CN115094044B - Hybridoma cell strain for producing monoclonal antibody of canine coronavirus, monoclonal antibody and application - Google Patents
Hybridoma cell strain for producing monoclonal antibody of canine coronavirus, monoclonal antibody and application Download PDFInfo
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- CN115094044B CN115094044B CN202210924338.0A CN202210924338A CN115094044B CN 115094044 B CN115094044 B CN 115094044B CN 202210924338 A CN202210924338 A CN 202210924338A CN 115094044 B CN115094044 B CN 115094044B
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Abstract
The invention provides a hybridoma cell strain for producing a monoclonal antibody of canine coronavirus, the monoclonal antibody and application thereof, and belongs to the technical field of biological detection. In order to provide a method for detecting canine coronavirus with high sensitivity and high specificity. The invention provides a pair of hybridoma cell strains for generating monoclonal antibodies of canine coronavirus, wherein the hybridoma cell strains are hybridoma cell strains 1B2 and 3C2, the preservation number of the hybridoma cell strain 1B2 is CGMCC No.45200, and the preservation number of the hybridoma cell strain 3C2 is CGMCC No.45199. The hybridoma cell lines 1B2 and 3C2 for the monoclonal antibody are prepared by a monoclonal antibody technology aiming at CCV viruses, the monoclonal antibody has higher specificity aiming at different CCV virus strains, and the prepared colloidal gold detection test strip has high sensitivity and good specificity, so that the canine coronavirus can be detected more accurately and rapidly.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a hybridoma cell strain for producing a monoclonal antibody of canine coronavirus, the monoclonal antibody and application thereof.
Background
The Canine Coronavirus (CCV) belongs to coronaviridae coronavirus, is a single-stranded positive strand RNA virus, has similar morphological characteristics, has fibrous processes similar to crowns, has a capsule membrane, is round or oval, and has the size of 80-120nm. The canine coronavirus has 4 structural proteins, namely, fibronectin (S, spike Protein,180-220 kD) as receptor binding sites; a small Envelope glycoprotein (E, envelope Protein,9-12 kD) which is less bound to the Envelope; the transmembrane protein M protein (M, transmembrane Protein,29-32 kD) is responsible for transmembrane transport of nutrients and budding release of the nascent virus and formation of the viral outer envelope; the nuclear protein N protein (N, nucleocapsid Protein,50-60 kD) is tightly bound to the viral RNA into the viral core shell.
Canine coronavirus (canine coronavirus, CCV) is a source of viral infectious disease that is a serious hazard to the canine industry, economic animal farming and wild animal protection. Canine coronavirus can cause high-grade contagious diseases characterized by canine vomiting, diarrhea, dehydration and easy recurrence, and can occur all the year round. The disease is especially serious to puppies, and the death rate is high. The sick dogs and the dogs with the viruses are main infectious agents, and the viruses are transmitted to healthy dogs and other susceptible animals through the respiratory tract and the digestive tract by direct contact and indirect contact. Infected dogs can detoxify by feces for 2 weeks or more, and the environment contaminated by infectious feces is the main cause of infection.
At present, no effective vaccine is used for preventing CCV, and no specific therapy exists, so early diagnosis is very important. Common clinical symptoms of CCV are not readily distinguishable from other enteropathogens such as CPV-2, canine rotavirus and canine adenovirus. Diagnosis of CCV therefore requires laboratory diagnosis. Diagnostic techniques for the detection of CCV in fecal samples include Electron Microscopy (EM), cell culture isolation of Virus (VI), virus gene detection (RT-PCR), and colloidal gold test strips.
In the Electron Microscope (EM), cell culture and Virus Isolation (VI), virus gene detection (RT-PCR) detection method, special equipment and professional technicians are required, the detection time is long, and the cost is high. 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.
Disclosure of Invention
The invention aims to provide a method for detecting canine coronavirus with high sensitivity and high specificity.
The invention provides a hybridoma cell strain for producing a monoclonal antibody of canine coronavirus, wherein the hybridoma cell strain is 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, the preservation time is 2022, 7 months and 1 day, or the hybridoma cell strain is hybridoma cell strain 3C2, the preservation number of the hybridoma cell strain 3C2 is CGMCC No.45199, the preservation address is 2022, 7 months and 1 day.
The invention provides a monoclonal antibody of canine coronavirus, which is an antibody 1B2, wherein the antibody 1B2 is an antibody generated by a hybridoma cell strain 1B2 or the monoclonal antibody is an antibody 3C2, and the antibody 3C2 is an antibody generated by a hybridoma cell strain 3C 2.
The invention provides a pair of hybridoma cell strains for generating monoclonal antibodies of canine coronavirus, wherein the hybridoma cell strains are hybridoma cell strains 1B2 and 3C2, 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.
The invention provides a pair of monoclonal antibodies of canine coronavirus, wherein the pair of monoclonal antibodies are antibodies 1B2 and 3C2, and the antibody 1B2 is an antibody generated by a hybridoma cell strain 1B 2; the antibody 3C2 is an antibody produced by hybridoma cell line 3C 2.
Further defined, the monoclonal antibody 1B2 is a labeled antibody and the monoclonal antibody 3C2 is a detection antibody.
The invention provides application of the hybridoma cell strain, the monoclonal antibody, the pair of hybridoma strains or the pair of monoclonal antibodies in preparation of a kit for detecting canine coronavirus.
The invention provides a canine coronavirus colloidal gold detection test strip, 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 is coated with monoclonal antibody 3C2, and the quality control line is coated with goat anti-mouse IgG; the colloidal gold pad is coated with a monoclonal antibody 1B2 marked by colloidal gold.
Further defined, the preparation method of the colloidal gold-labeled monoclonal antibody 1B2 comprises:
step 1: stirring the colloidal gold solution, regulating the pH value to 8.5, slowly adding the canine coronavirus monoclonal antibody 1B2 dropwise to ensure that the final concentration of the canine coronavirus monoclonal antibody 1B2 is 20 mug/ml, and standing for 120 minutes at room temperature;
step 2: adding 10% BSA solution into the monoclonal antibody-added colloidal gold solution to make the final concentration be 1%, uniformly mixing, sealing for 30 minutes or overnight, centrifuging for 20 minutes at 7500r/min, discarding the supernatant, and re-suspending the centrifuged precipitate by using a gold-labeled buffer solution to make the volume be 1/10 of the original volume to obtain the gold-labeled conjugate.
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; adjusting the pH value to 8.5 by using 0.2mol/L potassium carbonate solution; the gold-labeled buffer solution in the step 2 is composed of the following components: pH8.5 Tris-HCl, 1% BSA, 1% sucrose and 0.5% trehalose.
Further limiting, adding 300 μl of gold-labeled buffer solution into each 1ml of gold-labeled conjugate obtained by centrifuging colloidal gold, mixing, and spraying onto glass fiber membrane with a spraying amount of 25 μl/cm 2 。
After diluting the canine coronavirus monoclonal antibody 3C2 with a membrane coating liquid, spraying the diluted canine coronavirus monoclonal antibody on a nitrocellulose membrane according to the concentration of 1.0 mug/cm to serve as a detection line; sheep anti-mouse IgG was sprayed onto nitrocellulose membrane at 1.0. Mu.g/cm as a quality control line.
The beneficial effects are that: the hybridoma cell lines 1B2 and 3C2 for the monoclonal antibody are prepared for the CCV virus by a monoclonal antibody technology, so that the monoclonal antibody has higher specificity for a single gram of the monoclonal antibody for different CCV virus strains, and the prepared colloidal gold detection test strip has high sensitivity and good specificity, so that the canine coronavirus can be detected more accurately and rapidly.
The collection number of the canine coronavirus hybridoma cell strain 1B2 is CGMCC No.45200, the collection address is China general microbiological culture Collection center (China Committee for culture Collection), and the collection address is: the preservation time of the national institute of microbiology of the national academy of sciences of China is 2022, 7 months and 1 day; the collection number of the canine coronavirus hybridoma cell strain 3C2 is CGMCC No.45199, the collection address is the China general microbiological culture Collection center, and the collection address is: the preservation time of the national institute of microbiology of the national academy of sciences of China is 2022, 7 months and 1 day.
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 is a block diagram of a test strip.
Detailed Description
Example 1 preparation of 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, tail blood is cut and taken simultaneously after three immunization for 7 days to measure the antibody titer of serum in the mouse body, the mouse myeloma cell SP2/0 is fused with spleen lymphocytes, positive fusion holes are screened, and individual treatment is carried out to obtain hybridoma cell strains.
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 3000r/min for 20 minutes to remove larger host cell debris and other larger impurities; the virus supernatant was centrifuged at 35000r/min for 2 hours by sucrose density gradient centrifugation, and the precipitated band was dissolved in PBS at pH 7.2 to obtain purified immunogen. 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, the protein content 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 an antigen intraperitoneal injection of 50 μg protein of non-emulsified antigen per mouse 3 days prior to fusion.
3. Mouse serum titer 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 titer 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 transfer on SP2/0 mouse myeloma cells HGPRT without generating immunoglobulin and through mycoplasma detection, using an IMDM complete culture medium (containing 20% serum) to obtain blood from the eyeballs of the mice with highest canine coronavirus titer, killing the eyeballs, putting the mice into 75% alcohol for soaking for 5 minutes, pouring a small amount of serum-free IMDM into a plate, putting a cell sieve and an inner core of a syringe into the plate, taking down the spleen of the mice with scissors and forceps, putting 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 mice with scissors and forceps, grinding the ground thymus cells into a centrifuge tube of 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 centrifugal tube, sucking the prepared splenic lymphocyte cells into a centrifugal 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 stock medium was replaced with IMDM medium containing HAT,20% fbs, and after 16 days of fusion, 100 μl of cell supernatant was aspirated into ELISA plates previously coated with purified canine coronavirus CCV2015 strain antigen, while 100 μl 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 dilution corresponding to minimum OD reading greater than maximum OD/2.
The canine coronavirus CCV2015 strain is coated, fusion cell holes are detected by ELISA, 9 positive hybridoma cell holes are screened together, namely 1B2,1E8, 1F3, 2B5,2C10,2D3,3C1,3C2 and 3H11 are respectively selected, 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 screening of canine coronavirus antibody positive cells first plate (OD 450nm Value of
Table 3 canine coronavirus antibody positive cell screening second plate (OD 450nm Value of
TABLE 4 screening of canine coronavirus antibody positive cells third plate (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 monoclonal antibody was obtained
1. Expanding and freezing hybridoma cell lines 3C2 and 1B2, namely after CCV hybridoma cell lines 3C2 and 1B2 grow to form a monolayer, discarding the culture medium, blowing the cells with 10ml IMDM complete culture medium (containing 20% serum) to separate from the bottom of a cell bottle, and obtaining a ratio of 1:3In the example, 1 cell flask of hybridoma cells was equally distributed to 3 cell flasks, and 30 to 40ml of IMDM complete medium (containing 20% serum) was injected into each cell flask, and the culture was continuously observed and the growth of cells was recorded daily. Cells were passaged every 4 days, serially passed for 3 passages, and the results were recorded. The 1 st generation CCV hybridoma cells 3C2 and 1B2 are continuously transferred for 5 generations under the condition of containing 5% 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, -frozen at 70℃for 4 hours, and stored in liquid nitrogen. According to the method, the 3 rd to 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
Hybridoma cell line secretion antibody assay results: after the hybridoma cell line 1B2,3C2 had grown on the bottom of the flask, the supernatant was collected and assayed for secreted antibodies, 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 carried out, supernatant fluid is collected for measuring secreted antibodies, the properties of the secreted antibodies are stable, and the results are shown in Table 12.
TABLE 12 determination of hybridoma cell passage stability
Cell cryopreservation assay results:
(1) Cell viability assay results: 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.
(2) Growth characterization of resuscitated cells: resuscitates the P5 and P10 generation cells, and then performs growth characteristic measurement, cultures the cells for 5 days, and counts the cells to obtain P5 generation cells with a number of 5.5X10 5 6.1X10 th generation cell per ml, 10 th generation cell 5 And each ml.
The detection result of the exogenous factors is that the hybridoma cell strains 1B2 and 3C2 are negative in bacterial and mould detection, and the detection result is negative in mycoplasma detection 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.
Results: the CCV hybridoma cells 1B2 and 3C2 are tumor cells, are stable in continuous passage, can be frozen for resuscitation, and have good proliferation performance, and are derived from mouse myeloma cells and mouse lymphocytes.
The identification of the CCV hybridoma cells 1B2 and 3C2 can generate specific immune binding reaction with the CCV strain, does not react with canine parvovirus, canine distemper virus, canine adenovirus, canine parainfluenza virus and F81 cells, has good specificity and high sensitivity, and is suitable for being used as a CCV diagnosis raw material.
CCV hybridoma cells 1B2 and 3C2 were free of bacterial, mold, mycoplasma and exogenous viral contamination. The hybridoma cell strain can continuously transmit 15 generations, and the antibody secretion performance is stable.
Identification of monoclonal antibodies:
hybridoma cell and ascites titer test: preparing mouse ascites by screening monoclonal hybridoma cells, respectively taking cell supernatant and the mouse ascites as primary antibodies, taking purified CCV2015 strain virus as an antigen coating ELISA plate, and performing titer test by adopting an indirect ELISA method, 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, adding TMB substrate in the well (100 μl/well), developing color at 37deg.C for 5-10 min under dark condition, adding 2: 2M H deg.C for 15 min 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 neutralization 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 at 37 deg.C in 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 control.
The neutralizing titer of antibodies in mAb-1B2 culture supernatant was determined to be 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 450nm 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 . The respective 50% OD of the 4 curves was found out in the 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 And, when belonging to 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 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, respectively, at a dilution ratio, are added to the susceptible 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 subjected to 2-fold specific dilution, 50-fold, 100-fold, 200-fold, 400-fold. Cndot. 819200-fold, 15 dilution gradients in total, were sequentially added to 96-well plates, 50. Mu.l/well, and incubated for 1 hour at 37 ℃. PBST wash plate, 100. Mu.l/wellThe plate was washed 3 times for 3 minutes each, and then the liquid in the wells was scraped. 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 result of indirect immunofluorescence by fold dilution showed that the fluorescent 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: the ascites of each monoclonal antibody strain was diluted to a saturation working concentration (OD 450nm The value does not increase with increasing antibody concentration), one monoclonal antibody was added, incubated for 1.5 hours at 37 ℃, then another monoclonal antibody was added after 4 times of washing, incubated for 1.5 hours at 37 ℃, then enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG) was added after 4 times of washing, and the test was performed 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
7. Pairing test of coating and detection antibody: the 4 purified mAbs are respectively marked with colloidal gold at the same concentration (10 mug/mu l), NC films are coated at the same concentration, after drying 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.
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
8. Preparation of hybridoma cell ascites: taking hybridoma cell strains 3C2 and 1B2 for production in frozen storage, re-tempering in a water bath kettle at 37 ℃ for 2-5 minutes, and adding the 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 reconstituted solution by using a maximum range 1ml syringeHybridoma cell suspension, abdominal cavity 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. 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.
9. 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, elution buffer (0.1M sodium citrate, pH 4.0) was added to elute the binding protein, and finally, pH adjustment was performed with neutralization buffer (1M Tris-HCl, pH 9.0).
10. Purity test (SDS-PAGE method) 5 volumes of purified antibody and 1 volume of Buffer protein were mixed and boiled for 5 minutes, and the supernatant was loaded after centrifugation: 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.
11. Titer test 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 was added per well, and incubated 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
And (3) screening 9 anti-canine coronavirus monoclonal hybridoma cell strains with higher affinity, namely 1B2,1E8, 1F10,2B5,2C10,2D3,3C1,3C2 and 3H11 respectively, and performing an indirect ELISA test to prove that 4 monoclonal antibodies are directed against different epitopes of the antigen, and performing a pairing test to find that the mark 1B2 and the coating 3C2 are the optimal combination, wherein the detection line signals are stronger than other combinations, and are respectively used as the mark monoclonal antibodies and the detection monoclonal antibodies and used for colloidal gold marking and the detection line on the coating nitrocellulose membrane.
Example 3 preparation of colloidal gold test strip for detecting Canine coronavirus
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. Preparation of gold-labeled conjugate: the colloidal gold solution is placed on a magnetic stirrer, stirred at the rotating speed of 300g/min, the pH value is regulated to 8.5 by using 0.2mol/L potassium carbonate solution, the canine coronavirus monoclonal antibody 1B2 is slowly added dropwise, the final concentration is 20 mug/ml, and the mixture is kept stand for 120 minutes at room temperature.
Adding 10% BSA solution into the monoclonal antibody-added colloidal gold solution to make the final concentration be 1%, uniformly mixing, sealing for 30 min or overnight, centrifuging for 20 min at 7500r/min, discarding supernatant, and using gold-labeled buffer solution (pH8.5 Tris-HCl, 1% BSA, 1% sucrose and 0.5% trehalose) to resuspend the centrifuged precipitate to make its volume be 1/10 of original volume, namely the gold-labeled conjugate, and cold-storing for later use.
3. Preparation of gold-labeled binding pad: adding 300 μl of gold-labeled antigen heavy suspension (0.02 moL/L Tris-HCl, 1% BSA,5% sucrose, 5% trehalose, pH 8.5) into 1ml colloidal gold centrifugation gold-labeled conjugate, mixing, and spraying onto glass fiber membrane (spraying amount of gold-labeled conjugate is 25 μl/cm) 2 ) Placed in a forced air drying oven at 37℃for 5 hours. After drying, the gold mark bonding pad is cut into strips with the length of 4mm multiplied by 300mm, and the strips are sealed and stored.
4. Coating a nitrocellulose membrane: the canine coronavirus monoclonal antibody 3C2 was diluted with a membrane-coating solution (0.02 mol/L PBS,1.5% sucrose) and sprayed onto nitrocellulose membrane at 1.0. Mu.g/cm as a detection line. Sheep anti-mouse IgG is diluted by a film coating liquid (0.02 mol/L PBS,1.5% sucrose) and sprayed on a nitrocellulose film at a position 5mm below a detection line according to 1.0 mug/cm to serve as a quality control line. And (3) drying the coated nitrocellulose membrane at 37 ℃ for 3 hours, and then sealing and preserving.
5. Preparation of large plates: as shown in fig. 5, each component (sample pad, colloidal gold pad, 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 mixture was stored in a sealed state.
6. 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.
And (3) specificity test: taking 5 specific reference substances for detection, wherein 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 detected by the test strip, the F81 cell culture liquid is directly detected by 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 negative.
The test strip has high sensitivity and good purposefulness.
Claims (10)
1. A hybridoma cell strain for producing a monoclonal antibody of canine coronavirus, which is characterized in that one hybridoma cell strain is a hybridoma cell strain 1B2, the preservation number of the hybridoma cell strain 1B2 is CGMCC No.45200, the preservation address is the China general microbiological culture collection center, the preservation time is 2022, 7 months and 1 day, or one hybridoma cell strain is a hybridoma cell strain 3C2, the preservation number of the hybridoma cell strain 3C2 is CGMCC No.45199, the preservation address is 2022, 7 months and 1 day, the preservation number is the China general microbiological culture collection center.
2. A monoclonal antibody of canine coronavirus, wherein the monoclonal antibody is antibody 1B2, wherein the antibody 1B2 is the antibody produced by the hybridoma cell line 1B2 of claim 1 or the monoclonal antibody is antibody 3C2, and wherein the antibody 3C2 is the antibody produced by the hybridoma cell line 3C2 of claim 1.
3. A pair of hybridoma cell lines for generating monoclonal antibodies of canine coronavirus, which is characterized in that the hybridoma cell lines are hybridoma cell lines 1B2 and 3C2, the preservation number of the hybridoma cell line 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.
4. A pair of monoclonal antibodies against canine coronavirus, wherein the pair of monoclonal antibodies are antibodies 1B2 and 3C2, and wherein the antibody 1B2 is an antibody produced by the hybridoma cell line 1B2 of claim 1; the antibody 3C2 is an antibody produced by the hybridoma cell line 3C2 of claim 1.
5. The antibody pair of claim 4, wherein the monoclonal antibody 1B2 is a labeled antibody and the monoclonal antibody 3C2 is a detection antibody.
6. Use of the hybridoma cell line of claim 1, the monoclonal antibody of claim 2, the pair of hybridoma strains of claim 3, or the pair of monoclonal antibodies of claim 4 or 5 in the preparation of a kit for detecting canine coronavirus.
7. The canine coronavirus colloidal gold detection test strip 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, the monoclonal antibody 3C2 of claim 2 is coated on the detection line, and goat anti-mouse IgG is coated on the quality control line; the monoclonal antibody 1B2 of claim 2 marked by colloidal gold is coated on the colloidal gold pad.
8. The canine coronavirus colloidal gold detection test strip of claim 7, wherein the preparation method of the colloidal gold labeled monoclonal antibody 1B2 comprises the steps of:
step 1: stirring the colloidal gold solution, regulating the pH value to 8.5, slowly adding the canine coronavirus monoclonal antibody 1B2 dropwise to ensure that the final concentration of the canine coronavirus monoclonal antibody 1B2 is 20 mug/ml, and standing for 120 minutes at room temperature;
step 2: adding 10% BSA solution into the monoclonal antibody-added colloidal gold solution to make the final concentration be 1%, uniformly mixing, sealing for 30 minutes or overnight, centrifuging for 20 minutes at 7500r/min, discarding the supernatant, and re-suspending the centrifuged precipitate by using a gold-labeled buffer solution to make the volume be 1/10 of the original volume to obtain the gold-labeled conjugate.
9. The canine coronavirus colloidal gold test strip of claim 8, wherein the method for preparing the colloidal gold solution in step 1 comprises: 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, stirred at the rotating speed of 300g/min, and the pH value is regulated to 8.5 by using 0.2mol/L potassium carbonate solution; the gold-labeled buffer solution in the step 2 is composed of the following components: pH8.5 Tris-HCl, 1% BSA, 1% sucrose and 0.5% trehalose.
10. The canine coronavirus colloidal gold detection test strip according to claim 8, wherein 300 μl of gold-labeled buffer is added to each 1ml of gold-labeled conjugate obtained by centrifugation of colloidal gold, and after mixingSpraying on the glass fiber film with the spraying amount of 25 mu l/cm 2 ；
After diluting a canine coronavirus monoclonal antibody 3C2 with a film coating liquid, spraying the diluted canine coronavirus monoclonal antibody on a nitrocellulose film according to the film coating liquid of 1.0 mu g/cm to serve as a detection line; sheep anti-mouse IgG is sprayed on a nitrocellulose membrane according to the volume of 1.0 mug/cm and is used as a quality control line.
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