CN115094043A

CN115094043A - Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application

Info

Publication number: CN115094043A
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: 2022-09-23
Anticipated expiration: 2042-08-02
Also published as: CN115094043B

Abstract

The invention provides a hybridoma cell strain for canine coronavirus and canine parvovirus, a monoclonal antibody and application, and belongs to the technical field of biological detection. To provide a hybridoma cell line for detecting canine coronavirus and canine parvovirus and a monoclonal antibody produced by the same. 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 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.

Background

The Canine Parvovirus (CPV) disease and the Canine coronavirus (Canine coronavirus) disease are main epidemic diseases which endanger the health of dogs, the clinical symptoms of the two diseases caused in the onset period are similar and are often mixed infection, and the specific virus infection is difficult to identify by the clinical symptoms alone, so that a laboratory detection means is required. There are three common detection methods specified in the current national standards, namely, an immunoenzyme method, an immunohistochemical method and an RT-PCR method. There is also a clinical test of colloidal gold technology that can detect only one virus.

The method applied in national standard is used for detecting the canine parvovirus and the canine coronavirus, which needs special equipment and professional technical personnel, 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 professionals, convenience for veterinary personnel to use in clinic or field, low cost and the like.

At present, colloidal gold test strips for separately detecting canine coronavirus and canine parvovirus exist in the market, but the detection sensitivity is low, and the viruses with high concentration can be detected, so that detection omission and false negative results appear in the detection process; and only a single colloidal gold detection method is adopted, so that two viruses cannot be detected simultaneously, and the use cost is still high.

Disclosure of Invention

The invention aims to provide a hybridoma cell strain for detecting canine coronavirus and canine parvovirus and a monoclonal antibody produced by the hybridoma cell strain.

The invention also aims to provide a colloidal gold triple test strip for detecting canine coronavirus and canine parvovirus, which can be used for detecting the canine coronavirus and the canine parvovirus simultaneously.

The invention provides a hybridoma cell strain for producing a monoclonal antibody of canine parvovirus, which is 3G4 or 5H 8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No. 45197; or the hybridoma cell strain 5H8 has the preservation number of CGMCC No.45198, the preservation address of China general microbiological culture Collection center, and the preservation time of 2022 years, 7 months and 1 day.

The invention provides a pair of hybridoma cell strains for generating a monoclonal antibody of canine parvovirus, wherein the hybridoma cell strains are 3G4 and 5H 8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No. 45197; the hybridoma cell strain 5H8 has the preservation number of CGMCC No.45198, the preservation address of China general microbiological culture Collection center, and the preservation time of 2022 years, 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 preparing a triple detection kit for detecting canine coronavirus and canine parvovirus or a canine parvovirus detection kit.

The invention provides a dog coronavirus and dog parvovirus triple colloidal gold detection test strip, which consists of the following components: a sample pad, a colloidal gold pad, absorbent filter paper and a nitrocellulose membrane; the nitrocellulose membrane is provided with a quality control line and a detection line, wherein 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 was coated with colloidal gold labeled antibodies 1B2 and 3G 4.

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

step 1: stirring the colloidal gold solution, adjusting the pH value to 8.5, averagely dividing into 2 bottles, dropwise and slowly adding the canine coronavirus monoclonal antibody 1B2 into one bottle to enable the final concentration of the canine coronavirus monoclonal antibody 1B2 to be 10 mu G/ml, dropwise and slowly adding the canine parvovirus monoclonal antibody 3G4 into the other bottle to enable the final concentration of the canine parvovirus monoclonal antibody 3G4 to be 10 mu G/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.

And 2, step: adding 10% BSA solution into colloidal gold solution of two bottles of monoclonal antibody to make the final concentration 1%, mixing uniformly, sealing for 30 min, centrifuging at 8000r/min for 20 min, discarding supernatant, re-suspending the centrifuged precipitate with gold-labeled buffer solution to make the volume of the precipitate be 1/10, 1B2 gold-labeled suspension and 3G4 gold-labeled re-suspension according to the ratio of 1:1 ratio to obtain colloidal gold labeled monoclonal antibodies 1B2 and 3G 4.

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

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

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

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

Has the advantages that:

after purifying the canine parvovirus WQ8401 cell culture solution, immunizing a female BALB/c mouse with the age of 6-8 weeks, screening 6 strains of cell strains of a monoclonal well with positive for more than 3 times through indirect ELISA detection, preparing ascites, and identifying a monoclonal antibody after purification. The affinity assay of 6 monoclonal antibodies is 1A4>3G4>5H8>4H2>2G8>1A4 from high to low, the subclasses of the monoclonal antibodies are identified as 3G4 and 5H8 belonging to IgG2a k subclass, 1A4 and 2G3 belonging to IgG2b k subclass, and 2G8 and 4H2 belonging to IgG1 k subclass. The indirect immunofluorescence shows that the canine parvovirus monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 can detect the fluorescence signal with the multiple no less than 6400 times.

Monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 all demonstrated an AI of greater than 50% after addition of the 4 mAbs, indicating that they act at different antigenic sites, by an additive ELISA assay.

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

[ BIOLOGICAL PRESERVATION CERTIFICATION ] canine parvovirus hybridoma cell lines 3G4 and 5H8, wherein the hybridoma cell lines are 3G4 or hybridoma cell lines 5H 8; 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 years, 7 months and 1 day; or the hybridoma cell strain 5H8 has the preservation number of CGMCC No.45198, the preservation address of China general microbiological culture Collection center, and the preservation time of 2022 years, 7 months and 1 day.

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

The address of the depository: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

Drawings

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

FIG. 2 is a graph of OD coated with different CCV antigen concentrations _450nm And (6) detecting a result.

FIG. 3 shows the results of indirect immunofluorescence, wherein A is the results of indirect immunofluorescence with monoclonal antibody 1B 2; b is a blank cell control; c is monoclonal antibody 3C2 indirect immunofluorescence result; d is a blank cell control; e is the result of indirect immunofluorescence of monoclonal antibody 2B 5; f is a blank cell control; g is the indirect immunofluorescence result of the monoclonal antibody 2D 3; h is a blank cell control.

FIG. 4 shows the purity check of the electrophoresis of the purified ascites fluids 1B2 and 3C 2.

FIG. 5 shows the results of indirect immunofluorescence, wherein A is the results of indirect immunofluorescence with monoclonal antibody 1A 4; b is a blank cell control; c is the monoclonal antibody 3G4 indirect immunofluorescence result; d is a blank cell control; e is the result of monoclonal antibody 4H2 indirect immunofluorescence; f is a blank cell control; g is the result of monoclonal antibody 5H8 indirect immunofluorescence; h is a blank cell control.

FIG. 6 is a schematic diagram of 4 monoclonal antibody pairing experiments; 1 to 12 are 1 respectively, and the label 3G4 is coated with 1A 4; 2. label 4H2 coated 1a 4; 3. label 5H8 coating 1a 4; 4. label 1a4 coating 3G 4; 5. label 4H2 coating 3G 4; 6. marker 5H8 coated 3G 4; 7. label 1a4 coated with 4H 2; 8. marker 3G4 coated with 4H 2; 9. marker 5H8 coated with 4H 2; 10. label 1a4 coated with 5H 8; 11. marker 3G4 coated with 5H 8; 12. marker 4H2 was coated with 5H 8.

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

Detailed Description

Example 1 preparation of Canine coronavirus hybridoma cells

A purified canine coronavirus CCV2015 strain (obtained by autonomous separation and identification of West Nuo biology, Changchun) is adopted as an immunogen to immunize a BALB/c mouse with the age of 6-8 weeks, a tail is cut simultaneously after immunization for 7 days for three times to take blood and measure the in-vivo serum antibody titer of the mouse, a mouse myeloma cell SP2/0 is fused with spleen lymphocytes, and a positive fusion hole is screened and is subjected to single strain treatment.

1. Purifying and detecting the immune source: the method adopts a purified canine coronavirus CCV2015 strain as an immune source and comprises the following specific steps: centrifuging at a low speed of 2000-3000 r/min for 20-30 minutes to remove larger host cell fragments and other larger impurities; and centrifuging the virus supernatant for 2 hours at 35000r/min by adopting a sucrose density gradient centrifugation method, and dissolving a precipitate zone by using PBS (phosphate buffer solution) with the pH value of 7.2 to obtain the purified immunogen. Detection was performed according to the BCA kit detection procedure, and OD was measured at 562 nm. Meanwhile, standard 2.0mg/ml, 1.0mg/ml, 0.5mg/ml, 0.25mg/ml and 0.125mg/ml protein solutions are prepared to prepare a standard curve. Deducing the protein content of the protein to be detected according to the standard curve.

Protein quantification is carried out on the purified virus by using a Pierce BCA protein Assay Kit protein content detection Kit. Y2.4553X +0.185(Y represents absorbance, X represents protein content, R ² 0.9828). When the absorbance of the purified viral protein after 50-fold dilution was 1.232, the protein content was 0.385mg/ml, and the original concentration was 19.25mg/ml, and the results are shown in FIG. 1.

2. Animal immunization: purified canine coronavirus CCV2015 strain was used as the source of immunization at 200 μ g protein per mouse. Firstly, exempting from: mixing antigen and Freund's complete adjuvant at volume ratio of 1:1, making into emulsion, and injecting subcutaneously at multiple points on mouse abdomen. And (2) avoiding: after the first immunization for 14 days, the antigen and Freund's incomplete adjuvant are mixed in a volume ratio of 1:1 to prepare emulsion, and immunization is carried out according to the immunization method of the first immunization. And (3) three-step (I): after 14 days of the secondary immunization, immunization was performed using the same procedure as the secondary immunization. After 7 days, the mice are subjected to tail breaking and blood drawing, the serum neutralizing antibody titer is measured by an indirect ELISA method, and when the antibody titer meets the requirement, the mice are subjected to one-time antigen intraperitoneal injection by using 50 mu g of protein per mouse of non-emulsified antigen 3 days before fusion.

3. Mouse serum potency assay

Establishment of indirect ELISA detection method

Diluting the purified canine coronavirus antigen with a coating solution to final concentrations 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 respectively, coating the antigen at 4 ℃ overnight, washing the antigen with a washing solution for 3 times, sealing the antigen with 5% skim milk sealing solution, incubating the antigen at 200 mu l/well for 2 hours at 37 ℃, washing the antigen with a washing solution for 3 times, using mouse positive serum as a positive control, adding PBS buffer solution into a blank control, using mouse negative serum as a negative control, incubating the antigen in each well for 100 mu l at 37 ℃ for 1 hour; then, the cells were washed 3 times with a washing solution, a secondary antibody (HRP-labeled goat anti-mouse IgG) diluted 4000 times with PBS was added thereto at 100. mu.l/well, incubated at 37 ℃ for 1 hour, then, after taking out the cells, the cells were washed 3 times with a washing solution, a developing solution was added thereto at 100. mu.l/well, and developed for 5 minutes, 50. mu.l of a stop solution was added to each well to terminate the incubation, and OD was measured at 450nm, and the storage data was recorded, and as can be seen from FIG. 2, the optimum coating concentration of the purified canine coronavirus was measured at 0.25. mu.g/. mu.l.

4. Mouse serum potency assay

Diluting with purified canine coronavirus antigen with coating solution to a final concentration of 0.25. mu.g/. mu.l, 100. mu.l/well, coating overnight at 4 ℃, washing with washing solution for 3 times, blocking with 5% skim milk blocking solution, incubating at 37 ℃ for 2 hours, washing with washing solution for 3 times, performing 2-fold gradient dilution of mouse positive serum from 200-fold, adding PBS buffer into blank control, and diluting negative control with negative serum 200-fold. All are 100 mul/well, incubated for 1 hour at 37 ℃; then washing with a washing solution for 3 times, adding a secondary antibody (HRP-labeled goat anti-mouse IgG) diluted by 4000 times with PBS, incubating at 37 ℃ for 1 hour, taking out, washing with a washing solution for 3 times, adding a developing solution for 100. mu.l/well, developing for 5 minutes, adding 50. mu.l of a stop solution into each well, stopping, measuring the OD value at 450nm, and recording and storing data. The titer test is carried out on the mice after 4 immunizations by using the canine coronavirus ELISA method, wherein the dilution corresponding to the minimum OD reading with the titer being more than the maximum OD/2 is determined as the final canine coronavirus serum titer, the mouse with the canine coronavirus serum ELISA titer being the highest is No.4, and the result is shown in Table 1.

TABLE 1 ELISA titer results after mouse immunization

5. Preparation of myeloma cells, splenic lymphocytes and thymocytes: SP2/0 mouse myeloma cell HGPRT is deleted, immunoglobulin is not generated, mycoplasma detection is carried out, IMDM complete culture medium (containing 20% serum) is used for culture and passage, mouse eyeballs with the highest canine coronavirus titer are selected, blood is taken, killed, the mouse eyeballs are placed into 75% alcohol for soaking for 5 minutes, a small amount of serum-free IMDM is poured into a flat dish, a cell sieve and an inner core of an injector are placed into the flat dish, the spleen of the mouse is taken down by scissors and tweezers and placed on the cell sieve, the spleen is gently and fully crushed by the inner core of the injector, the crushed cells are sucked into a centrifuge tube, the mouse is washed for 3 times by PBS for standby use, the thymus of the mouse is taken down by the scissors and the tweezers and crushed, and the crushed thymocytes are put into a 15ml centrifuge tube for standby use.

6. Fusion test: gently blowing off well-conditioned SP2/0 cells from the wall of a culture flask, sucking the cells into a 50ml centrifuge tube, sucking the prepared splenic lymphocyte cells into the centrifuge tube filled with SP2/0, and centrifuging for 5 minutes at 1500 r/min; and pouring off the supernatant of the centrifuged cells, gently and uniformly blowing the cells by serum-free IMDM, centrifuging at 1500r/min for 5 minutes, and pouring off the supernatant of the centrifuged cells as much as possible. Beating the bottom of the centrifugal tube to fully suspend the cells, putting the centrifugal tube into warm water at 37 ℃, slowly adding 1ml of PEG within 1 minute, standing in the warm water for 1 minute after the addition is finished, then slowly adding 2ml of serum-free IMDM within 2 minutes, then slowly adding 8ml of serum-free IMDM within 2 minutes, and centrifuging at 1000r/min for 5 minutes. The supernatant was decanted, 45ml of HAT complete medium was added, the cells were carefully blown down, the previously prepared thymocytes were poured in, and mixed well. The cells were added to 3 96-well cell culture plates, 150. mu.l per well, using a dispenser, and the cell culture plates were placed in a wet box and then incubated in an incubator.

7. Screening of positive fusion wells: after 7 days of HAT complete medium culture, half of the original culture medium was replaced with IMDM medium containing HAT, 20% FBS, and after 16 days of fusion, 100. mu.l of cell supernatant was pipetted into an ELISA plate previously coated with purified canine coronavirus CCV2015 strain antigen, while 100. mu.l of HT complete medium was supplemented into a 96-well plate. If the ELISA test result is positive, subcloning and individualizing are performed.

8. And (3) carrying out hybridoma cell titer test: performing ELISA screening on the fused hybridoma cells to determine positive cell strains, wherein the steps are as follows: diluting the purified virus of the canine coronavirus CCV2015 by coating liquid to obtain a final concentration of 0.25 mu g/mu l and 100 mu l/hole, and standing overnight at 4 ℃; then washing with washing liquor for 3 times; 5% skim milk blocking solution is sealed, 200 mu l/hole is incubated for 2 hours at 37 ℃, then washed by washing liquor for 3 times, primary antibody (cell culture supernatant or ascites), negative control (SP2/0 culture supernatant), blank control (PBS) and positive control (positive serum PBS is diluted 1000 times) are added, 100 mu l/hole are added, incubation is carried out for 1 hour at 37 ℃, then washed by washing liquor for 3 times, secondary antibody (HRP-labeled goat anti-mouse IgG) diluted 4000 times is added, 100 mu l/hole is incubated for 1 hour at 37 ℃, after being taken out, washing liquor is used for 3 times, color developing liquor is added for 100 mu l/hole, color development is carried out for about 5 minutes, 50 mu l of stop solution is added into each hole to terminate, OD value is measured at 450nm, data is recorded and stored, and the titer is the dilution corresponding to the minimum OD reading which is larger than the maximum OD/2.

The canine coronavirus CCV2015 strain was coated, fusion cell wells were tested by ELISA, and xx positive hybridoma cell wells were co-screened, namely 1B2, 1E8, 1F3, 2B5, 2C10, 2D3, 3C1, 3C2 and 3H11, wherein H12 is a positive control, G12 is a blank control, and F12 is a 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)

TABLE 3 Canine coronavirus antibody positive cell Screen 2 (OD) _450nm Value)

TABLE 4 Canine coronavirus antibody positive cell Screen 3 (OD) _450nm Value)

9. Monochorization of hybridoma cells: to achieve uniformity of the genetic background of the hybridoma cells, positive clone wells in a 96-well plate were limiting diluted to 2 cells/well. The total cell number was counted by a hemocytometer, diluted with 10ml of IMDM medium containing 20% serum to a cell number of about 200 cells, and added to a 96-well plate at 100. mu.l/well with 2 cells per well. Observing after 7-10 days, selecting holes with only 1 clonal colony, transferring to 24 holes after 14 days, and transferring to 25cm after 18 days ² The antibody hybridomas and the antibodies secreted therefrom were designated as 1B2, 1E8, 1F10, 2B5, 2C10, 2D3, 3C1, 3C2, and 3H11, as individual clones confirmed by microscopic observation in cell culture flasks.

Example 2 preparation of Canine coronavirus antibodies

1. Identification of monoclonal antibodies

Hybridoma cell and ascites titer test the screened monoclonal hybridoma cell is prepared into mouse ascites by a method of 2.10, cell supernatant and the mouse ascites are respectively used as primary antibodies, a purified CCV2015 strain virus is used as an antigen to coat an ELISA plate, and an indirect ELISA method is adopted to carry out titer test, wherein the titer is a dilution corresponding to the minimum OD reading which is more than the maximum OD/2.

The 9 monoclonal hybridoma cell supernatants 1B2, 1E8, 1F10, 2B5, 2C10, 2D3, 3C1, 3C2 and 3H11, and the correspondingly prepared mouse ascites were re-plated with purified CCV2015 strain viruses, and titer test was performed by ELISA method, and the results are shown in table 5.

TABLE 5 monoclonal hybridoma cell Canine coronavirus ELISA titer results

2. And (3) identifying the subtype of the monoclonal antibody: coating a 96-well enzyme label plate (100 mu l/well) with an optimized antigen concentration, acting at 37 ℃ for 2 hours, washing and drying by PBST, adding ascites (50 mu l/well) of monoclonal antibody to be detected diluted by 1:5000, incubating at 37 ℃ for 1 hour, washing and drying in the same way, respectively adding serum (50 mu l/well) of subclasses of goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Ig and Ig, respectively, acting at 37 ℃ for 1 hour, washing and drying, adding HRP-rabbit anti-goat IgG (100 mu l/well) diluted by 1:5000, acting at room temperature (15-25 ℃) for 15 minutes, adding TMB substrate into the wells, developing in the dark at 37 ℃ for 5-10 minutes (100 mu l/well), and adding 2M H ₂ SO ₄ Color development was stopped (100. mu.l/well) and OD read _450nm Values, antibody types with significantly higher readings than other wells were selected as mab subclasses.

And (4) carrying out subclass identification on the screened 9 positive cell strains to finally obtain 9 IgG type positive hybridoma cell strains. 1E8, 2B5, 2C10, 2D3 and 3C1 cell strains are IgG1 and kappa chains, and 1F10 and 3H11 cell strains are IgG1 and lambda chains; 1B2 cells were IgG2B, kappa chain; the 3C2 cells were IgG2b, lambda chain, and the results are shown in table 6.

TABLE 6 monoclonal cell subset identification (OD) _450nm Value)

3. Monoclonal antibody neutralization activity assay: the neutralizing activity of each monoclonal antibody is determined by adopting a fixed virus-diluted serum method: will 100TCID ₅₀ Mixing diluted canine coronavirus virus solution with culture supernatant (2-fold serial dilution) or ascites (10-fold serial dilution) of hybridoma cells of the same volume, placing in 37 deg.C incubator for 1 hr, inoculating the virus-antibody mixture into 96-well plate (100 μ l/well), uniformly spreading F81 cells, placing in 37 deg.C 5% CO ₂ The cells are cultured in an incubator for several days, the cytopathic effect is observed day by day, and anti-canine coronavirus positive serum, canine coronavirus and normal F81 cells are synchronously set as controls.

The neutralizing titer of the antibody in the culture supernatant of mAb-1B2 was determined to be 2 ⁶ The ascites has a neutralizing titer of 1.28X 10 ³ The other 8 monoclonal antibodies had no neutralizing activity.

4. Determination of affinity constant of clone antibody: the affinity of the antibodies was determined using a non-competitive ELISA. The coating antigen is purified canine coronavirus, and the enzyme label plate is coated according to the concentration of 1, 0.5, 0.25 and 0.125 mu g/mu l, each hole is 100 mu l, and the enzyme label plate is coated overnight at 4 ℃; adding 1% BSA, 150. mu.l per well, blocking at 37 ℃ for 2 hours; after PBST washing, the monoclonal antibody was diluted in multiples starting from 5. mu.g/. mu.l at the determined monoclonal antibody concentration, with the logarithm of the antibody concentration (moL/L) as abscissa and the corresponding OD ₄₅₀ The values are ordinate and 4 sigmoid curves are made in one coordinate system. Find the top of the S-curve and set it as OD _max . The 50% OD of each of the 4 curves was found out in the curve _max Corresponding antibody concentration. And (4) calculating the affinity constant of the monoclonal antibody according to a formula in a group of two by two concentration.

Ka＝(n-1)/2(n[Ab']t-[Ab]t)

n is the multiple of two coating antigen concentrations 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 of the canine coronavirus monoclonal antibody 1B2 is 2.67X 10 ⁷ M ^-1 3C2 had an average value of 6 Ka's of 2.6X 10 ⁷

M

^-1 6 Ka averages of 2B5The value was 2.3X 10 ⁷ M ^-1 2D3 had an average value of 6 Ka's of 1.6X 10 ⁷ M ^-1 1E8 had an average value of 6 Ka's of 0.8X 10 ⁶ M ^-1 1F10 had an average value of 6 Ka's of 1.6X 10 ⁶ M ^-1 2C10 had an average value of 6 Ka's of 3.0X 10 ⁶ M ^-1 3C1 had an average value of 6 Ka's of 1.2X 10 ⁶ M ^-1 And 6 Ka averages of 3H11 of 2.0X 10 ⁶ M ^-1 . The affinity of nine monoclonal antibodies is 1B2 from high to low>3C2>2B5>2D3>2C10>3H11> 1F10>3C1>1E8。

It is generally considered that the affinity constant is 10 ⁷ ～10 ¹² M ^-1 When it is a high affinity antibody, and the affinity constant is 10 ⁵ ～10 ⁷ M ^-1 When it is a low affinity antibody. Therefore, four monoclonal antibodies, 1B2, 3C2, 2B5 and 2D3, were selected for labeling and coating studies.

5. Indirect immunofluorescence assay: f81 cells were cultured at 2X 10 ⁵ mu/ul-3X 10 ⁵ Mu.l of cell suspension was added to a 96-well plate at 100. mu.l/well. Dilution of Canine coronavirus to 100TCID ₅₀ 100 mul/well, and adding 100 mul/well of canine distemper virus, canine parvovirus, canine adenovirus and canine parainfluenza virus into susceptible cells of the following viruses at a certain dilution ratio. Meanwhile, a blank cell control was set up at 37 ℃ with 5% CO ₂ The cell culture box was incubated for 48 hours. After the virus infects the cells for 48 hours, the virus solution is discarded, the cells are fixed by 80% cold acetone and 200 mul/hole, and the cells are fixed for 30 minutes at room temperature (15-25 ℃). PBST washing plate, 100u L/hole, washing plate 3 times, each time 3 minutes, then will be the hole liquid clean. The hybridoma cell culture supernatant was diluted 2-fold, 50-fold, 100-fold, 200-fold, 400-fold. cndot. 819200-fold, and 15 dilution gradients were added sequentially to a 96-well plate at 50. mu.l/well and incubated at 37 ℃ in an incubator for 1 hour. PBST washing plate, 100u L/hole, washing plate 3 times, each time 3 minutes, then will be the hole liquid clean. The secondary antibody is FITC labeled goat anti-mouse IgG, the secondary antibody diluent (FITC labeled goat anti-mouse IgG diluted 1:200 times and Evans blue diluted 1:100 times) is added into 96-well plate cells, and incubated for 1 hour at 37 ℃ in an incubator. PBST Wash plate, 100. mu.l/wellThe plate was washed 3 times for 3 minutes each, and then the wells were rinsed clean of liquid.

After the hybridoma cell lines 1B2, 3C2, 2B5 and 2D3 are full, the supernatants are taken for antibody property identification, and an indirect immunofluorescence method is used, so that the results show that 1B2, 3C2, 2B5 and 2D3 can be specifically combined with CCV strains and can not be combined with canine distemper virus, canine parvovirus, canine adenovirus and canine parainfluenza virus. The indirect immunofluorescence results by dilution at fold-by-fold showed that fluorescence signals were still detectable when the antibody was diluted 6400 fold, and the results are shown in FIG. 3.

6. Differential analysis of monoclonal antibody antigen recognition sites: the difference of the acting antigen sites of each monoclonal antibody is analyzed by an additive ELISA test: each monoclonal antibody ascites fluid was diluted to saturation working concentration (OD) _450nm Values did not increase with increasing antibody concentration), one monoclonal antibody was added, incubated at 37 ℃ for 1.5 hours, washed 4 times, the other monoclonal antibody was added, incubated at 37 ℃ for 1.5 hours, washed 4 times, and then an enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG) was added for the assay following the procedure of the indirect ELISA method. The total OD after mixing and addition was measured separately _450nm Value and OD of each independent reaction _450nm Values, which were averaged over 3 replicates per set of experiments. The AI values of the 2 monoclonal antibodies after being superimposed on each other were calculated according to the following formula: AI ═ AI (%) [ (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100，A ₁ And A ₂ Is the absorbance of each of the two monoclonal antibodies, A ₁₊₂ Is the absorbance measured after the addition of 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 monoclonal hybridoma cell strain, the calculation formula AI (%) ═ 2A is combined ₁₊₂ /(A ₁ +A ₂ )-1]X 100 analysis of antigen epitope, if AI is greater than or equal to 50%, the two monoclonal antibodies are different antigen epitopes, if AI is greater than or equal to 50%<50% of the two monoclonal antibodies are the same epitope. The additive ELISA test proves that the AI of 4 mAbs after addition is more than 50 percent, which indicates that the mAbs act on different antigenic sites, and the results are shown in the table7。

AI values determined by strain McAb addition test in Table 74

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

Coating and antibody detection matching test 4 purified mAbs are respectively labeled with colloidal gold at the same concentration (10 mug/mul), coated with NC membrane at the same concentration, dried at 37 ℃ for 5 hours, respectively cross-matched, and dropwise added with positive reference substances, and the one with the largest signal intensity is selected as the optimal combination of the captured monoclonal antibody and the coated monoclonal antibody.

The monoclonal antibodies 1B2, 3C2, 2B5 and 2D3 were matched with the corresponding labeled antibodies, respectively, and the results show that the enveloped monoclonal antibody 3C2 and the labeled monoclonal antibody 1B2 have the best effect on detecting the canine coronavirus sensitivity control, and the results are shown in Table 8.

TABLE 8 test results for different combinations of monoclonal antibodies

7. Preparation of ascites from hybridoma cells

Collecting hybridoma cell strains 3C2 and 1B2 for production in frozen storage, resuscitating in water bath at 37 deg.C for 2-5 min, adding into 75cm ² Cell culture flasks at 5% CO ₂ CO at 37 deg.C ₂ Growing in an incubator for 3-5 days. Taking hybridoma cells growing in a monolayer, removing supernatant, sucking 10ml of IMDM into a 10ml suction pipe, repeatedly blowing a cell bottle, collecting cell suspension, adding the cell suspension into a 50ml centrifuge tube, centrifuging at 1000rpm for 10 minutes, removing supernatant, and dissolving the precipitate again by using 2ml of IMDM. Using a syringe with the maximum range of 1ml to suck the hybridoma cell suspension after redissolution, and inoculating female BALB/c mice with the abdominal cavity age of 8-10 weeks, wherein each female BALB/c mouse is 1 multiplied by 10 ⁶ And (4) one cell. After 10 days of inoculation, the ascites of the mice are sucked by a syringe, and each mouse is collected for 1-3 times, 3-10 ml each time, until the mice die. Collecting 12000g ascites, centrifuging for 5 min, collectingCollect the supernatant and repeat once. Adding thimerosal with final concentration of 0.01% into ascites, subpackaging, and storing at-70 deg.C.

8. Antibody purification: mouse ascites antibody was affinity-purified using protein G, and the mouse ascites antibody was purified by loading the filler into a column, diluting the ascites with a buffer (20mM PBS, 300mM NaCl, pH 7.4-8.5) about 5-fold, and loading the column. After loading, removing the unbound protein fraction with an equilibration buffer (20mM PBS, 300mM NaCl, pH 7.4-8.5); then, elution buffer (0.1M sodium citrate, pH4.0) was added to elute the bound protein, and finally, pH adjustment was performed with neutralization buffer (1M Tris-HCl pH 9.0).

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

10. And (4) performing titer inspection: purified antibodies were titer tested using viral ELISA, coated: diluting and purifying the coating solution to obtain a canine coronavirus CCV2015 strain with a final concentration of 0.25 mu g/mu l and 100 mu l/hole, and keeping the strain in a constant temperature box at 37 ℃ for 1 hour; and (3) sealing: sealing 5% skim milk at 200/hole, sealing overnight, washing the plate for 3 times, diluting the purified antibody by 100 times, diluting by PBS (phosphate buffer solution) at a multiple ratio, and washing the plate for 3 times in a 37 ℃ thermostat for 1 hour; diluting HRP-labeled goat anti-mouse IgG (secondary antibody) at a ratio of 1: 4000PBS, adding 100 μ l of the diluted solution into each hole of an ELISA plate, and keeping the temperature at 37 ℃ for 0.5 hour; washing the plate for 3 times; color development: TMB 100PBS room temperature for 3 minutes; 2mol/L H ₂ SO ₄ And (4) stopping the solution.

Monoclonal antibodies 1B2 and 3C2 mouse ascites were prepared and affinity purified using protein G. The purified sample is subjected to purity detection by an SDS-PAGE method, content detection by BCA protein and titer detection by canine coronavirus ELISA, and the result is shown in figure 4 and table 9.

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

Expansion and cryopreservation of hybridoma cell lines 3C2 and 1B 2: after the CCV hybridoma cells 3C2 and 1B2 grow to a monolayer, removing the culture medium, blowing and beating the cells by using 10ml of IMDM complete culture medium (containing 20% serum) to separate the cells from the bottom of a cell bottle, distributing the hybridoma cells in 1 cell bottle into 3 cell bottles on average according to the ratio of 1:3, injecting 30-40 ml of IMDM complete culture medium (containing 20% serum) into each cell bottle, continuously observing and culturing, and recording the growth condition of the cells every day. Cells were passaged every 4 days for 3 consecutive passages and the results were recorded. Continuously passing the 1 st generation CCV hybridoma cells 3C2 and 1B2 for 5 generations in the presence of 5% CO ₂ The cells were cultured in an incubator at 37 ℃ for 5 days, and the growth of the cells was recorded daily.

Digesting the cells, adding a small amount of serum-free IMDM, centrifuging at 1000rpm for 5 min, collecting the bottom layer cells, dispersing with 10% bovine serum IMDM, continuously washing for 3 times, collecting the bottom layer cells, and adjusting the cell concentration to 10 ⁶ Mu.l, adding a freezing medium (IMDM 95% containing 20% fetal bovine serum, dimethyl sulfoxide 5%, antibiotics), freezing at 20 ℃ for 2 hours, freezing at-70 ℃ for 4 hours, and storing in liquid nitrogen. The hybridoma cells of 3 rd to 10 th generations are preserved according to the method, the times of cell generation and the preparation date are noted, and the cells are preserved in liquid nitrogen.

The CCV hybridoma cells 3C2 and 1B2 proliferate 5-10 times after passage, the cell morphology is mouse myeloma cells, and the cell shows good proliferation characteristic results shown in Table 10.

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

Hybridoma cell line antibody secretion assay results hybridoma cell lines 1B2, 3C2 were grown to fill the bottom of the cell vial, and the supernatant was collected for antibody secretion assay, the results of which are shown in table 11.

TABLE 11 measurement results of antibody secretion from hybridoma cell lines

Hybridoma cell passage stability P6 generation cells of hybridoma cell strains 1B2 and 3C2 are continuously passaged for 15 generations, the cells can stably proliferate, and supernatant is collected for secretory antibody determination, so that the secretory antibody property is stable, and the result is shown in Table 12.

TABLE 12 hybridoma cell passage stability assay

Results of cell cryopreservation assay

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

3.17.2 identification of growth characteristics of recovered cells after recovery of P5 generation and P10 generation cells, growth characteristics were measured, cells were cultured for 5 days, and cell count was carried out to obtain 5.5X 10 generation cells of P5 generation ⁵ Cell/ml, passage 10 cells 6.1X 10 ⁵ Each/ml.

The detection results of the exogenous factors are negative when the hybridoma cell strains 1B2 and 3C2 carry out bacterial and mould tests, and the results are negative when the liquid culture medium is adopted for mycoplasma detection. The fluorescent antibodies for resisting the canine parvovirus, the rabies virus and the bovine viral diarrhea virus are used for detection, 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 excrement of a suspected canine parvovirus-suffering dog collected clinically is separated and identified, and the separated canine parvovirus is identified by means of a microscope observation method, a physicochemical characteristic identification method, an erythrocyte hemagglutination spectrum test method, a specificity test detection, a nucleic acid detection and the like.

The results show that: the separated pathogen is canine parvovirus and is named as canine parvovirus WQ8401 strain (obtained by autonomous separation and identification of Changchun West Nuo biology, Inc.), after synchronous virus inoculation of F81 cells, the canine parvovirus WQ8401 strain has obvious reticuloid lesion, virus particles can be seen under an electron microscope, the virus has the most obvious phenomenon of porcine red blood cell agglutination when the pH value is 7.2, specificity detection proves that the canine parvovirus WQ8401 strain can be specifically combined with canine parvovirus fluorescent monoclonal antibody, and nucleic acid detection proves that the canine parvovirus WQ8401 strain is a canine parvovirus positive result. Therefore, the WQ8401 strain is a canine parvovirus strain.

2. After the canine parvovirus WQ8401 virus liquid is purified, a female BALB/c mouse with the age of 6-8 weeks is immunized, and after two weeks of secondary immunization, a tail is cut simultaneously to take blood to measure the titer of serum antibodies in the mouse and an indirect ELISA detection method is established. 6 strains of cell strains of the positive monoclonal hole which is detected by indirect ELISA for more than 3 times are screened out, ascites is prepared, the anti-canine parvovirus monoclonal antibody is purified, identification such as monoclonal antibody subtype identification and affinity constant determination is carried out, and the label and the coating of the monoclonal antibody are selected by combining the preparation process of a test strip. And screening 4 anti-canine parvovirus monoclonal hybridoma cell strains with high affinity, namely 1A4, 3G4, 5H8 and 4H2 respectively, and through indirect ELISA tests, proving that the 4 monoclonal antibodies are directed at different epitopes of the antigen, through pairing tests, finding that the marked 3G4 and the coated 5H8 are optimal combinations, and the detection line signals are stronger than other combinations, and respectively serving as the capture monoclonal antibody and the detection monoclonal antibody for detection lines of colloidal gold marking and coated nitrocellulose membranes.

3. Concentration and purification of antigen: repeatedly freezing and thawing the canine parvovirus WQ8401 strain third-generation virus culture solution for 3 times at 37 +/-20 ℃. After the freeze-thaw liquid is centrifuged for 35 minutes at 4 ℃ and 3500r/min, the supernatant is collected. PEG8000 was added to the collected supernatant so that the final concentration was 12%, and NaCl was added so that the final concentration was 0.5 mol/L. The mixture was stirred once every 15 minutes, repeated four times, and the virus solution was placed in a refrigerator at 4 ℃ overnight. Centrifuging overnight virus solution at 4 deg.C and 15000r/min for 50 min, carefully taking out, discarding supernatant, adding appropriate amount of PBS according to precipitation amount, resuspending, and freezing at-80 deg.C.

4. Immunization of animals: 5 female BALB/c mice with the age of 6-8 weeks are selected, canine parvovirus purified virus and equivalent Freund's complete adjuvant emulsion are injected into the mice, and 200 mu l of the canine parvovirus purified virus and the equivalent Freund's complete adjuvant emulsion are injected into each mouse at multiple subcutaneous points. Two weeks later, the mice were immunized with the canine parvovirus-enriched purified virus and an equivalent amount of Freund's incomplete adjuvant emulsion in the same manner as the initial injection amount. The third immunization is carried out two weeks after the second immunization, and the immunization method and the virus injection amount are the same as the second immunization. After three-time immunization and two weeks, mice with high serum titer after tail clipping blood collection are selected and injected with purified virus solution in the abdominal cavity.

5. The indirect ELISA method is used for measuring the in vivo serum titer of the mice: the optimal coating concentration of CPV-VLPs and the optimal dilution of serum were determined by a square-matrix method. The purified CPV-VLPs were diluted to different concentrations, 10, 8, 7, 6, 5, 4, 3, 1ug/ml using coating solutions, and 12 wells, 100u L/well were coated at each concentration. CPV positive serum and negative serum diluted by confining liquid 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 the conventional indirect ELISA detection method.

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

TABLE 1 Indirect ELISA Titers of monoclonal antibodies

6. Screening of mouse myeloma cells (SP2/0)

(1) The SP2/0 cells were revived around the first two weeks of cell fusion: 8-azaguanine (8-AG) was prepared at 20. mu.g/ml and filtered through a 0.22 μm filter for further use.

SP2/0 cells (round and transparent cells) with good state are selected and added into RPMI-1640 cell culture solution containing 8-AG for screening. One week later, the cells were replaced with normal cell culture medium.

(2) Cell fusion: preparation of feeder cells

BALB/c mice aged about 8 weeks were sacrificed after neck-breaking and soaked in 75% alcohol for 10 minutes. The sacrificed mice were mounted belly-up in an ultra-clean bench. The skin of the abdomen of the mouse is lifted by the forceps with the left hand, a small opening is cut at the lifted position by the surgical scissors with the right hand, and the skin of the mouse is torn along the cut small opening to expose the abdomen of the mouse. After the exposed abdomen of the mouse was sterilized with an alcohol cotton ball, the incomplete culture medium RPMI-1640 was aspirated with a 10ml syringe, gently injected into the abdominal cavity of the mouse, and the abdomen of the mouse was gently kneaded with the alcohol cotton ball. The syringe gently pushes the liquid into the abdominal cavity, and then gently withdraws the liquid, and the liquid is sucked twice back and forth. The injection liquid was gently aspirated and transferred to a sterile centrifuge tube. Centrifuging the liquid containing the feeder cells at 1500r/min for 8 minutes, discarding the supernatant, adding the RPMI-1640 incomplete culture solution, gently blowing and beating the incomplete culture solution by using a pipette once, centrifuging the incomplete culture solution again under the same centrifugation condition, and discarding the supernatant. After cell pellets were suspended and counted in 12% FBS RPMI-1640 complete medium containing HAT, they were added to 96-well cell culture plates at 100. mu.l/well. Feeder cells prepared from one mouse can be used for 3-4 96-well plates. The 96-well plate was placed in a cell incubator for 24 hours.

(3) Preparation of SP2/0 cells

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

7. Preparation of splenocytes from immunized mice

And selecting BALB/c immune mice with high titer for cell fusion. BALB/c immunized mice were blood-sampled from the eyeballs, sacrificed, and soaked in a beaker containing 75% alcohol for 10 minutes. The immunized mice were fixed in a clean bench. The skin of the mice was cut open with surgical scissors, respectively, to expose the abdomen, and the abdomen of the mice was sterilized with an alcohol cotton ball. The surgical scissors cut the abdomen of the mouse. The immunized spleen (enlarged spleen, dark red) was isolated at the anatomical site of the spleen with forceps. Placed in a sterile cell culture dish. Immediately, the spleen was washed with an incomplete RPMI-1640 culture medium, and the connective tissue and the envelope were detached from the outside of the spleen with forceps, and then placed in a sterile cell culture dish, respectively. Spleen was squeezed with a disposable syringe plunger to allow splenocytes to enter the cell culture dish. The spleen was washed with incomplete medium RPMI-1640 and more spleen cells were allowed to pass from the spleen into the cell culture dish. And respectively filtering the splenocyte suspension obtained by extrusion punching through a 200-mesh sterilized cell sieve into a new marked sterile cell culture dish to form single splenocyte suspension. The single spleen cell suspension was pipetted into a 50ml sterile centrifuge tube. Centrifuging the spleen single cell suspension at 1300r/min for 6 minutes, discarding the supernatant, adding 25ml of RPMI-1640 incomplete cell culture solution for washing, centrifuging under the same conditions, and discarding the supernatant. Washed twice under the same conditions. The suspension was cell counted.

8. Fusing: mixing immune spleen cells and SP2/0 at a ratio of 6: 1 in a sterile centrifuge tube, and adding RPMI-1640 incomplete culture solution to 35 ml. And (4) gently blowing and beating the two cells by using a sterile pipette to fully and uniformly mix the two cells, centrifuging the mixture for 6 minutes at 1300r/min, discarding supernatant, and completely sucking residual liquid. Gently tap the bottom of the centrifuge tube to create a void between the sedimented cells. While pre-heating in a 37 ℃ water bath, 0.8ml of the 37 ℃ pre-heated 50% PEG4000 was pipetted using a 1ml pipette. The mixture was added dropwise to the centrifuge tube over 60 seconds, and the centrifuge tube was gently shaken at all times during the addition. After adding the 37 ℃ preheated RPMI-1640 incomplete culture solution at a speed from high to low by using a sterile pipette, the fusion tube is placed in a 37 ℃ incubator for 25 minutes. The settled fusion tube 1300r/min 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 previously plated with feeder cells was removed, and 100. mu.l/well of the fused cell suspension was added and labeled. The 96-well plate was placed in a cell 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 culture was replaced with HAT, 20% FBS-containing RPMI-1640 culture medium. HAT was changed to HT medium 12 days after fusion. The cells were changed to normal cell culture medium 17 days after the fusion. In the whole culture screening process, the growth change of cells in each hole is closely observed, when cell fusion clusters appear and the number of fused cells reaches enough, the cells are marked and the antibody secretion condition of the cells is timely detected. And marking the fusion hole indirect ELISA for more than three times continuously to detect positive holes, and cloning in time to avoid loss.

Cloning of positive wells: feeder cells were prepared the day before positive well cloning, and fusion well cells with positive results detected were pipetted into a 15ml sterile centrifuge tube containing 1ml of cell culture medium and labeled. The aspirated cells were counted per well. According to the cell counting condition, the cells in each hole are diluted to 5/ml-10/ml, 10/ml-20/ml, 20/ml-30/ml and three different dilutions. Cell suspensions of different dilutions of each well of cells were added to feeder cells in 96-well plates at 100. mu.l/well and labeled. The 96-well plate was placed in a cell incubator for culture. The growth of each well of cells was observed daily during the colony culture and labeled. And (4) detecting the cell hole with a single clone in time, and marking to protect the cell strain. Wells of more than two clones were discarded. Cloning of single clones which were positive in detection was continued as described above.

10. Enlarging and freezing the canine parvovirus positive monoclonal well cell strain: the cells in the wells which have been cloned twice more than twice and always have positive results are subjected to scale-up culture and frozen storage. Canine parvovirus positive monoclonals are transferred from 96-well plates to 24-well plates with feeder cells for expanded culture. After the cells grew over the cell culture plate, the cells were transferred from the 24-well plate to a 12-well plate containing feeder cells, and the secretion of the antibody from each well was measured. Positive-secreting cell well cells were transferred from 12-well plates to large volume cell culture flasks (75 cm) ² ) And (4) culturing. The secretion of antibody by the cells is detected at any time. And (3) freezing and storing the monoclonal cell strain with the amplification culture detection positive result according to a conventional method, wherein the frozen and stored cell sap is prepared according to the proportion of RPMI-1640: FBS: DMSO: 7: 2: 1. Marking the code of the frozen cell strain and the freezing date.

Example 4 preparation of Canine parvoVirus antibody

1. Preparation of monoclonal antibody: and (4) taking the frozen hybridoma cells, recovering and carrying out expanded culture on the hybridoma cells by using complete RPMI1640 culture medium, harvesting the hybridoma cells, and counting the hybridoma cells. Intraperitoneal inoculation of 8-10 week-old female BALB/c mice, 1 × 10 for each ⁶ And (4) one cell. And (3) after 10 days of inoculation, sucking ascites of the mice by using an injector, and collecting 3-10 ml of ascites for each mouse 1-3 times until the mice die. Collecting ascites, centrifuging at 2400r/min for 5 min, and collecting supernatant; and repeating the steps once. Adding thimerosal with final concentration of 0.01% into ascites, subpackaging, and storing at-70 deg.C.

2. Purification of monoclonal antibodies: each ascites fluid was purified by precipitation. Sequentially adding NaCl with final concentration of 0.2mol/L and CaCl with final concentration of 0.025mol/L ₂ And (3) solution. After filtration through a filter paper, 100 times by volume of sterilized purified water was added, and the filtrate was dialyzed at 4 ℃ for 12 hours, during which time water was changed 1 time. Centrifuging the filtrate at 22000r/min for 30 min, and removing the supernatant; resuspend the pellet 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 10 mg/ml. Filtering with 0.22 μm filter to remove bacteria, storing at-70 deg.C, and preserving for 1 year.

3. Identification of monoclonal antibodies: monoclonal antibody subtype identification

Coating a 96-well enzyme label plate (100 mu l/well) with optimized antigen concentration, acting at 37 ℃ for 2 hours, washing and drying by PBST, adding ascites (50 mu l/well) to be detected diluted by 1:5000, incubating at 37 ℃ for 1 hour, washing and drying by the same method, respectively adding goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Ig, and Ig, serum (50 mu l/well) of each subclass of Ig, acting at 37 ℃ for 1 hour, washing and drying by the same method, adding HRP-rabbit anti-goat IgG (100 mu l/well) diluted by 1:5000, acting at room temperature (15-25 ℃) for 15 minutes, adding OPD substrate into the well, shading and developing at 37 ℃ for 5-10 minutes (100 mu l/well), and adding 2M H ₂ SO ₄ Color development was stopped (100. mu.l/well) and OD read _450nm Values, antibody types with significantly higher readings than other wells were selected as mab subclasses.

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

4. Determination of neutralizing activity of monoclonal antibody: the neutralizing activity of each monoclonal antibody is determined by adopting a fixed virus-diluted serum method: will 100TCD ₅₀ Mixing the diluted canine parvovirus virus solution with culture supernatant (2-fold serial dilution) or ascites (10-fold serial dilution) of hybridoma cells of the same volume, placing the mixture in a 37 ℃ incubator for acting for 1 hour, taking the virus-antibody mixed solution to inoculate in a 96-well plate (100 mu l/well), uniformly paving F81 cells, placing the mixture in 5% CO at 37 ℃ and uniformly mixing ₂ The cells are cultured in an incubator for several days, the cytopathic effect is observed day by day, and anti-canine parvovirus positive serum, canine parvovirus and normal F81 cells are synchronously set as controls.

The neutralizing titer of the antibody in the culture supernatant of mAb-4H2 was determined to be 2 ⁸ The neutralization titer of ascites is 10 ⁴ The other 5 monoclonal antibodies had no 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, an ELISA plate is coated according to 1, 0.5, 0.25 and 0.125 mu g/ml, each hole is 100 mu l, and the coating is carried out overnight at 4 ℃; adding 1% BSA, 150. mu.l per well, blocking at 37 ℃ for 2 hours; after PBST washing, the monoclonal antibody was diluted in multiples starting from 5. mu.g/ml, according to the determined monoclonal antibody concentration, with the logarithm of the antibody concentration (mol/L) as the abscissa and the corresponding OD ₄₅₀ The values are ordinate and 4 sigmoid curves are made in one coordinate system. Find the top of the S-curve and set it as OD _max . The 50% OD of each of the 4 curves was found out in the curve _max Corresponding antibody concentration. And (4) calculating the affinity constant of the monoclonal antibody according to a formula in pairs at 4 concentrations.

Ka＝(n-1)/2(n[Ab']t-[Ab]t)

The average value of 6 Ka of the canine parvovirus monoclonal antibody 1A4 is 6.5X 10 ⁷ M ^-1 2G3 had an average value of 6 Ka's of 4.8X 10 ⁴ M ^-1 2G8 with 6 Ka average values of 1.310 ⁵ M ^-1 3G4 had an average value of 6 Ka's of 3.1X 10 ⁷ M ^-1 4H2 had an average value of 6 Ka's of 0.7X 10 ⁷ M ^-1 And 6 Ka averages of 5H8 of 2.8X 10 ⁷ M ^-1 . The affinity of the six monoclonal antibodies is 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 it is a high affinity antibody, and the affinity constant is 10 ⁵ ～10 ⁷ M ^-1 When it is a low affinity antibody. Therefore, four monoclonal antibodies 1A4, 3G4, 5H8 and 4H2 were selected for labeling and coating studies.

6. Indirect immunofluorescence assay

F81 cells were cultured at 2X 10 ⁵ one/ml-3X 10 ⁵ One/ml of the cell suspension was added to a 96-well plate at 100. mu.l/well. Diluting canine parvovirus to 100TCID ₅₀ 100 mul/well, and adding 100 mul/well of canine distemper virus, canine coronavirus and canine adenovirus with certain dilution factor into susceptible cells of the following viruses. Simultaneously, a blank cell control was set up with 5% CO at 37 deg.C ₂ The cell culture box was incubated for 48 hours. After the virus infects the cells for 48 hours, the virus solution is discarded, the cells are fixed by 80 percent cold acetone at 200 mu l/hole for 30 minutes at room temperature (15-25 ℃). PBST washing plate, 100u L/hole, washing plate 3 times, each time 3 minutes, then will be the hole liquid clean. The hybridoma cell culture supernatant was diluted 2-fold, 50-fold, 100-fold, 200-fold, 400-fold. cndot. 819200-fold, and 15 dilution gradients were added sequentially to a 96-well plate at 50. mu.l/well and incubated at 37 ℃ in an incubator for 1 hour. PBST wash plate, 100u l/hole, wash plate 3 times, each time for 3 minutes, then will hole liquid clean. The secondary antibody is FITC-labeled donkey anti-mouse IgG, a secondary antibody diluent (FITC-labeled donkey anti-mouse IgG is diluted 1:200 times and Evans blue is diluted 1:300 times), and the secondary antibody diluent is added into cells of a 96-well plate and incubated for 1 hour at 37 ℃ in an incubator. PBST wash plate, 100u l/hole, wash plate 3 times, each time for 3 minutes, then will hole liquid clean.

The indirect immunofluorescence is used for detecting the monoclonal antibodies 1A4, 3G4, 4H2 and 5H8, the result shows that the four monoclonal antibodies can generate specific reaction with canine parvovirus and do not react with other virus and blank cell controls, and the result of dilution of the indirect immunofluorescence by the multiple ratio shows that when the antibodies are diluted by 6400 times, fluorescence signals can still be detected.

7. Differential analysis of monoclonal antibody antigen recognition sites

The difference of the acting antigen sites of each monoclonal antibody is analyzed by an additive ELISA test: ascites of each monoclonal antibody was diluted to saturation working concentration (OD) _450nm The value did not increase with the increase of antibody concentration), one monoclonal antibody was added, incubated at 37 ℃ for 1.5 hours, washed four times, the other monoclonal antibody was added, incubated at 37 ℃ for 1.5 hours, washed four times, and then enzyme-labeled secondary antibody was added to conduct the experiment according to the procedure of indirect ELISA. The total OD after mixing and addition was measured separately _450nm Value and OD of each independent reaction _450nm Values, which were averaged in triplicate for each set of experiments. The AI values of the 2 monoclonal antibodies after being superimposed on each other were calculated according to the following formula: AI ═ AI (%) - (2A) ₁₊₂ /A ₁ +A ₂ )-1]×100，A ₁ And A ₂ Is the absorbance of each of the two monoclonal antibodies, A ₁₊₂ Is the absorbance measured after the addition of 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 monoclonal hybridoma cell strain, the calculation formula AI (%) - (2A) is combined ₁₊₂ /(A ₁ +A ₂ )-1]X 100 analysis of antigen epitope, if AI is greater than or equal to 50%, the two monoclonal antibodies are different antigen epitopes, if AI is greater than or equal to 50%<50% of the two monoclonal antibodies are the same epitope. The AI values after 4 mAbs were added were all greater than 50% as confirmed by additive ELISA, indicating that they act on different antigenic sites.

AI values determined by McAb addition test of 144 strains in Table

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

8.4 monoclonal antibodies as coating and detecting antibody pairing test

Marking 4 purified mAbs with the same concentration (10 mu g/ml) with colloidal gold, respectively, coating an NC membrane with the same concentration (75 mu g/ml), drying at 37 ℃ for 5 hours, respectively carrying out cross pairing, dropwise adding a positive control substance, and selecting the monoclonal antibody with the maximum signal intensity as the optimal combination of the captured monoclonal antibody and the coated monoclonal antibody.

The monoclonal antibodies 1A4, 3G4, 4H2 and 5H8 were each subjected to a pairing test with the corresponding labeled antibody, and the results are shown in Table 15.

TABLE 15 ELISA test results for different monoclonal antibody combinations

Example 5 preparation of colloidal gold triple test strip for Canine coronavirus and Canine parvovirus

1. Preparing colloidal gold: adding purified water into 1g of chloroauric acid to a constant volume of 100ml, filtering with a 0.22 mu m filter membrane, placing into a clean glass container, and refrigerating and storing in dark place. Adding 1% chloroauric acid solution into appropriate 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 in one step under a stirring state, continuously heating for 6-8 minutes, cooling to room temperature, recovering to 100ml with purified water, filtering with a 0.22 mu m filter membrane, placing the solution into a clean glass bottle, storing the colloidal gold solution at 2-8 ℃, observing the appearance of the colloidal gold before use, displaying wine red, having no oily floaters on the liquid surface, and having no black particles agglutinated at the bottom of the container.

2. Coating of nitrocellulose membrane: placing the colloidal gold solution on a magnetic stirrer, stirring at the rotating speed of 300G/min, adjusting the pH value to 8.5 by using a 10% potassium carbonate solution, evenly dividing into 2 bottles, dropwise and slowly adding the canine coronavirus monoclonal antibody 1B2 into one bottle to enable the final concentration of the canine coronavirus monoclonal antibody 1B2 to be 10 mu G/ml, dropwise and slowly adding the canine parvovirus monoclonal antibody 3G4 into the other bottle to enable the final concentration of the canine parvovirus monoclonal antibody 3G4 to be 10 mu G/ml, standing at room temperature for 30 minutes, adding 0.01ml of 10% NaCl solution into the two bottles, standing for 1 hour after uniformly mixing, and observing that the red color is kept unchanged.

Adding the two kinds of colloidal gold solutions added with the monoclonal antibody into a 10% bovine serum albumin solution while stirring until the final concentration is 1%, stirring for 5 minutes at room temperature (15-25 ℃), and standing for 30 minutes. Starting stirring, slowly dropwise adding 10% polyethylene glycol-6000 to a final concentration of 1%, stirring at room temperature (15-25 ℃) for 5 minutes, and standing for 30 minutes.

Taking the gold-labeled conjugate at 8000r/min, refrigerating and centrifuging for 20 minutes, collecting supernatant, refrigerating and centrifuging at 12000r/min again for 20 minutes, discarding supernatant, resuspending the twice-centrifuged precipitate with gold-labeled buffer solution (0.02mol/L PBS pH7.2, bovine blood albumin with final concentration of 1%, sucrose with final concentration of 0.5%, and sodium azide with final concentration of 0.01%) and collecting the precipitate in the same container, wherein the volume of the precipitate is 1/10, the gold-labeled suspension with 1B2 and the gold-labeled resuspension solution with 3G4 are calculated according to the ratio of 1: mixing at a ratio of 1 to obtain purified gold-labeled conjugate, and refrigerating for later use.

Adding equal volume of gold-labeled buffer solution (0.02mol/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%) into purified gold-labeled conjugate, mixing, and spraying onto glass fiber membrane (spraying amount of gold-labeled conjugate is 60 μ L/100 mm) ² I.e. 36ml per glass fibre spray), placed in a 37 c forced air drying cabinet for 5 hours. After drying, cutting the gold mark bonding pad into strips of 5mm × 300mm, sealing and storing.

Diluting canine coronavirus monoclonal antibody 3C2 to 20 μ g/ml with membrane coating solution (0.8% sucrose, 0.02mol/L PBS pH7.2), spraying onto nitrocellulose membrane with membrane spraying apparatus, and detecting line T ₁ (ii) a The canine parvovirus monoclonal antibody 5H8 is diluted to 75 mu g/ml by a membrane coating solution (0.8% sucrose, 0.02mol/L PBS pH7.2), sprayed on a nitrocellulose membrane by using a membrane spraying instrument, and used as a detection line T ₂ . The goat anti-mouse IgG was diluted to 200. mu.g/ml with a membrane coating solution (0.8% sucrose, 0.02mol/L PBS pH7.2), and sprayed onto nitrocellulose using a membrane sprayerAnd 5mm below the detection line on the membrane to form a quality control line. Drying the coated nitrocellulose membrane at 37 ℃ for 12 hours, and sealing and storing.

3. Preparation of the large plate: as shown in FIG. 7, the components (sample pad, two colloidal gold pads, absorbent filter paper, NC membrane, etc.) were sequentially attached to a clean PVC base plate, and after the attachment, a desiccant was added, and the plate was sealed and stored.

4. Finished product assembly

And cutting a large plate into test paper strips with the width of 4 mm.

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

The single test paper strip, the drying agent and the plastic dropper which are packaged inside are put into an aluminum foil bag together and sealed by a sealing machine.

And (3) spraying information such as batch numbers, production dates and expiration dates on the packaging box according to requirements, folding the packaging box, and filling 10 test paper strips, 10 sample treatment solutions, 10 cotton swabs and 1 part of instructions into each box.

Sensitive test takes sensitive reference substance, re-dissolves with 1.0ml sample treatment solution attached in the product, and then dilutes with sensitive test sample treatment solution according to its calibrated titer until the virus content is TCID ₅₀ ＝10 ^-5.0 0.1ml, as a sensitive control solution, and then according to the sensitive control (the virus content is TCID) ₅₀ ＝10 ^-5.0 0.1 ml): sensitivity test sample treatment fluid was diluted 1:10,1:100,1:1000 Times (TCID) ₅₀ Is 10 ^-2.0 0.1ml) and each dilution was used as a sample to be tested. 10 tests per dilution, sensitive controls: the detection results of the samples diluted in the ratio of 1:10 to 1:100 are positive. Compared with PCR detection samples, the coincidence rate is 100%.

The specificity test takes 6 kinds of specificity control articles to test, wherein, the dog coronavirus cell culture, the dog distemper virus cell culture, the dog parvovirus cell culture and the dog parainfluenza virus cell culture are respectively re-dissolved by 1ml of sample treatment fluid and then detected by the test strip, the F81 cell culture fluid is directly detected by the test strip after being melted, 5 healthy dog excrement samples are dipped by a cotton swab, each kind of specificity control article is repeatedly detected for 5 times, and the detection result is as follows: when the canine parvovirus test paper is used for detecting, the canine parvovirus cell culture is detected to be positive, other samples are negative, when the canine coronavirus test paper is used for detecting, the canine coronavirus cell culture is detected to be positive, and other samples are negative.

The test strip has high sensitivity and good specificity.

Claims

1. A hybridoma cell strain for producing a monoclonal antibody of canine parvovirus, which is characterized in that the hybridoma cell strain is 3G4 or hybridoma cell strain 5H 8; the hybridoma cell strain 3G4 has a preservation number of CGMCC No.45197, a preservation address of China general microbiological culture Collection Center (CCM) and a preservation time of 2022 years, 7 months and 1 day; or the hybridoma cell strain 5H8 has the preservation number of CGMCC No.45198, the preservation address of China general microbiological culture Collection center, and the preservation time of 2022 years, 7 months and 1 day.

2. A pair of hybridoma cell lines for producing monoclonal antibodies of canine parvovirus is characterized in that the hybridoma cell lines are 3G4 and hybridoma cell line 5H 8; the preservation number of the hybridoma cell strain 3G4 is CGMCC No. 45197; the hybridoma cell strain 5H8 has a preservation number of CGMCC No.45198, a preservation address of China general microbiological culture Collection center, and a preservation time of 2022 years, 7 months and 1 day.

3. The monoclonal antibody of the canine parvovirus is characterized in that the monoclonal antibody is canine parvovirus antibodies 3G4 and 5H8, and the antibody 3G4 is an antibody produced by a hybridoma cell line 3G 4; the antibody 5H8 is an antibody produced by hybridoma cell line 5H 8.

4. The use of the hybridoma cell strain of claim 1, the pair of hybridoma cell strains of claim 2, or the monoclonal antibody of claim 3 in the preparation of a triple detection kit for detecting canine coronavirus and canine parvovirus, or a canine parvovirus detection kit.

5. The test strip for detecting the canine coronavirus and the canine parvovirus by the triple colloidal gold is characterized by comprising the following components in parts by weight: a sample pad, a colloidal gold pad, absorbent filter paper and a nitrocellulose membrane; the nitrocellulose membrane is provided with a quality control line and a detection line, wherein 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 was coated with colloidal gold labeled antibodies 1B2 and 3G 4.

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

Step 2: adding 10% BSA solution into colloidal gold solution of monoclonal antibody in two bottles respectively to make the final concentration of the BSA solution 1%, uniformly mixing, sealing for 30 minutes, centrifuging at 8000r/min for 20 minutes, discarding the supernatant, re-suspending the centrifuged precipitate with gold-labeled buffer solution to make the volume of the precipitate equal to the original volume of 1/10, 1B2 gold-labeled suspension and 3G4 gold-labeled re-suspension according to the ratio of 1:1 ratio to obtain colloidal gold labeled monoclonal antibodies 1B2 and 3G 4.

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

8. The canine coronavirus and canine parvovirus colloidal gold test strip of claim 6, wherein the gold-labeled buffer in step 2 consists of the following components: 0.02mol/L PBS pH7.2, bovine blood albumin final concentration of 1% (m/V), sucrose final concentration of 0.5% (m/V) and sodium azide final concentration of 0.01% (m/V).

9. The canine coronavirus and canine parvovirus colloidal gold test strip of claim 5, wherein 1ml of colloidal gold-labeled antibodies 1B2 and 3G4 are sprayed on a glass fiber membrane in an amount of 60 μ l/100mm ² 。

10. The canine coronavirus and canine parvovirus colloidal gold test strip of claim 5, wherein the canine coronavirus monoclonal antibody 3C2 is diluted by a membrane coating solution and sprayed on a nitrocellulose membrane at a concentration of 1.0 μ g/cm to serve as a test line 1; diluting the canine parvovirus antibody 5H8 with a membrane coating solution, and spraying the diluted solution on a nitrocellulose membrane according to the concentration of 1.0 mu g/cm to serve as a detection line 2; spraying goat anti-mouse IgG on a nitrocellulose membrane according to the concentration of 1.0 mu g/cm to serve as a quality control line; the concentration of the canine coronavirus monoclonal antibody 3C2 is 20 mu g/ml, and the concentration of the canine parvovirus monoclonal antibody 5H8 is 75 mu g/ml.