Preparation of SP2/0 cells

SP2/0 cells screened by 8-AG were passaged for expansion culture 48 hours prior to fusion, making them in the log phase of growth. On the day of fusion, SP2/0 cells were gently blown off the walls of the cell culture flask with a sterile pipette and collected in a 50ml sterile centrifuge tube. The collected SP2/0, 1300r/min was centrifuged for 5 minutes, the supernatant was discarded, 25ml of the incomplete RPMI-1640 culture solution was added for washing, and the discarded supernatant was centrifuged under the same conditions. SP2/0 was washed twice more under the same conditions. SP2/0 was counted after washing.

7. Preparation of spleen cells of immunized mice

And selecting BALB/c immunized mice with high titers for cell fusion. BALB/c immunized mice were first collected from the eyes, sacrificed, and placed in a 75% alcohol beaker for 10 minutes. Immunized mice were fixed in a super clean bench. The skin of the mice was cut off with surgical scissors, the abdomen was exposed, and the abdomen of the mice was sterilized with alcohol cotton balls. Surgical scissors cut the abdomen of the mice. The immune spleen (splenomegaly, dark red in color) was isolated from the anatomical site where the spleen was located with forceps. Placed in a sterile cell culture dish. Immediately, the spleen was rinsed with an incomplete RPMI-1640 culture solution, and after the connective tissue and the capsule outside the spleen were peeled off with forceps, they were placed in sterile cell culture dishes, respectively. The spleen was pressed with a disposable plunger to allow spleen cells to enter the cell culture dish. The spleen was rinsed by adding the RPMI-1640 incomplete culture solution, and more spleen cells were allowed to enter the cell culture dish from the spleen. The extruded spleen cell suspensions were filtered through 200 mesh sterile cell sieves, respectively, into labeled new sterile cell culture dishes to make individual spleen cell suspensions. The individual spleen cell suspensions were pipetted into a 50ml sterile centrifuge tube. The spleen single cell suspension 1300r/min was centrifuged for 6 min, the supernatant was discarded, washed with 25ml of RPMI-1640 incomplete cell culture broth, and the discarded supernatant was centrifuged under the same conditions. The same conditions were used for washing twice. Cell counts were performed on the suspensions.

8. Fusion: mixing immune spleen cells with SP2/0 at ratio of 6:1 in sterile centrifuge tube, and adding RPMI-1640 incomplete culture solution to 35ml. The two cells were thoroughly mixed by gentle blowing with a sterile pipette, centrifuging at 1300r/min for 6 min, discarding the supernatant, and pipetting the residual liquid. The bottom of the centrifuge tube was gently tapped to create a gap between the pelleted cells. While being preheated in a 37℃water bath, 0.8ml of 50% PEG4000 preheated at 37℃was pipetted into a 1ml pipette. The solution was added dropwise to the centrifuge tube over 60 seconds, and the tube was gently shaken at all times during the addition. After adding 37℃pre-warmed RPMI-1640 incomplete broth with a sterile pipette at a rate from rapid to slow, the fusion tube was placed in a 37℃incubator for 25 minutes. The fusion tube 1300r/min after standing was centrifuged for 6 minutes, and the supernatant was discarded. The fused cell pellet was suspended by adding RPMI-1640 complete medium containing HAT 12% FBS. The 96-well plate with feeder cells laid in advance was removed, 100. Mu.l/well of the fused cell suspension was added, and labeling was performed. The 96-well plate was placed in a cell culture incubator for culture, and the cell change was observed every day.

9. Screening of positive fusion wells: after 5 days of cell fusion, half of the original medium was replaced with RPMI-1640 medium containing HAT,20% FBS. HAT was changed to HT medium 12 days after fusion. After 17 days of fusion, the cells were changed to normal cell culture. In the whole culture screening process, the growth change of cells in each hole is closely observed, and when cell fusion clusters appear and the fused cells reach a sufficient quantity, the conditions of labeling and timely detecting the secretion of antibodies are well done. And marking the holes which are detected to be positive by the fusion hole indirect ELISA method for more than three times in succession, and timely cloning to avoid losing.

Cloning of the positive well: feeder cells were prepared the day before the positive Kong Ke clone, and the cells of the fusion wells for positive detection were pipetted into a 15ml sterile centrifuge tube containing 1ml of cell culture medium and labeled. The aspirated cells were counted per well. The cells of each well are diluted to 5 to 10, 10 to 20, 20 to 30, and three different dilutions according to the cell count. Cell suspensions of different dilutions per well of cells were added to 96-well plates with feeder cells, 100 μl/well, and labeled. The 96-well plate was placed in a cell incubator for culture. The growth of cells per well was observed daily and marked during the cloning culture. And detecting the cell holes with single clone in time, and marking to protect the cell strain. More than two cloned cell wells were discarded. The cloning was continued as described above for single clones that were positive for detection.

10. Expansion and freezing of canine parvovirus positive monoclonal pore cell strain: the cells in the wells which are cloned twice or more in succession and have been positive are subjected to expansion culture and cryopreservation. Canine parvovirus positive monoclonal was transferred from 96-well plates to 24-well plates with feeder cells added for expansion culture. After the cells were grown on the cell culture plate, the cells were transferred from the 24-well plate to a 12-well plate with feeder cells added thereto, and antibody secretion was detected at each well. Secretion positive cell-well cells were transferred from a 12-well plate to a large-volume cell culture flask (75 cm) ² ) Culturing. The secretion of antibodies by the cells was detected at any time. Freezing the monoclonal cell strain with positive result according to conventional method, and preparing frozen cell solution according to the ratio of RPMI-1640:FBS/DMSO=7:2:1. The code of the frozen cell strain is marked, and the frozen date is marked.

EXAMPLE 4 canine parvovirus antibody preparation

1. Preparation of monoclonal antibodies: the frozen hybridoma cells were harvested by resuscitating and expanding culture in complete RPMI1640 medium, and counted. Abdominal inoculation of 8-10 week old female BALB/c mice, 1X 10 each ⁶ Individual cells. After 10 days of inoculation, the ascites of the mice are sucked by a syringe, each mouse is collected 1-3 times, 3-10 ml each time, until the mice die. Collecting ascites, centrifuging at 2400r/min for 5 min, and collecting supernatant; repeating once. Adding merthiolate with final concentration of 0.01% into ascites, subpackaging, and preserving at-70deg.C.

2. Purification of monoclonal antibodies: the ascites is purified by precipitation. Adding NaCl with the final concentration of 0.2mol/L and CaCl with the final concentration of 0.025mol/L successively ₂ A solution. Filtering with filter paper, adding100 volumes of sterilized purified water were dialyzed against the filtrate at 4℃for 12 hours, during which time water was changed 1 time. Centrifuging the filtrate at 22000r/min for 30 min, and discarding the supernatant; the pellet was resuspended in 0.1mol/L Tris-HCl solution (pH 8.0) containing 1mol/L NaCl; repeating the dialysis and centrifugation for 1 time; the precipitated protein concentration was adjusted to 10mg/ml. Filtering and sterilizing with 0.22 μm filter, preserving at-70deg.C, and keeping for 1 year.

3. Identification of monoclonal antibodies: monoclonal antibody subtype identification

Coating 96-well ELISA plates (100 μl/well) with optimized antigen concentration, washing with PBST for 2 hours at 37 ℃, drying, adding 1:5000 diluted monoclonal antibody ascites to be detected (50 μl/well) after washing with PBST, incubating for 1 hour at 37 ℃, after washing and drying, respectively adding 1:1000 diluted goat anti-mouse IgG1, igG2a, igG2b, igG3, igM, igA, ig k, ig-on-light-shielding subclasses serum (50 μl/well) after washing and drying, adding 1:5000 diluted HRP-rabbit anti-sheep IgG (100 μl/well) after washing and drying, reacting for 15 minutes at room temperature (15-25 ℃), adding OPD substrate in the well, developing color at 37 ℃ for 5-10 minutes (100 μl/well), adding 2M H ₂ SO ₄ Termination color development (100. Mu.l/well), reading OD _450nm The antibody type with a significantly higher read than the other wells was chosen as the mab subclass.

3G4 and 5H8 belong to IgG2a _к Subclasses 1A4 and 2G3 belong to IgG2b _к Subclasses, 2G8 and 4H2 belong to IgG1 _к Subclasses.

4. Monoclonal antibody neutralization activity assay: the neutralizing activity of each monoclonal antibody was determined by the immobilized virus-diluted serum method: will be 100TCD ₅₀ Mixing diluted canine parvovirus virus liquid with culture supernatant (2 times serial dilution) or ascites (10 times serial dilution) of equal volume hybridoma cells, standing in 37 deg.C incubator for 1 hr, inoculating the virus-antibody mixture into 96 well plate (100 μl/hole), spreading into F81 cells, standing at 37 deg.C in 5% CO ₂ Culturing in incubator for several days, observing cytopathy day by day, and synchronously setting anti-canine parvovirus positive serum, canine parvovirus and normal F81 cells as controls.

The neutralizing titer of antibodies in mAb-4H2 culture supernatant was determined to be 2 ⁸ Neutralization of ascitesHas a potency of 10 ⁴ None of the other 5 monoclonal antibodies had neutralizing activity.

5. Monoclonal antibody affinity constant determination

The affinity of the antibodies was determined using a non-competitive ELISA. The coating antigen is purified canine parvovirus-VLPs protein, and the ELISA plate is coated according to the dosage of 1, 0.5, 0.25 and 0.125 mug/ml, 100 mug/well is coated overnight at 4 ℃; 1% BSA was added and each well was blocked at 37℃for 2 hours in 150. Mu.l; after PBST washing, the monoclonal antibody is diluted by a multiple ratio from 5 mug/ml according to the determined concentration of the monoclonal antibody, the logarithmic value of the concentration of the antibody (mol/L) is taken as the abscissa, and the corresponding OD is taken as the corresponding OD ₄₅₀ The values are on the ordinate, and 4S-curves are made in one coordinate system. Find the top of the S-curve, set to OD _max . Finding out 50% OD of each of the 4 curves _max Corresponding antibody concentration. The affinity constants of the monoclonal antibodies were calculated according to the formula, with 4 concentrations in pairs.

Ka＝(n-1)/2(n[Ab']t-[Ab]t)

n is the multiple of the concentration of two coating antigens in each group, [ Ab ]']t and [ Ab ]t is two 50% OD in each group _max Corresponding antibody concentration (mol/L).

The average value of 6 Ka's of the canine parvovirus monoclonal antibody 1A4 was 6.5X10 ⁷ M ^-1 6 Ka averages of 2G3 are 4.8X10 ⁴ M ^-1 6 Ka averages of 2G8 are 1.3X10 ⁵ M ^-1 6 Ka averages of 3G4 are 3.1X10 ⁷ M ^-1 6 Ka averages of 4H2 are 0.7X10 ⁷ M ^-1 And 6 Ka averages of 5H8 are 2.8X10 ⁷ M ^-1 . The affinity of the six monoclonal antibodies is sequentially 1A4 from high to low>3G4>5H8>4H2>2G8>1A4。

It is generally considered that the affinity constant is 10 ⁷ ～10 ¹² M ^-1 When belonging to high affinity antibodies, the affinity constant is 10 ⁵ ～10 ⁷ M ^-1 And, when belonging to low affinity antibodies. Four strains of mab 1A4, 3G4, 5H8 and 4H2 were therefore selected as markers and coating studies.

6. Indirect immunofluorescence assay

F81 cells were grown at 2X 10 ⁵ Per ml to 3X 10 ⁵ Each/ml of the cell suspension was added to a 96-well plate at 100. Mu.l/well. Dilution of canine parvovirus to 100TCID ₅₀ 100 μl/well, and 100 μl/well of canine distemper virus, canine coronavirus and canine adenovirus, each of which has a dilution, are added to the cells of the following viruses. Blank cell control was also established at 37℃with 5% CO ₂ The cells were cultured in an incubator for 48 hours. After 48 hours of virus infection of cells, the virus solution was discarded, and the cells were fixed with 80% cold acetone at 200. Mu.l/well for 30 minutes at room temperature (15 to 25 ℃). PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, and then the wells were rinsed. Hybridoma cell culture supernatants were diluted 2-fold, 50-fold, 100-fold, 200-fold, 400-fold, 819200-fold, and 15 dilution gradients were added sequentially to 96-well plates, 50. Mu.l/well, and incubated for 1 hour at 37 ℃. PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, followed by pipetting the wells. The secondary antibody is FITC marked donkey anti-mouse IgG, and secondary antibody diluent (FITC marked donkey anti-mouse IgG 1:200 times dilution and Evan blue 1:300 times dilution) is added into 96-well plate cells, and the mixture is incubated for 1 hour at 37 ℃. PBST plates were washed, 100. Mu.l/well, 3 times for 3 minutes each, followed by pipetting the wells.

The indirect immunofluorescence detection of monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 shows that the four monoclonal antibodies can generate specific reaction with canine parvovirus, do not react with other viruses and blank cell contrast, and the indirect immunofluorescence result is diluted by a double ratio, so that the fluorescence signal can be still detected when the antibodies are diluted by 6400 times.

7. Monoclonal antibody antigen recognition site differential analysis

The differences in the action antigen sites of the monoclonal antibodies of each strain are analyzed by adopting additive ELISA test: the ascites of each monoclonal antibody strain was diluted to a saturation working concentration (OD _450nm The value does not increase with the increase of the concentration of the antibody), adding one monoclonal antibody, incubating for 1.5 hours at 37 ℃, adding the other monoclonal antibody after washing for four times, incubating for 1.5 hours at 37 ℃, washing for four times, and then adding the enzyme-labeled secondary antibody for experiment according to the steps of an indirect ELISA method. Determination of the total OD after mixing and addition, respectively _450nm Value and OD of each independent reaction _450nm Values, each set of experiments were repeated three times and averaged. The AI value of 2 strains of monoclonal antibodies superimposed on each other was calculated according to the following formula: ai=ai (%) = [ (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100，A ₁ And A ₂ Is the absorption value of each of two monoclonal antibodies, A ₁₊₂ Is the absorbance measured after the addition of the two monoclonal antibodies. If AI <50, then both monoclonal antibodies bind to the same antigenic site, if AI>50, the two monoclonal antibodies bind to different antigenic sites.

According to the OD value result of the detected monoclonal hybridoma cell strain and the calculation formula AI (%) = [2A ₁₊₂ /(A ₁ +A ₂ )-1]X100 to analyze epitope, if AI is more than or equal to 50%, two monoclonal antibodies are different epitopes, if AI<50% of the two monoclonal antibodies are the same epitope. The addition ELISA test shows that the AI of the 4 mAbs after addition is greater than 50%, which indicates that the mAbs act on different antigen sites.

TABLE 14 determination of AI values by McAb addition test

Note that: AI (%) = [ (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100

8.4 pairing test of monoclonal antibodies as coating and detection antibodies respectively

The 4 purified mAbs are respectively marked with colloidal gold at the same concentration (10 mug/ml), NC films are coated at the same concentration (75 mug/ml), after the NC films are dried for 5 hours at 37 ℃, positive control substances are respectively crossed and paired, and the optimal combination of the capture monoclonal antibody and the coating monoclonal antibody with the maximum signal intensity is selected.

The monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 were paired with the corresponding labeled antibodies, respectively, and the results are shown in table 15.

TABLE 15 ELISA test results for different combinations of monoclonal antibodies

Example 5 preparation of Canine coronavirus and Canine parvovirus colloidal gold Triplex test strips

1. Preparation of colloidal gold: adding purified water into 1g of chloroauric acid to a volume of 100ml, filtering with a 0.22 μm filter membrane, placing into a clean glass container, refrigerating, and preserving in dark place. Adding 1% chloroauric acid solution into a proper amount of purified water to make the concentration of chloroauric acid solution be 0.01%. Adding 0.01% chloroauric acid solution into a siliconized glass container, placing the container on a magnetic heating stirrer, boiling for 3 minutes, rapidly adding 1.2ml of 1% trisodium citrate aqueous solution at one time under stirring, continuously heating for 6-8 minutes, cooling to room temperature, recovering to 100ml with purified water, filtering with a 0.22 μm filter membrane, placing into a clean glass bottle, placing the colloidal gold solution at 2-8 ℃ for preservation, visualizing the appearance of the colloidal gold before use, and obtaining a wine red liquid level oil-free floating object, wherein black particles do not coagulate at the bottom of the container.

2. Coating a nitrocellulose membrane: the colloidal gold solution is placed on a magnetic stirrer, the rotating speed is 300G/min, the pH value is regulated to 8.5 by using 10% potassium carbonate solution, the solution is averagely divided into 2 bottles, the canine coronavirus monoclonal antibody 1B2 is slowly added dropwise into one bottle to ensure that the final concentration of the canine coronavirus monoclonal antibody 1B2 is 10 mu G/ml, the canine parvovirus monoclonal antibody 3G4 is slowly added dropwise into the other bottle to ensure that the final concentration of the canine parvovirus monoclonal antibody 3G4 is 10 mu G/ml, the solution is kept stand for 30 minutes at room temperature, 0.01ml of 10% NaCl solution is added into the two bottles, and the solution is kept stand for 1 hour after uniform mixing, so that the observation result is kept red.

The two kinds of colloidal gold solutions added with the monoclonal antibodies are added with 10% bovine blood albumin solution under stirring to a final concentration of 1%, stirred for 5 minutes at room temperature (15-25 ℃), and then kept stand for 30 minutes. Stirring is started, 10% polyethylene glycol-6000 is slowly added dropwise to reach a final concentration of 1%, and after stirring for 5 minutes at room temperature (15-25 ℃), the mixture is allowed to stand for 30 minutes.

Taking gold-labeled conjugate, refrigerating and centrifuging for 20 minutes at 8000r/min, collecting supernatant, refrigerating and centrifuging again at 12000r/min for 20 minutes, discarding supernatant, re-suspending the twice centrifuged precipitate by gold-labeled buffer (0.02 mol/L PBS pH7.2, bovine serum albumin final concentration of 1%, sucrose final concentration of 0.5% and sodium azide final concentration of 0.01%) and collecting into the same container, so that the volume of gold-labeled heavy suspension with original volume of 1/10,1B2 and gold-labeled heavy suspension with 3G4 are mixed according to 1: and mixing in proportion to obtain the purified gold-labeled conjugate, and refrigerating and preserving for later use.

Adding equal volume of gold-labeled buffer (0.02 mol/L PBS pH7.2, bovine serum albumin final concentration 1%, sucrose final concentration 0.5%, and sodium azide final concentration 0.01%) into purified gold-labeled conjugate, mixing, and spraying onto glass fiber membrane (spraying amount of gold-labeled conjugate is 60 μl/100 mm) ² I.e. 36ml of glass fibres per sheet) was placed in a forced air drying oven at 37 c for 5 hours. After drying, the gold mark bonding pad is cut into strips with the length of 5mm multiplied by 300mm, and the strips are sealed and stored.

Diluting canine coronavirus monoclonal antibody 3C2 to 20 mug/ml with film coating liquid (0.8% sucrose, 0.02mol/L PBS pH 7.2), spraying onto nitrocellulose film with film spraying instrument to obtain detection line T ₁ The method comprises the steps of carrying out a first treatment on the surface of the The canine parvovirus monoclonal antibody 5H8 is diluted to 75 mug/ml by a film coating liquid (0.8 percent sucrose, 0.02mol/L PBS pH 7.2), and is sprayed on a nitrocellulose film by a film spraying instrument to be a detection line T ₂ . The goat anti-mouse IgG is diluted to 200 mug/ml by a film coating liquid (0.8% sucrose, 0.02mol/L PBS pH 7.2), and sprayed on a nitrocellulose film 5mm below a detection line by using a film spraying instrument to form a quality control line. And (3) drying the coated nitrocellulose membrane at 37 ℃ for 12 hours, and then sealing and preserving.

3. Preparation of large plates: as shown in fig. 7, each assembly (sample pad, two colloidal gold pads, water-absorbing filter paper, NC film, etc.) was sequentially stuck on a clean PVC base plate, and after the sticking was completed, a desiccant was added, and the sealed storage was performed.

4. Assembly of finished products

The strip was cut into large plates, which were cut into test strips 4mm wide.

Assembling the cut test paper strip to a base groove of the plastic card, covering an upper cover of the plastic card, compacting, and pressing the card by using a shell pressing machine to form the test paper strip.

The single test paper strip, the desiccant and the plastic dropper are packaged in the aluminum foil bag together, and the aluminum foil bag is sealed by a sealing machine.

The external packing box is sprayed with information such as batch number, production date, expiration date and the like according to requirements, and the packing box is folded, and 10 test strips, 10 sample treatment fluids, 10 cotton swabs and 1 instruction book are filled in each box.

Sensitivity test A sensitivity control is taken, 1.0ml of sample treatment liquid attached to the product is used for re-dissolving, and then the sample treatment liquid is diluted to the virus content of TCID according to the calibrated titer ₅₀ ＝10 ^-5.0 0.1ml of the sample was used as a sensitive control solution, and the sample was then treated according to the sensitive control (virus content: TCID ₅₀ ＝10 ^-5.0 0.1 ml): sensitivity test sample treatment fluid = 1:10,1:100,1:1000 fold dilution (TCID ₅₀ Is 10 ^-2.0 0.1 ml), each dilution was used as the sample to be examined. Each dilution was tested 10 times, sensitive control: the test results of the diluted 1:100 samples should be positive for the sensitivity test sample treatment fluid=1:10. Compared with a PCR detection sample, the coincidence rate is 100 percent.

The specific test is carried out by taking 6 specific reference substances for test, wherein, the canine coronavirus cell culture, the canine distemper virus cell culture, the canine parvovirus cell culture and the canine parainfluenza virus cell culture are respectively re-dissolved by 1ml of sample treatment liquid and then are detected by adopting the test strip, F81 cell culture liquid is directly detected by adopting the test strip after being melted, 5 healthy canine fecal samples are dipped by a cotton swab, each specific reference substance is repeatedly detected for 5 times, and the detection result is that: the canine parvovirus test paper is positive for detecting canine parvovirus cell cultures, other samples are negative, and the canine coronavirus test paper is positive for detecting canine coronavirus cell cultures, and other samples are negative.

The test strip has high sensitivity and good purposefulness.

Claims

1. A hybridoma cell line producing a monoclonal antibody to canine parvovirus, wherein the hybridoma cell line is 3G4 or hybridoma cell line 5H8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No.45197, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day; or the preservation number of the hybridoma cell strain 5H8 is CGMCC No.45198, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

2. A pair of hybridoma cell lines producing monoclonal antibodies against canine parvovirus, wherein said hybridoma is fine

The cell line is the hybridoma cell line 3G4 of claim 1 and the hybridoma cell line 5H8 of claim 1.

3. A monoclonal antibody of canine parvovirus, wherein the monoclonal antibodies are canine parvovirus antibodies 3G4 and 5H8, and the antibody 3G4 is an antibody produced by the hybridoma cell line 3G4 of claim 1; the antibody 5H8 is an antibody produced by the hybridoma cell line 5H8 of claim 1.

4. Use of the hybridoma cell line of claim 1, the pair of hybridoma cell lines of claim 2 or the monoclonal antibody of claim 3 in preparing a triple detection kit for detecting canine coronavirus and canine parvovirus or a canine parvovirus detection kit.

5. The test strip for detecting the triple colloidal gold of the canine coronavirus and the canine parvovirus is characterized by comprising the following components: sample pad, colloidal gold pad, water-absorbing filter paper, nitrocellulose membrane; a quality control line and a detection line are arranged on the nitrocellulose membrane, an antibody 3C2 is coated on the detection line 1, an antibody 5H8 of claim 3 is coated on the detection line 2, and goat anti-mouse IgG is coated on the quality control line; the colloidal gold pad is coated with a colloidal gold-labeled antibody 1B2 and 3G4 of claim 3, wherein the antibody 1B2 is produced by a hybridoma cell strain 1B2, the preservation number of the hybridoma cell strain 1B2 is CGMCC No.45200, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day; the antibody 3C2 is produced by a hybridoma cell strain 3C2, the preservation number of the hybridoma cell strain 3C2 is CGMCC No.45199, the preservation address is China general microbiological culture Collection center, and the preservation time is 2022, 7 months and 1 day.

6. The test strip for detecting canine coronavirus and canine parvovirus triple colloidal gold according to claim 5, wherein the preparation method of the colloidal gold labeled antibodies 1B2 and 3G4 comprises the steps of:

step 1: stirring the colloidal gold solution, regulating the pH value to 8.5, equally dividing into 2 bottles, slowly adding the canine coronavirus monoclonal antibody 1B2 dropwise into one bottle to ensure that the final concentration of the canine coronavirus monoclonal antibody 1B2 is 10 mug/ml, slowly adding the canine parvovirus monoclonal antibody 3G4 dropwise into the other bottle to ensure that the final concentration of the canine parvovirus monoclonal antibody 3G4 is 10 mug/ml, standing at room temperature for 30 minutes, adding 0.01ml of 10% NaCl solution into the two bottles, uniformly mixing, and standing for 1 hour;

7. The test strip for detecting canine coronavirus and canine parvovirus triple colloidal gold according to claim 6, wherein the method for preparing the colloidal gold solution in step 1 comprises the steps of: adding 0.01% chloroauric acid solution into a siliconized glass container, placing the container on a magnetic heating stirrer, boiling for 3 minutes, rapidly adding 1.2ml of 1% trisodium citrate aqueous solution at one time under stirring, continuously heating for 6-8 minutes, cooling to room temperature, recovering to 100ml with purified water, filtering with a 0.22 μm filter membrane, and filling into a clean glass bottle; the colloidal gold solution in the step 1 is placed on a magnetic stirrer and stirred at the rotating speed of 300 g/min; the pH was adjusted to 8.5 with a 0.2mol/L potassium carbonate solution.

8. The test strip for detecting canine coronavirus and canine parvovirus triple colloidal gold according to claim 6, wherein the test strip comprises

In step 2, the gold-labeled buffer consists of the following components: 0.02mol/L PBS pH7.2, bovine serum albumin final concentration 1% (m/V), sucrose final concentration 0.5% (m/V) and sodium azide final concentration 0.01% (m/V).

9. The test strip for detecting the triple colloidal gold of canine coronavirus and canine parvovirus according to claim 5, wherein each 1ml of the colloidal gold-labeled antibodies 1B2 and 3G4 are sprayed on the glass fiber film in an amount of 60 μl/100mm ² 。

10. The test strip for detecting the triple colloidal gold of the canine coronavirus and the canine parvovirus according to claim 5, wherein after the canine coronavirus monoclonal antibody 3C2 is diluted by a film coating liquid, the diluted solution is sprayed on a nitrocellulose film according to 1.0 mug/cm to be used as a detection line 1; after diluting a canine parvovirus antibody 5H8 with a film coating liquid, spraying the diluted canine parvovirus antibody on a nitrocellulose film according to the concentration of 1.0 mug/cm to serve as a detection line 2; spraying goat anti-mouse IgG on a nitrocellulose membrane according to the volume of 1.0 mug/cm, and taking the goat anti-mouse IgG as a quality control line; the concentration of the canine coronavirus monoclonal antibody 3C2 is 20 mug/ml, and the concentration of the canine parvovirus monoclonal antibody 5H8 is 75 mug/ml.