CN116819075A - Test strip for detecting canine parvovirus, hybridoma cells, monoclonal antibody and application - Google Patents
Test strip for detecting canine parvovirus, hybridoma cells, monoclonal antibody and application Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a fluorescent test strip for detecting canine parvovirus, hybridoma cells, monoclonal antibodies and application. The test strip of the invention is coated with fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent marked rabbit IgG in the binding pad; a detection line and a quality control line are arranged on the surface of the nitrocellulose membrane, a canine parvovirus monoclonal antibody is coated in the detection line, and rabbit anti-IgG is coated in the quality control line; wherein, the canine parvovirus monoclonal antibody is secreted by hybridoma cell with accession number of C202355. The test strip can be used for simultaneously detecting the genotypes of the feline parvovirus, the canine parvovirus and the canine parvovirus, has higher sensitivity and specificity, and has the technical advantages of accuracy, rapidness and simplicity.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a canine parvovirus test strip, hybridoma cells, monoclonal antibodies and application.
Background
Canine parvovirus viral disease Canine Parvovirus (CPV) is an acute, highly infectious viral disease caused by infection of puppies with canine parvovirus. CPV is a small, non-enveloped, single-stranded negative-strand DNA virus with an icosahedral symmetrical structure and a genome length of about 5kb, encoding mainly two nonstructural proteins (NS 1 and NS 2) and two structural proteins (VP 1, VP2 and VP 3). VP2 is the main capsid protein, accounting for 90% of the total nucleocapsid, and is also the main structural component and immunogenic protein, and can promote the body to produce neutralizing antibodies. The disease appears in 1970 at the earliest, and is reported for the first time in 1982 in China, and then gradually spread to the world. The incubation period of the disease is about 3-7 days, and clinically, the disease is manifested by severe gastroenteritis, vomiting, diarrhea, fever and other symptoms, puppies and pure dogs are most susceptible, and the death rate is relatively high, so that the disease has serious influence on the canine industry and the pet industry in China and even worldwide. Early prevention and detection is therefore of great practical importance for the control of this disease.
Common canine parvovirus disease detection methods include Hemagglutination Assay (HA) and hemagglutination inhibition assay (HI), enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection technique (IFA), polymerase Chain Reaction (PCR), loop-mediated isothermal amplification detection technique (LAMP), immunochromatography detection technique, and the like. The above detection methods have advantages and disadvantages, and compared with immunochromatography detection techniques, the other methods are relatively sensitive in response, but are complex in overall operation, long in detection time, and unsuitable for on-site rapid diagnosis because of the need of specific instruments and specialized technicians for operation.
The immunochromatography technology is a novel diagnosis detection technology based on antigen-antibody specific reaction which is newly developed in recent years, and the markers mainly comprise colloidal gold, quantum dots, time-resolved fluorescent microspheres and the like. At present, CPV is detected mainly by a colloidal gold immunochromatography detection method, but the method has the defects of poor stability, low sensitivity and the like, and results such as false positive, false negative and the like are easy to appear, so that the early detection and prevention of the CPV are not facilitated.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides a fluorescent test strip for detecting canine parvovirus, a hybridoma cell, a monoclonal antibody and application, and the test strip can detect all genotypes of feline parvovirus, canine parvovirus and canine parvovirus simultaneously.
The invention provides a fluorescent test strip for detecting canine parvovirus, which comprises a PVC bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate; the binding pad is internally coated with fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent marked rabbit IgG; the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, a canine parvovirus monoclonal antibody is coated in the detection line, and rabbit anti-IgG is coated in the quality control line; the canine parvovirus monoclonal antibody is secreted by hybridoma cells with accession number C202355.
Optionally, in the detection line: the coating concentration of the canine parvovirus monoclonal antibody is 0.4-1.0 mg/mL, preferably 0.6mg/mL, and the coating amount is 0.5-2 mu L/cm, preferably 1 mu L/cm; in the quality control line: the coating concentration of the rabbit anti-IgG is 0.4-1.0 mg/mL, preferably 0.5mg/mL, and the coating amount is 0.5-2 mu L/cm, preferably 1 mu L/cm.
Optionally, the coating amount of the fluorescent microsphere marked canine parvovirus monoclonal antibody in the binding pad is 4-10 mug, preferably 6 mug; preferably, the mass ratio of the fluorescent-labeled canine parvovirus monoclonal antibody to the rabbit anti-IgG is 1:1.
optionally, the mass ratio of the fluorescent microsphere to the canine parvovirus monoclonal antibody is 1:20 to 40, preferably 1:30; the mass ratio of the fluorescent microsphere to the rabbit IgG is 1:40 to 80, preferably 1:50.
the invention also provides a preparation method of the fluorescent test strip, which at least comprises the following steps:
s1, respectively preparing a fluorescent microsphere marked canine parvovirus monoclonal antibody and a fluorescent microsphere marked rabbit IgG;
s2, respectively sealing a binding pad and a sample pad, and spraying fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent microsphere marked rabbit IgG on the binding pad;
s3, spraying detection lines and quality control lines on the surface of the nitrocellulose membrane respectively; the detection line is internally coated with canine parvovirus monoclonal antibody; the quality control line is internally coated with rabbit anti-IgG;
and S4, assembling the sample pad, the bonding pad, the nitrocellulose membrane and the water absorption pad to obtain the test strip.
Optionally, S1 includes: after activating fluorescent microspheres, respectively adding canine parvovirus monoclonal antibodies and rabbit IgG for incubation, and adding BSA solution for blocking after incubation; preferably, the activation is performed using EDC and NHS solutions; preferably, the incubation time is 1 to 3 hours and the incubation temperature is 20 to 25 ℃.
Optionally, S2 includes: sealing the sample pad by adopting a sample pad sealing liquid, wherein the sample pad sealing liquid comprises the following components: boric acid solution of 0.75% Tween-20, 1% PEG20000, 3% BSA; the bonding pad is sealed by bonding pad sealing liquid, and the bonding pad sealing liquid comprises the following components: boric acid solution of 3% trehalose, 2% BSA, 0.75% Tween-20, 0.5% Triton X-100; preferably, the closing time is 1 to 3 hours and the temperature is 36 to 38 ℃.
The invention also provides a hybridoma cell strain secreting the virus monoclonal antibody, and the preservation number is CCTCC NO: C202355.
The invention also provides a canine parvovirus monoclonal antibody which is secreted by hybridoma cells with the preservation number of CCTCC NO: C202355.
The invention also provides application of the canine parvovirus monoclonal antibody in preparation of preparations, kits or vaccines for detecting canine parvovirus.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the test strip can be used for simultaneously detecting the feline parvovirus and the canine parvovirus, has higher sensitivity and specificity, and has the technical advantages of accuracy, rapidness and simplicity.
The canine parvovirus monoclonal antibody has the technical advantages of strong specificity and strong binding capacity.
Drawings
FIG. 1 shows the result of SDS-PAGE experiments of VP2 recombinant proteins;
FIG. 2 shows the results of SDS-PAGE performed by collecting the eluate of antibody purification;
FIG. 3 shows the results of Western-blot with CPV using 6A8 mAb as primary antibody;
FIG. 4 is an indirect immunofluorescence assay (IFA) assay result;
FIG. 5 is a graph showing the results of the hemagglutination reaction;
FIG. 6 is a graph showing experimental results of comparison of T-line 6A8 mAb coating amounts;
FIG. 7 is an experimental result of comparison of fluorescent microsphere-labeled 6A8 mAb in a binding pad;
FIG. 8 is a graph showing experimental results of comparison of reaction times;
FIG. 9 shows the experimental results of the detection limit;
FIGS. 10 and 11 show the results of the specificity experiments;
FIGS. 12 and 13 are experimental results of sensitivity;
FIG. 14 shows the results of a specific experiment for each genotype of canine parvovirus;
fig. 15 is an experimental result of stability.
Preservation information
The hybridoma cell strain CPV VP2 6A8 Hybridoma cell line CPV VP2 6A8 is preserved in China center for type culture collection (CCTCC NO: C202355) with the number of 299 in Wuchang district of Wuhan, hubei province on 3-month 8 days.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
In the embodiment of the invention, a segment of VP2 protein gene sequence is obtained from GenBank: MK 332007.1. The recombinant protein is utilized to immunize experimental animals, and a hybridoma cell strain with the best performance is obtained through screening. BALB/c mice were immunized 4 times with VP2 recombinant protein as immunogen. Complete Freund's adjuvant emulsified with 50. Mu.g protein for dose 1. 50 μg of protein emulsified incomplete Freund's adjuvant was inoculated at times 2 and 3. The last boost, 100 μg protein was injected via the peritoneal cavity. Three days after boost, cell fusion was performed. And (3) taking VP2 recombinant protein as an antigen, and screening out cell culture supernatant from the hybridoma cell culture supernatant by using a specific enzyme-linked immunosorbent assay. Subcloning hybridoma clone producing VP2 specific antibody into single cell clone, preparing purified monoclonal antibody from ascites, and screening to obtain hybridoma cell strain with high titer by monoclonal antibody titer. The specificity was determined by indirect ELISA and detected by indirect immunofluorescence assay (IFA) and immunoblotting.
The embodiment of the invention creatively adopts a Time-resolved fluorescence immunochromatography detection Technology (TRFIA), utilizes the long service life and ultrasensitivity of lanthanide chelates, can measure fluorescence by using the Time-resolved technology, can effectively eliminate the interference of nonspecific fluorescence and the like, and takes common europium and other elements as labels. Therefore, compared with a colloidal gold test strip, the sensitivity and the specificity are greatly improved.
Based on the characteristics of TRFIA, the embodiment of the invention combines the obtained recombinant VP2 protein to provide a time-resolved fluorescence immunochromatography test strip capable of rapidly detecting CPV, so that CPV can be rapidly and sensitively detected, the spread and the epidemic of the disease can be controlled, and an accurate, rapid and simple detection method is provided for preventing and controlling canine parvovirus diseases.
The test strip for detecting canine parvovirus in the embodiment of the invention comprises a PVC bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate; the binding pad is internally coated with fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent marked rabbit IgG; the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line (T line) is internally coated with canine parvovirus monoclonal antibody, and the quality control line (C line) is internally coated with rabbit anti-IgG. If the detection line (T line) and the quality control line (C line) are both red, the detection line (T line) and the quality control line (C line) are positive; the detection line does not develop color, and the color development of the quality control line (C line) is negative; the failure of the test strip can be judged by the fact that the quality control line (C line) does not develop. When the sample to be detected reaches the binding pad through capillary action forward chromatography, if CPV antigen is contained in the sample to be detected, the fluorescent marked antibody on the binding pad can react with the corresponding antigen to generate specific antigen-antibody reaction to form an antigen-antibody complex, and then the two complexes continue to be subjected to forward chromatography to reach the detection line T to form a red strip. Meanwhile, the fluorescence labeled rabbit IgG antibody is also combined with the goat anti-rabbit IgG antibody to form a red band by capillary action and forward chromatography to reach the position of the quality control line C.
As an improvement of the embodiment of the invention, the coating amount of the fluorescent marked canine parvovirus monoclonal antibody in the detection line is 0.5-2 mu L/cm, preferably 1 mu L/cm; the coating amount of the rabbit anti-IgG in the quality control line is 0.5-2 mu L/cm, preferably 1 mu L/cm. If the coating amount is too large, the defect of nonspecific reaction exists; if the coating amount is too small, a defect of insufficient color development may occur.
As an improvement of the embodiment of the invention, the coating amount of the fluorescent marked canine parvovirus monoclonal antibody in the binding pad is 0.5-2 mu L/cm, preferably 1 mu L/cm; if the coating amount is too large, the defect of nonspecific reaction exists; if the coating amount is too small, a defect of insufficient color development may occur.
As an improvement of the embodiment of the invention, the mass ratio of the fluorescent-labeled canine parvovirus monoclonal antibody to the rabbit anti-IgG is 1:1.
as an improvement of the embodiment of the invention, fluorescent microsphere markers are Eu-time-resolved fluorescent nanoparticle purchased from Nanjing micro-assay biotechnology Co.
As an improvement of the embodiment of the invention, in the fluorescent-labeled canine parvovirus monoclonal antibody, the mass ratio of the fluorescent microsphere to the canine parvovirus monoclonal antibody is 1:20 to 40, preferably 1:30.
as an improvement of the embodiment of the invention, the mass ratio of the fluorescent microsphere to the rabbit IgG is 1:40 to 80, preferably 1:50. the embodiment of the invention also relates to a preparation method of the test strip, which at least comprises the following steps:
s1, respectively preparing a fluorescent microsphere marked canine parvovirus monoclonal antibody and a fluorescent microsphere marked rabbit IgG;
s2, respectively sealing a binding pad and a sample pad, and spraying fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent microsphere marked rabbit IgG on the binding pad;
s3, spraying detection lines and quality control lines on the surface of the nitrocellulose membrane respectively; the detection line is internally coated with canine parvovirus monoclonal antibody; the quality control line is internally coated with rabbit anti-IgG;
and S4, assembling the sample pad, the bonding pad, the nitrocellulose membrane and the water absorption pad to obtain the test strip.
As an improvement of the embodiment of the present invention, S1 includes: after activating fluorescent microspheres, respectively adding canine parvovirus monoclonal antibodies and rabbit IgG for incubation, and adding BSA solution for blocking after incubation; wherein EDC and NHS solution are adopted for activation, the incubation time is 1-3 hours, and the incubation temperature is 20-25 ℃.
As an improvement of the embodiment of the present invention, S2 includes:
the sample pad is sealed by adopting sample pad sealing liquid, and the sample pad sealing liquid can adopt: boric acid solution of 0.75% Tween-20, 1% PEG20000, 3% BSA; the bonding pad is sealed by adopting bonding pad sealing liquid, and the bonding pad sealing liquid can be adopted: boric acid solution of 3% trehalose, 2% BSA, 0.75% Tween-20, 0.5% Triton X-100; preferably, the closing time is 1 to 3 hours and the temperature is 36 to 38 ℃.
The embodiment of the invention also relates to a hybridoma cell strain secreting the virus monoclonal antibody, and the preservation number is CCTCC NO: C202355.
The embodiment of the invention also relates to a canine parvovirus monoclonal antibody which is secreted by hybridoma cells with the preservation number of CCTCC NO: C202355. The canine parvovirus monoclonal antibody obtained through screening has the technical advantages of high titer and strong antigen binding capacity.
The embodiment of the invention also relates to application of the canine parvovirus monoclonal antibody in preparation of preparations, kits or vaccines for detecting canine parvovirus.
Examples
Materials and methods
1.1 proteins and serum
Feline kidney cell line cells (F81), SP2/0 cells, 60 clinical specimens, canine parvovirus Canine Parvovirus (CPV), canine coronavirus canine coronavirus (CCoV), canine adenovirus Canine Adenovirus (CAV), canine distemper virus Canine Distemper Virus (CDV) were all provided by the national institute of agriculture, national institutes of health. BABLC mice were purchased from animal centers at the animal research institute of Lanzhou; rabbit IgG antibodies and sheep anti-rabbit IgG antibodies were purchased from zemoeid.
1.2 Main reagents, apparatus and kit
Chloroauric acid was purchased from Sigma-Aldrich; trisodium citrate is purchased from the national drug group; sample pad, absorbent pad, conjugate pad, nitrocellulose membrane (NC), polyvinyl chloride (pvc) were purchased from Shanghai Jiegen biotechnology limited; potassium carbonate was purchased from national pharmaceutical chemicals limited; 1% carboxyl modified time-resolved fluorescent microspheres and fluorescent quantitative rapid detectors were purchased from Nanjing microassay.
[1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride ] and bovine serum albumin were purchased from Sigma-Aldrich.
Ultrapure water systems are available from Milli-QILLIPORE, france; biodottad 3200 diagnostic spot system was purchased from Biodot inc; biodotCM4000 flat-pushing automated cutter was purchased from Biodot in the united states; JY200 type electronic scale was purchased from Shanghai precision scientific instruments Inc.
Both the Biodot AD3200 gold standard diagnostic positioning system and the Biodot CM4000 flat-top automatic stripper were purchased from Biodot.
Example 1
This example is used to illustrate the process of obtaining an antigenic protein:
1. identification of VP2 proteins
1.1 obtaining VP2 protein gene sequence:
1.2 the VP2 protein gene sequence was concatenated with the SUMO tag and His tag. The VP2 gene sequences (GenBank: MK332007.1SEQ ID NO: 1) and SUMO were then concatenated with a His tag, sent to the Biotechnology Co., ltd. (Shanghai, china) for synthesis, the synthesized VP2-SUMO-His gene fragment was cloned into E.coli expression vector pET-28a, and the recombinant plasmid was named pET-28a-VP2.
SEQ ID NO:1
atgagtgatggagcagttcaaccagacggtggtcagcctgctgtcagaaatgaaagagctacaggatctgggaacgggtctggaggcgggggtggtggtggttctgggggtgtggggatttctacgggtacttttaataatcagacggaatttaaatttttggaaaacggatgggtggaaatcacagcaaactcaagcagacttgtgcatttaaatatgccagaaagtgaaaattatagaagagtggttgtaaataatttggataaaactgcagttaacggaaacatggctttagatgatactcatgcacaaattgtaacaccttggtcattggttgatgcaaatgcttggggagtttggtttaatccaggagattggcaactaattgttaatactatgagtgagttgcatttagttagttttgaacaagaaatttttaatgttgttttaaagactgtttcagaatctgctactcagccaccaactaaagtttataataatgatttaactgcatcattgatggttgcattagatagtaataatactatgccatttactccagcagctatgagatctgagacattgggtttttatccatggaaaccaaccataccaactccatggagatattattttcaatgggatagaacattaataccatctcatactggaactagtggcacaccaacaaatatataccatggtacagatccagatgatgttcaattttacactattgaaaattctgtgccagtacacttactaagaacaggtgatgaatttgctacaggaacattttattttgattgtaaaccatgtagactaacacatacatggcaaacaaatagagcattgggcttaccaccatttctaaattctttgcctcaagctgaaggaggtactaactttggttatataggagttcaacaagataaaagacgtggtgtaactcaaatgggaaacacaaacattattactgaagctactattatgagaccagctgaggttggttatagtgcaccatattattcttttgaggcgtctacacaagggccatttaaaacacctattgcagcaggacgggggggagcgcaaacagatgaaaatcgagcagcagatggtgatccaagatatgcatttggtagacaacatggtcaaaaaactaccacaacaggagaaacacctgagagatttacatatatagcacatcaagatacaggaagatatccagaaggagattggattcaaaatattaactttaaccttcctgtaacagaagataatgtattgctaccaacagatccaattggaggtaaaacaggaattaactatactaatatatttaatacttatggtcctttaactgcattaaataatgtaccaccagtttatccaaatggtcaaatttgggataaagaatttgatactgacttaaaaccaagacttcatgtaaatgcaccatttgtttgtcaaaataattgtcctggtcaattatttgtaaaagttgcacctaatttaacaaatgaatatgatcctgatgcatctgctaatatgtcaagaattgtaacttactcagatttttggtggaaaggtaaattagtatttaaagctaaactaagagcctctcatacttggaatccaattcaacaaatgagtattaatgtagataaccaatttaactatgtaccaagtaatattggaggtatgaaaattgtatatgaaaaatctcaactagcacctagaaaattatattaa
1.3 transforming the recombinant plasmid into competent cells of Escherichia coli BL21 to obtain recombinant Escherichia coli named E.coli-CPV-VP2.
1.4 expressing target protein with isopropyl beta-d-1-thiopyran galactose glycoside induced recombinant engineering bacteria.
VP2 recombinant protein purification was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and using Western blotting.
The experimental results obtained by SDS-PAGE are shown in FIG. 1. According to SDS-PAGE analysis, VP2-SUMO-His recombinant protein (hereinafter referred to as VP2 recombinant protein) has good reactivity and specificity, and the size is about 76kDa.
Example 2
This example is used to illustrate the preparation and identification process of monoclonal antibodies:
1. production of VP2 monoclonal antibodies
Using the VP2 recombinant protein prepared in example 1 as an immunogen, BABL/c mice were immunized in 4 times. The first immunization is performed after the Freund's complete adjuvant is emulsified with 50 mug of VP2 recombinant protein, and the second and third immunization are performed after the Freund's incomplete adjuvant is emulsified with 50 mug of VP2 recombinant protein. The final boost was performed and BABL/c mice were intraperitoneally injected with 100. Mu.g of VP2 recombinant protein. Three days after boost.
Cell culture supernatants from wells containing hybridoma colonies were screened by ELISA using VP2 recombinant protein prepared in example 1 as antigen. Hybridoma clones producing VP 2-specific antibodies were subcloned into single cell clones (monoclonal) and purified mabs were prepared from ascites fluid.
And preserving the hybridoma cell strain obtained by screening, wherein the preservation number is CCTCC NO: C202355. The monoclonal antibody secreted by this hybridoma cell line was designated 6a8 mAb.
2. Identification of VP2 monoclonal antibodies
The results of SDS-PAGE of the collected eluate of the antibody purification are shown in FIG. 2, and it is clear from FIG. 2 that the relative molecular weights of the heavy chain and the light chain of the purified VP2 antibody are about 55 kDa and about 25kDa, respectively, indicating that the purified antibody has a correct band and high purity.
The result of Western-blot analysis of 6A8 mAb as primary antibody with CPV is shown in FIG. 3, and it is clear from FIG. 3 that 6A8 mAb reacts only with VP2 protein.
3. Indirect immunofluorescence assay (IFA)
The VP2 recombinant protein was analyzed for reactivity with 6A8 mAb, and the VP2-SUMO-His recombinant protein was separated by SDS-PAGE, and then transferred onto NC membrane. 1.5 hours of ambient temperature blocking with 5% (w/v) skim milk in TBST followed by 1 hour of ambient temperature incubation with 6A8 mAb (1:2000 dilution). After 3 washes with TBST (containing 0.1% Tween-20), membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:20000; abcam, cambridge, mass., USA) for 1 hour at room temperature. Protein expression was then detected using an Electrochemiluminescence (ECL) kit (englin biosystems, beijing, china) and exposed to a developer (Azure c300, usa). After 3 washes with PTBST, protein expression was detected using an Electrochemiluminescence (ECL) kit (englin biosystems, beijing, china) and a developer (Bio-Rad, usa). The results of the indirect immunofluorescence assay (IFA) are shown in fig. 4.
As can be seen from FIG. 4, the specific fluorescent staining was seen with 6A8 mAb, whereas no fluorescence was found in the blank cell group.
Example 3
The present example is for illustrating the preparation method of the test strip
1. Preparation of fluorescent microsphere-labeled 6A8 mAb (prepared in example 2) and rabbit IgG
Taking two parts of 800 mu L of 0.5M boric acid buffer solution (pH 8.5) and respectively adding into a 2mL centrifuge tube, adding 200 mu L of 1% fluorescent microsphere, rapidly mixing, adding 20 mu L of 10mg/mL EDC and 50 mu L of 10mg/mL NHS, blowing and mixing by using a gun, activating 15min,14000r 10min at room temperature and centrifuging; adding 1000 mu L of boric acid buffer solution for resuspension, repeating the steps twice, and if obvious precipitation incapable of being uniformly mixed exists, performing ultrasonic treatment for 1min at 100W3s and 3s until the fluorescent microspheres are completely dispersed; 60 mug of 6A8 mAb (prepared in example 2) and 100 mug of rabbit IgG are added into the solution respectively, incubated for 2 hours at normal temperature, 100 mug of 10% BSA solution is added respectively for sealing, incubated for 2 hours at normal temperature, centrifuged 14000r for 10min, the supernatant is discarded, finally 1000 mug of fluorescent microsphere marker protective agent (0.1% BSA 0.05M boric acid buffer solution, pH8.2) is added respectively for resuspension, the steps are repeated twice, if there is obvious sediment which cannot be mixed evenly, 100W, 3s and 3s are processed by ultrasound for 1min, and the solution is preserved at 4 ℃ for standby.
5.2 assembling of fluorescent test strips
The test strip consists of a nitrocellulose membrane (NC membrane), a combination pad, a sample pad, a water absorption pad and PVC. The sample pad was blocked with a sample pad blocking solution (boric acid solution containing 0.75% Tween-20, 1% PEG20000, 3% BSA) at 37℃for 2 hours, taken out, dried in an oven, and sealed in an aluminum foil bag for use. The conjugate pad was blocked with a conjugate pad blocking solution (3% trehalose, 2% BSA, 0.75% Tween-20, 0.5% Triton X-100 in boric acid) at 37℃for 2 hours, taken out, and dried in an oven. Fluorescence labelled 6a8 mAb and rabbit IgG were prepared using a three-dimensional spray film apparatus according to 1:1 are evenly mixed and sprayed on the sealed bonding pad according to the ratio of 1 mu L/cm, wherein the coating amount of the fluorescent-labeled 6A8 mAb on the bonding pad of each test strip is 6 mu g, and the coating amount of the fluorescent-labeled rabbit IgG is 6 mu g. Drying at 37deg.C for 3 hr, and sealing in aluminum foil bag. Rabbit anti-IgG (0.5 mg/mL) was coated on NC membrane at control line C, 6A8 mAb (19. Mu.g/mL) was coated on nitrocellulose membrane at detection line T, the coating amounts were 1. Mu.L/cm, and the membrane was dried at 37℃for 1 hour. And finally, sequentially adhering the NC film, the bonding pad, the sample pad and the water absorption pad on a PVC bottom plate, cutting the PVC bottom plate into test strips with the width of 4mm by using a chopper, and sealing the test strips in an aluminum foil bag for later use.
Example 4
The embodiment is used for explaining the using method of the test strip:
samples to be tested were prepared using PBS solution according to 1:5 dilution, namely, 100 mu L of diluted sample solution is dripped into a sample groove of the test strip prepared in the example 3, and the result is observed after reaction is carried out for 10 minutes at normal temperature.
If the detection line (T) and the quality control line (C line) are both red, the detection line (T) and the quality control line (C line) are positive; the detection line does not develop color, and the color development of the quality control line (C line) is negative; the failure of the test strip can be judged by the fact that the quality control line (C line) does not develop.
Experimental example 1
This experimental example is used to demonstrate the optimal labelling amount of monoclonal antibody 6a8 mAb in T-line when preparing fluorescent test strips:
the example was prepared according to the method of example 3, except that the concentration of 6A8 mAb at the T line was 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, respectively.
Positive samples (t+): tcid50=10 3.9 A sample diluted by 10 times by a sample diluent;
negative sample (T-): diluting the sample with a sample diluent;
respectively dripping a positive sample and a negative sample, detecting by adopting a fluorescence analyzer, and measuring a positive control T+ by using a reader; and healthy dog serum T-value, marking T+/T-as P, and taking the corresponding coating concentration as the optimal coating concentration when the P value is maximum. The experimental results obtained are shown in fig. 6.
As shown in FIG. 6, when the concentration of the monoclonal antibody 6A8 mAb at the T line was 0.6mg/mL, the P value was maximized, and thus 0.6mg/mL was determined as the optimal coating concentration.
Experimental example 2
The experimental example is used for explaining the optimal coating amount of the monoclonal antibody 6A8 mAb marked by the fluorescent microsphere in the binding pad when the fluorescent test strip is prepared;
the examples were prepared according to the method of example 3, except that the amount of fluorescent microsphere-labeled monoclonal antibody 6A8 mAb in the conjugate pad was 2. Mu.g, 4. Mu.g, 6. Mu.g, 8. Mu.g, 10. Mu.g, respectively.
Positive samples (t+): tcid50=10 3.9 A sample diluted by 10 times by a sample diluent;
negative sample (T-): diluting the sample with a sample diluent;
and respectively dripping a positive sample and a negative sample, detecting by adopting a fluorescence analyzer, marking T+/T-as N, and setting the corresponding marking quantity as the optimal marking quantity when the N value is maximum. The experimental results obtained are shown in fig. 7.
As can be seen from FIG. 7, the N value reached the highest value when the fluorescent microsphere-labeled 6A8 mAb was 6. Mu.g.
Experimental example 3
This experimental example is used to illustrate the determination of reaction time
The test was performed as in example 4, with the difference that:
positive samples (t+): tcid50=10 3.9 A sample diluted by 10 times by a sample diluent;
negative sample (T-): diluting the sample with a sample diluent;
and respectively dripping a positive sample and a negative sample, detecting at intervals of 2, 4, 6, 8, 10, 12, 14 and 16 minutes in sequence by adopting a fluorescence analyzer, recording fluorescence values in time, measuring T values, and simultaneously drawing a trend chart of the T values along with the change of time. The experimental results obtained are shown in fig. 8.
As can be seen from fig. 8, the fluorescence value at T line increases with time. When the time exceeds 10min, the fluorescence value becomes gentle after a short decrease. Thus, 10min was chosen as the optimal reaction time.
Experimental example 4
The specificity, sensitivity and stability of the test strip prepared in example 3 were respectively verified.
1. Limit of detection:
50 CPV negative samples were tested under the conditions of example 4, T-line fluorescence was read, and the results were statistically analyzed to calculate an average1195, standard Deviation (SD) 566, < ->Thus, the fluorescence value of the sample to be detected is positive when the fluorescence value is not lower than 2893, and is negative when the fluorescence value is lower than 2893, and the result is shown in FIG. 9.
4.3 specificity
The test strip of the embodiment of the invention is adopted to detect CPV, CCoV, CDV, CAV positive samples, the detection result shows that the test strip is positive with CPV positive samples only, and has no non-specific cross reaction with other samples, and the result is shown in fig. 10 and 11.
4.4 sensitivity
CPV positivity (TCID) 50 ＝10 3.9 0.1 mL) of the sample was diluted to 3200 times, and the sample was tested using the test strip according to the example of the present invention, and the test results are shown in FIG. 12 and FIG. 13.
As can be seen from fig. 12 and 13, the test result of the fluorescent test strip is still positive, so the sensitivity of the fluorescent test strip is 1:3200.
4.5 specific experiments for each genotype of canine parvovirus:
the test strip provided by the embodiment of the invention is used for detecting FPV, MEV, CPV-2c, CPV-2 and CPV-2a positive samples, and the detection result shows that the test strip can be positive to each genotype of a plurality of canine parvoviruses, and the result is shown in figure 14.
4.6 stability
The test strips were stored at room temperature (18-25 ℃) and 4℃for 7 and 5 months, respectively. The experimental results are shown in FIG. 15.
As can be seen from FIG. 15, the test strip of the embodiment of the present invention can be stably stored at room temperature (18 to 25 ℃) and 4℃for 7 and 5 months.
4.6 compliance test
The fluorescent test strip provided by the embodiment of the invention is used for respectively detecting 60 clinical samples simultaneously with qPCR, and the detection result shows that the qPCR detects positive 38 parts of positive and 22 parts of negative; the fluorescent test strip detected 37 positive and 23 negative. The experimental results obtained are shown in Table 1. FPV, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, CPV-2b were all detected as positive.
TABLE 1
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The fluorescent test strip for detecting the canine parvovirus is characterized by comprising a PVC bottom plate, wherein a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate;
the binding pad is internally coated with fluorescent microsphere marked canine parvovirus monoclonal antibody and fluorescent marked rabbit IgG;
the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is internally coated with canine parvovirus monoclonal antibodies, and the quality control line is internally coated with rabbit anti-IgG;
the canine parvovirus monoclonal antibody is secreted by hybridoma cells with the preservation number of CCTCC NO: C202355.
2. The fluorescent test strip of claim 1, wherein the fluorescent test strip comprises a fluorescent material,
in the detection line, the coating concentration of the canine parvovirus monoclonal antibody is 0.4-1.0 mg/mL, preferably 0.6mg/mL, and the coating amount is 0.5-2 mu L/cm, preferably 1 mu L/cm;
in the quality control line, the coating concentration of the rabbit anti-IgG is 0.4-1.0 mg/mL, preferably 0.5mg/mL, and the coating amount is 0.5-2 mu L/cm, preferably 1 mu L/cm.
3. The fluorescent test strip according to claim 1, wherein the fluorescent microsphere-labeled canine parvovirus monoclonal antibody in the binding pad has a coating amount of 4-10 μg, preferably 6 μg;
preferably, the mass ratio of the fluorescent-labeled canine parvovirus monoclonal antibody to the rabbit anti-IgG is 1:1.
4. the fluorescent test strip according to any one of claims 1-3, wherein,
the mass ratio of the fluorescent microsphere to the canine parvovirus monoclonal antibody is 1:20 to 40, preferably 1:30;
the mass ratio of the fluorescent microsphere to the rabbit IgG is 1:40 to 80, preferably 1:50.
5. the method for preparing a fluorescent test strip according to any one of claims 1 to 4, comprising at least the steps of:
s1, respectively preparing a fluorescent microsphere marked canine parvovirus monoclonal antibody and a fluorescent microsphere marked rabbit IgG;
s2, respectively sealing a binding pad and a sample pad, wherein the binding pad is sprayed with the fluorescent microsphere marked canine parvovirus monoclonal antibody and the fluorescent microsphere marked rabbit IgG;
s3, spraying detection lines and quality control lines on the surface of the nitrocellulose membrane respectively; the canine parvovirus monoclonal antibody is coated in the detection line; the quality control line is internally coated with rabbit anti-IgG;
and S4, assembling the sample pad, the bonding pad, the nitrocellulose membrane and the water absorption pad to obtain the test strip.
6. The method of claim 5, wherein S1 comprises:
after activating fluorescent microspheres, respectively adding the canine parvovirus monoclonal antibody and rabbit IgG for incubation, and adding BSA solution for blocking after incubation;
preferably, the activation is performed with EDC and NHS solution;
preferably, the incubation time is 1 to 3 hours and the incubation temperature is 20 to 25 ℃.
7. The method of claim 5, wherein S2 comprises:
sealing the sample pad by adopting a sample pad sealing liquid, wherein the sample pad sealing liquid comprises the following components: boric acid solution of 0.75% Tween-20, 1% PEG20000, 3% BSA;
the bonding pad is sealed by bonding pad sealing liquid, and the bonding pad sealing liquid comprises the following components: boric acid solution of 3% trehalose, 2% BSA, 0.75% Tween-20, 0.5% Triton X-100; preferably, the closing time is 1-3 hours, and the temperature is 36-38 ℃.
8. Hybridoma cell strain secreting virus monoclonal antibody, and the preservation number is CCTCC NO: C202355.
9. A monoclonal antibody of canine parvovirus is secreted by hybridoma with the preservation number of CCTCC NO: C202355.
10. Use of a canine parvovirus monoclonal antibody according to claim 9 in the preparation of a formulation, kit or vaccine for the detection of canine parvovirus.
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