CN109627293B - Seikaga valley virus structural protein epitope polypeptide and application thereof - Google Patents
Seikaga valley virus structural protein epitope polypeptide and application thereof Download PDFInfo
- Publication number
- CN109627293B CN109627293B CN201910028355.4A CN201910028355A CN109627293B CN 109627293 B CN109627293 B CN 109627293B CN 201910028355 A CN201910028355 A CN 201910028355A CN 109627293 B CN109627293 B CN 109627293B
- Authority
- CN
- China
- Prior art keywords
- enzyme
- sequence
- solution
- structural protein
- valley virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a seneca valley virus structural protein epitope polypeptide and application thereof. The kit comprises an ELISA plate, positive control serum, negative control serum, an ELISA secondary antibody, a sample diluent, a 20-time concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, wherein the ELISA plate is coated with the Seneca valley virus structural protein epitope polypeptide composition. The antigen epitope polypeptide composition is one or any combination of more than two of a polypeptide shown as a sequence 1 in a sequence table, a polypeptide shown as a sequence 2 in the sequence table, a polypeptide shown as a sequence 3 in the sequence table or a polypeptide shown as a sequence 4 in the sequence table. The kit uses chemically synthesized antigen peptide to coat the ELISA plate, has less antigen dosage and high sensitivity and specificity, and can efficiently detect whether the structural protein antibody infected by the Seneca valley virus exists. The kit has the advantages of high sensitivity, good specificity, convenient operation and good market prospect.
Description
The application is a divisional application of a patent application with the application number of ' 201710688944.6 ' and the invention name of ' a Seneca valley virus structural protein antibody enzyme-linked immunoassay kit
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a seneca valley virus structural protein antibody enzyme-linked immunoassay kit.
Background
Seneka Valley Virus (SVV), also known as SenecavirusA (SVA), is the major etiological agent of swine essential herpes disease (SIVD). After SVV infects a swinery, although great political and economic losses are not caused, vesicular lesions caused by SVV are similar to lesions caused by foot-and-mouth disease virus, swine vesicular disease, vesicular stomatitis, swine water herpes and the like, and cause certain difficulties for clinical differential diagnosis. Currently, SIVD has been reported in swine herds in north america, south america, and australia. SVV infects swinery and has serious clinical and death symptoms for several times in 2015, and Brazil, which causes serious economic loss, and the SVV is first separated in 2016 in China.
SVV is the only member of the genus Senecavirus (Senecavirus) of the family Picornaviridae (Picornaviridae), and SVV is a single-stranded, plus-stranded, non-segmented RNA virus. The genetic relationship between the genus Seikavirus and the genus myocarditis was found to be closest by whole genome sequence alignment of 12 representative viruses of the picornaviridae family. SVV is an icosahedral, membrane-free, single-stranded, positive-stranded, non-segmented RNA virus of about 27nm diameter. The viral genome contains 7280 nucleotides, an open reading frame contains 6543 nucleotides, and encodes a 2181-amino acid polyprotein which can be divided into 12 polypeptides, constituting the standard L-4-3-4 pattern of picornaviridae.
Like all members of the picornaviridae family, the P1 polypeptide is cleaved by the 3C protease into VP0, VP3 and VP1, constituting the viral nucleocapsid. Cleavage of mature VP0 yields VP2 and VP 4. The structures of the viral VP 1-VP 4 subunit proteins are conserved, and may be related to the cell tropism of the virus. Certain regions of VP2 and VP4 interact to participate in nucleic acid packaging.
As the SVV is firstly separated in 2016 in China, the SVV antibody detection method is still in a research stage at present, and the research related to the SVV antibody detection method mainly comprises indirect ELISA and competitive ELISA.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting a Seneka valley virus structural protein antibody, which utilizes Seneka valley virus structural protein VP1, VP2 and VP3 epitope polypeptides as coating antigens to establish an indirect ELISA method with good specificity, sensitivity and repeatability and is used for detecting whether a porcine serum contains the Seneka valley virus structural protein antibody.
In order to achieve the purpose, the invention firstly screens and obtains the Seineka valley virus epitope polypeptide composition with excellent performance. The seneca valley virus structural protein antigen epitope polypeptide composition provided by the invention is one or any combination of more than two of a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table, a polypeptide shown in a sequence 3 in the sequence table or a polypeptide shown in a sequence 4 in the sequence table. When the polypeptide composition is two of the polypeptide shown in a sequence 1, the polypeptide shown in a sequence 2, the polypeptide shown in a sequence 3 and the polypeptide shown in a sequence 4, the mass ratio of the two polypeptides is (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1; when the polypeptide composition is three of a polypeptide shown in a sequence 1, a polypeptide shown in a sequence 2, a polypeptide shown in a sequence 3 and a polypeptide shown in a sequence 4, the mass ratio of any three polypeptides is (0.5-1.5): (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1: 1;
when the polypeptide composition consists of a polypeptide shown in a sequence 1, a polypeptide shown in a sequence 2, a polypeptide shown in a sequence 3 and a polypeptide shown in a sequence 4, the mass ratio of the polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1: 1: 1.
the invention relates to a seneca valley virus structural protein antibody enzyme-linked immunoassay kit, which comprises an enzyme-linked reaction plate, positive control serum, negative control serum and enzyme-labeled secondary antibody; wherein the enzyme-linked reaction plate is coated with the Sernica valley virus structural protein epitope polypeptide composition.
The enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the polypeptide in the seneca valley virus structural protein epitope polypeptide composition is obtained by chemical artificial synthesis.
The optimal preparation method and conditions of the enzyme-linked reaction plate are that the Sernica valley virus structural protein epitope polypeptide composition is dissolved in a carbonate solution with the pH value of 9.6, then the mixture is added into a 96-hole polystyrene enzyme-linked reaction plate, 200ng of polypeptide is added into each hole, the mixture is placed for 8-12 hours at the temperature of 2-8 ℃, so that the polypeptide antigen is fully combined with the enzyme-linked reaction plate, then PBS buffer solution containing 1% (g/ml) Bovine Serum Albumin (BSA) with the pH value of 7.4 is added into each hole according to 300 mu l, the sealing treatment is carried out for 2-3 hours at the temperature of 37 ℃, after being dried, the enzyme-linked reaction plate is sealed and stored at the temperature of 4 ℃.
The positive control serum is porcine serum collected after the artificial infection of the Seneca valley virus; the negative control serum is swine serum free of Specific Pathogens (SPF).
The enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-pig IgG antibody.
The kit also comprises a substrate solution A, a substrate solution B and a stop solution, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.06% (g/ml) of urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use. The stop solution is a 2mol/L sulfuric acid solution.
The kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent was a 0.01M, pH value 7.4 phosphate buffer containing 0.5% (g/100 ml) casein; the concentrated washing solution is 0.01M phosphate buffer solution with pH value of 7.4 and contains 0.8-1.2% (ml/ml) of Tween-20.
The detection program of the kit of the invention is as follows:
1. balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and (3) using the sample diluent to mix the serum of the sample to be detected, the negative control serum and the positive control serum according to the ratio of 1:20 in proportion.
5. Sample adding: and (4) respectively adding 100 mul diluted samples to be detected into each hole according to the preset value. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding the reaction solution, adding 300 mul of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying.
11. And adding 100 mul of substrate working solution into each well for color development (the substrate working solution is obtained by mixing the substrate solution A and the substrate solution B in equal amount, and is prepared at present), shaking and uniformly mixing, placing in a 37 ℃ incubator, and reacting for 15min in a dark place.
12. Adding 50 mul of chromogenic stop solution into each well, and oscillating, uniformly mixing and stopping the reaction.
13. The OD450nm value was determined for each well (the stopped plates should read the OD450nm value within 15 min).
And (3) judging a detection result:
1. the negative control OD450nm should have an average value of 0.15 or less, otherwise it is ineffective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: cut-off =0.17 × positive control OD450nm value mean.
The serum to be detected is determined to be positive if the OD450nm value is greater than or equal to the critical value; the test serum is judged to be negative if the OD450nm value is less than the critical value.
The kit can be used for detecting the structural protein antibody of the Seneca valley virus so as to judge whether the animal to be detected has the structural protein antibody of the Seneca valley virus generated after infection.
The application of the kit in preparing the kit for detecting whether the virus is infected with the Seneca Valley virus also belongs to the protection scope of the invention.
The invention has the positive effects that: the invention adopts a bioinformatics method to accurately analyze the epitope of the structural protein of the Seneca valley virus, and selects the peptide segment suitable for ELISA detection from the main epitope on the VP1, VP2 and VP3 proteins. The peptide fragment concentrates the epitope and has the advantages of high sensitivity and strong specificity.
Meanwhile, the advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, because the coating antigen used in the kit is chemically synthesized polypeptide, does not contain hybrid protein and has high purity, the efficiency of detecting the structural protein antibody of the Seneca valley virus is further improved, so as to judge whether the detected animal is infected with the Seneca valley virus.
In a word, the kit adopts the antigen peptide of the main antigen sites of the chemically synthesized structural proteins VP1, VP2 and VP3 to coat the enzyme-linked reaction plate, has the advantages of small antigen dosage, high sensitivity and strong specificity, and can effectively detect the structural protein antibody generated after the Seneca valley virus is infected so as to judge whether the detected animal is infected with the Seneca valley virus. The experimental result shows that the kit has good repeatability, strong specificity and high sensitivity. Can meet the requirements of personnel at different levels, and has wide market prospect and good economic and social benefits.
The enzyme-linked immunoassay kit for the structural protein of the seneca valley virus is used for detecting whether animals are infected with the seneca valley virus, and is beneficial to establishing a seneca valley virus prevention and control system in China.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of envelope antigen of Seneca Valley Virus structural protein antibody enzyme-linked immunoassay kit
The test adopts a bioinformatics method to accurately analyze the main epitope of the structural proteins VP1, VP2 and VP3 of the Seineka valley virus, screens out proper peptide fragments, synthesizes four peptides respectively by using a full-automatic polypeptide synthesizer, and the sequences are respectively shown as sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table, so that the updated more complete envelope antigen with the purity of about 80% is prepared, the main neutralizing epitope of the Seineka valley virus can be covered, and the positive detection rate of the antibody is improved. The polypeptide synthesis method can be a conventional method, and the four polypeptides of the invention are synthesized by the following method as a coating antigen of the kit of the invention.
The coated antigen of the present invention can be prepared using an Applied Biosystem full-automatic polypeptide synthesizer (model 433A). The method applies a Merrifield solid phase synthesis method, adopts Fmoc (9-fluoromethylenecarbonyl, 9-fluorenylmethyloxycarbonyl) modified amino acid, and uses Rink Amide MBHA resin as a solid phase carrier. The production process comprises five parts of polypeptide antigen solid phase synthesis, polypeptide cracking and identification, antigen purification, freeze drying and storage. The following are described separately:
first, solid phase synthesis of envelope antigen
1. Preparation of synthetic reagents
The amino acid sequence of the synthetic coating antigen is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
Appropriate Fmoc-modified amino acids (purchased from NOVA) were prepared according to the coating antigen sequence and synthesis scale and added to the corresponding cartidge. Similarly, 5g of the resin was weighed on the desired synthesis scale, placed in the reaction chamber, the upper and lower caps were tightened, labeled, and the name of the synthesized peptide, the batch number, TARE in the reaction chamber and the weight of the resin weighed were recorded. The reaction chamber was loaded into the synthesizer. Appropriate amounts of synthesis reagents were prepared including 100% NMP, 3% AIM (hexanoyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc. and placed in corresponding reagent bottles.
2. Detection of synthesizer state
Checking 433A polypeptide synthesis instrument to determine whether it is normal, starting the instrument, running Run Self Test program, and Self-checking whether each index is normal. Further check if nitrogen is sufficient and system gauge pressure is normal (433A normal gauge pressure 10.2 psi). The flow rate of each synthesis reagent is measured because the performance of the instrument is known prior to synthesis. 433A synthesizer: sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test-finding Module A, Module D, Module I, Module A according to Prer or next-measuring or observing according to Start-measuring according to more, if the Flow is not proper, regulating lower valve pressure until reaching the requirement (the specific detection requirement is shown in Table 1).
TABLE 1 flow rate test standard table for polypeptide synthesizer
3. Initiation of coating antigen Synthesis
The amino acid sequence to be synthesized was sent to the Std Fmoc 1.0 Sol DIC90 on the synthesizer in the 433A synthesizer program. File-New-Sequence-editing the Sequence of the synthetic peptide, and storing. File-New-Run, check whether Chemistry is Std Fmoc 1.0 Sol DIC 90; whether Sequence is the stored name; setting Cycles; and (5) storing. And finally sending the data to a synthesizer.
Main Menu-Cycle Monitor-begin, run.
4. Coating antigen synthesis
The removal of Fmoc group and the electron-withdrawing action of fluorene ring system of Fmoc group make 9-H have acidity and be easily removed by weak base, during the reaction, piperidine (PIP) is used to attack 9-H, beta is eliminated to form dibenzofluorene alkene, and the dibenzofluorene alkene is easily attacked by secondary cyclic amine to form stable addition compound. Removal of the Fmov group exposes the "-NH 2" group for the synthesis reaction. The activated amino acid protected with the next Fmoc group and 1-Hydroxybenzotriazole (HOBT) were then added to the reactor.
The above polypeptide sequence is synthesized by repeating the synthesis steps (the synthesizer is automatically completed according to the program, and the specific cycle steps are as shown in the following table 2) from the C-terminal to the N-terminal in sequence according to a specific sequence. During which the recorded reagent dosage and the running condition were observed.
TABLE 2 coating antigen Synthesis cycle procedure
5. End of coating antigen Synthesis
The synthesizer will automatically stop after the synthesis of the coated antigen is complete and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then taking off the reactor from the polypeptide synthesizer, washing the peptide resin with 100% methanol for 3 times, drying in a fume hood, transferring all the polypeptide resin into a brown polyethylene bottle, placing into a refrigerator at-20 deg.C, and sealing with a sealing film for use.
Secondly, the cleavage and identification of the envelope antigen
1. Cleavage of polypeptide antigens
The polypeptide obtained by the above reaction is chemically bonded to the solid support, and the polypeptide must be separated from the solid support by acid hydrolysis using a specific strong organic acid. The acid hydrolysis also removes the protecting group on each amino acid function. The method comprises the following steps:
the synthesized polypeptide resin (meaning the peptide is also attached to the resin) was removed from the refrigerator, placed in a 2L round bottom flask, 90ml of Trifluoroacetic acid (TFA), 10ml of Tripropylsilane (TIS) and magnetic stir bar were added to the flask in a fume hood, and the flask was then placed on a magnetic stirrer steadily and stirred at room temperature for 1h until the reaction was complete. After the reaction is finished, continuously evaporating for 30-120 min by using a rotary evaporator with a cold trap to remove TFA in the crude product. Then using dimethyl formamide (DMF) to wash the crude product of the polypeptide antigen for multiple times, and finally filtering the mixed resin by using a sand core funnel to obtain the coating antigen.
2. Identification of coating antigens
After the polypeptide antigen is synthesized, qualitative and quantitative analysis is carried out by matrix-assisted laser desorption time of flight mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and the synthesized peptide is identified by common amino acid analysis.
3. Purification of coating antigen
The cyclized polypeptide antigen is ultrafiltered by using a circulating Tangential filtration membrane (a Tangential Flow Device circulating Tangential filtration membrane manufactured by PALL company and a peristaltic pump matched with the circulating Tangential filtration membrane), the polypeptide antigen as a macromolecule can not pass through a filtration membrane with a certain pore diameter, and small molecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filtration membrane. Then sterilizing through a filter with the pore diameter of 0.2 mu m, subpackaging the finally obtained solution into sterile plastic bottles, and labeling. The name, serial number, production lot number, concentration, production date, storage life and storage condition of the polypeptide are marked on the label, and the label is subpackaged and stored at-20 ℃ or-40 ℃ for later use.
4. Freeze drying of coated antigens
For long-term storage and transportation, the coated antigen needs to be freeze-dried to obtain the polypeptide in a solid state. And (3) placing the pre-frozen coated antigen on a freeze dryer of Labconco for drying to obtain the coated antigen in a solid state. And (5) labeling after packaging. The name, number, production lot number, concentration, production date, shelf life and storage conditions of the polypeptide are indicated on the label.
Example 2 preparation of a Seneca Valley Virus structural protein antibody enzyme-linked immunoassay kit
The seneca valley virus structural protein antibody enzyme-linked immunoassay kit comprises:
(1) a 96-well detachable polystyrene enzyme-linked reaction plate coated with Seneca valley virus antigen; 2X 96 wells.
(2) Positive control serum: porcine serum was collected after artificial infection with Seneca valley virus and used as positive control serum (1 tube, 1.5 ml/tube) for the kit.
(3) Negative control serum: is Specific Pathogen Free (SPF) porcine serum as negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: the preparation method is characterized in that horseradish peroxidase-labeled rabbit anti-pig IgG (purchased from sigma company, product number A5670) is used as a stock solution to be diluted by 1:30000 to prepare 2 bottles (12 ml/bottle).
(5) Sample diluent: 1 vial (24 ml/vial) of 0.01M, pH value 7.4 phosphate buffer containing 0.5% (g/100 ml) casein.
(6) Substrate solution A: is citrate phosphate buffer solution (1 bottle, 12 ml/bottle) containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a 0.2mg/ml solution of Tetramethylbenzidine (TMB) (1 vial, 12 ml/vial).
(8) Stopping liquid: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9) 20 times of concentrated washing solution: is 0.01M phosphate buffer solution (50 ml/bottle, 2 bottles) with pH value of 7.4 and containing Tween-20 with concentration of 0.8% -1.2% (ml/ml).
Serum dilution plates (2, 96 wells/block) may also be present in the kit for dilution of serum samples, as desired.
The preparation method of the 96-hole detachable polystyrene enzyme-linked reaction plate coated with the Seneca valley virus antigen comprises the following steps: 1. dissolving the polypeptide antigen prepared in the example 1 in a carbonate solution with pH of 9.6, adding the solution into a 96-hole polystyrene enzyme-linked reaction plate, placing 200ng of polypeptide in each hole (wherein 50ng of the polypeptide shown in a sequence 1 in a sequence table, 50ng of the polypeptide shown in a sequence 2 in the sequence table, 50ng of the polypeptide shown in a sequence 3 in the sequence table, and 50ng of the polypeptide shown in a sequence 4 in the sequence table) at 2-8 ℃ for 8-12 hours to fully combine the polypeptide antigen with the enzyme-linked reaction plate, adding PBS buffer solution containing 1% (g/ml) Bovine Serum Albumin (BSA) with pH of 7.4 into each hole according to 300 mu l, sealing the solution at 37 ℃ for 2-3 hours, drying the solution, and sealing and storing the enzyme-linked reaction plate at 4 ℃.
Example 3 susceptibility testing of the Seneca Valley Virus structural protein antibody enzyme-linked immunoassay kit
Application method of seneca valley virus structural protein antibody enzyme-linked immunoassay kit
1. Balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing liquid: diluting the 20 times of concentrated washing liquid with distilled water or deionized water by 20 times to obtain washing buffer liquid;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and (3) using the sample diluent to mix the serum of the sample to be detected, the negative control serum and the positive control serum according to the ratio of 1:20 in proportion.
5. Sample adding: and (4) respectively adding 100 mul diluted samples to be detected into each hole according to the preset value. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300 mul of washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding reaction liquid, adding 300 mul of washing buffer solution into each hole, soaking for 15s, throwing away the washing buffer solution, continuously washing the plate for 4 times, and then drying.
11. And adding 100 mul of substrate working solution into each well for color development (the substrate working solution is obtained by mixing the substrate solution A and the substrate solution B in equal amount, and is prepared at present), shaking and uniformly mixing, placing in a 37 ℃ incubator, and reacting for 15min in a dark place.
12. Adding 50 mul of chromogenic stop solution into each well, and oscillating, uniformly mixing and stopping the reaction.
13. Determination of OD per well450nmValue (reaction plate with stop solution should read OD within 15min450nmValue).
And (3) judging a detection result:
1. negative control OD450nmThe average value should be less than or equal to 0.15, otherwise it is not effective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: cut-off =0.17 × positive control OD450nmValue average.
Determination of OD in serum to be examined450nmValue greater than or equal to the critical valueJudging the result to be positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
Second, sensitivity test to known positive serum
Using three batches of seneca valley virus structural protein antibody enzyme-linked immunodetection kits (batches ZM2017001, ZM2017002, and ZM 2017003) prepared according to the method of example 2, 35 parts of seneca valley virus infected pig serum (pig serum collected at different times after clinical symptoms appear due to seneca valley virus infection) were subjected to sensitivity tests according to the method of using the seneca valley virus structural protein antibody enzyme-linked immunodetection kits, and the test results are shown in table 3.
TABLE 3 sensitivity test results of the enzyme-linked immunoassay kit for structural protein antibodies of Seneca valley virus
Third, the minimum detection limit test
Selecting 3 parts of swine serum positive to the structural protein antibody of the Seneca valley virus, carrying out dilution by times of 1:20 and 1: 40-1: 320, using three batches of enzyme-linked immunoassay kits (batches ZM2017001, ZM2017002 and ZM 2017003) of the structural protein antibody of the Seneca valley virus prepared in the example 2, and detecting the swine serum by times of dilution by times according to the using method of the enzyme-linked immunoassay kit of the structural protein antibody of the Seneca valley virus, wherein the result shows that the positive serum diluted by times of 1:160 can be detected by the kit, and the table 4 shows the experimental result of one batch. In table 4, positive control: porcine serum was collected after artificial infection with Seneca valley virus and used as positive control serum (1 tube, 1.5 ml/tube) for the kit. Negative control: is Specific Pathogen Free (SPF) pig serum as negative control serum for the kit (1 tube, 1.5 ml/tube). Cut-off value (Cut-off value) =0.17 × positive control OD450nmValue average.
TABLE 4 detection results of the minimum detection limit test of the seneca valley virus structural protein antibody enzyme-linked immunoassay kit
Example 4 specificity test of the Seneca Valley Virus structural protein antibody enzyme-linked immunoassay kit
50 healthy pig sera (provided by Lanzhou biological pharmacy, Zhongmu practical Co., Ltd.), 2 positive sera for swine foot-and-mouth disease virus type-A (FMD-A) (provided by Lanzhou biological pharmacy, Zhongmu practical Co., Ltd.), 2 positive sera for swine vesicular disease virus (SVD) (provided by Zhongmu practical Co., Ltd.), and 2 positive sera for swine Vesicular Stomatitis Virus (VSV) (provided by Zhongmu practical Co., Ltd.) were each assayed using the three kits described in example 2 in accordance with the method of using the Sainika valley virus structural protein antibody enzyme-linked immunodetection kit described in example 3.
The results of the specific detection of the kit are shown in the following table (table 5), and the results of the detection of 50 healthy pig sera show that the specificity of the ZM2017001 kit is 100.0%, the specificity of the ZM2017002 kit is 100.0%, and the specificity of the ZM2017003 kit is 100.0%. The detection results of 2 parts of swine foot and mouth disease virus type O (FMD-O) positive serum, 2 parts of swine foot and mouth disease virus type A (FMD-A) positive serum, 2 parts of Swine Vesicular Disease (SVD) positive serum and 2 parts of swine Vesicular Stomatitis Virus (VSV) positive serum are all shown to be negative, so that the detection specificity of the three kits on the 8 parts of related pathogen positive serum is 100%.
TABLE 5 detection results of specificity of enzyme-linked immunosorbent assay kit for structural protein antibody of Sernica valley virus
Example 5 compliance testing of the Seneca Valley Virus structural protein antibody enzyme-linked immunoassay kit
Method for Virus Neutralization Test (VNT)
The currently accepted detection method for Sernica Valley virus is the Virus Neutralization Test (VNT), so the kit is used for carrying out the coincidence rate test with the virus neutralization test.
1. Material
1.1, 24 parts of infected pig serum (serum number I1-I24) which is obtained after clinical symptoms appear after the infection of the Seneca valley virus, is inactivated at 56 ℃ for 30min and is provided by the middle-herding industry GmbH.
1.2 serum of healthy pig, 24 parts (serum number N1-N24), inactivated at 56 ℃ for 30min, provided by Zhongmu actual industry GmbH.
1.3 the test virus (SVV virus) is PK15 cell virus of Sernica valley virus, and its toxic valence is adjusted to 400 TCID500.1ml, and storing at-20 ℃ for later use.
2. Methods virus neutralization assay methods were as follows: after inactivation of the serum, dilutions (25 μ l/well) were performed in DMEM medium in multiples of 3 replicates per dilution; an equal volume (25. mu.l/well) of 100TCID was added to each well50(ii) SVV virus (excluding control), incubated at 37 ℃ for 1 hour; then 100. mu.l of PK15 cells (2-3X 10) diluted with DMEM medium were added to each well4) At 37 ℃ and 5% CO2Incubators were incubated and cytopathic effect (CPE) was observed after 72 hours. The final dilution at which CPE was inhibited (90-100% inhibition) was considered as the Virus Neutralization Test (VNT) titer, which was > 1:64 and was judged positive.
II, a coincidence rate test result:
24 parts of seneca valley virus porcine serum and 24 parts of healthy serum are respectively detected by using the seneca valley virus structural protein antibody enzyme-linked immunoassay kit prepared in example 2 and a Virus Neutralization Test (VNT).
The detection results of the enzyme-linked immunoassay kit for the structural protein antibody of the Seneca valley virus to 24 infected pig serums are shown in table 6, the sensitivity of the kit is 95.8 percent, the sensitivity of the virus neutralization test is 87.5 percent, and the detection results of the two methods are 22 parts in the 24 infected pig serums. Therefore, the coincidence rate of the kit and the virus neutralization test is 91.7%, and the kit has higher sensitivity. The test of the healthy pig serum has the coincidence rate of 100 percent, and the detection results are negative (see table 6).
The enzyme-linked immunoassay kit for the structural protein antibody of the Seneca valley virus and the virus neutralization test jointly detect 48 pig sera, wherein the detection results of the two methods of the 46 pig sera are consistent, the detection results of the 2 pig sera are different, and the coincidence rate is 95.8%.
TABLE 6 detection results of Saincavirus structure protein antibody ELISA kit and virus neutralization test method for infected pig serum and healthy pig serum
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Zhongmu industries GmbH
<120> Sernica valley virus structural protein antigen epitope polypeptide and application thereof
<130> WHOI190003
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 37
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 1
Ser Asp Leu Glu Val Thr Val Val Ser Leu Glu Pro Asp Leu Glu Phe
1 5 10 15
Ala Val Gly Trp Phe Pro Ser Gly Ser Glu Tyr Gln Ala Ser Ser Phe
20 25 30
Val Tyr Asp Gln Leu
35
<210> 2
<211> 36
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Thr Lys Ser Asp Pro Pro Ser Ser Ser Thr Asp Gln Pro Thr Thr Thr
1 5 10 15
Phe Thr Ala Ile Asp Arg Trp Tyr Thr Gly Arg Leu Asn Ser Trp Thr
20 25 30
Lys Ala Val Lys
35
<210> 3
<211> 38
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Pro Tyr Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser
1 5 10 15
Trp Thr Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly
20 25 30
Ala Thr Thr Asp Pro Glu
35
<210> 4
<211> 34
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 4
Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly
1 5 10 15
Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp
20 25 30
Tyr Arg
Claims (4)
1. An enzyme-linked immunoassay kit for a seneca valley virus structural protein antibody comprises an enzyme-linked reaction plate, positive control serum, negative control serum and an enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with a seneca valley virus structural protein antigen epitope polypeptide composition consisting of a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table, a polypeptide shown in a sequence 3 in the sequence table and a polypeptide shown in a sequence 4 in the sequence table; the enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-pig IgG antibody; the positive control serum is porcine serum collected after the artificial infection of the Seneca valley virus; the negative control serum is swine serum without specific pathogens.
2. The enzyme-linked immunoassay kit of claim 1, characterized in that: the enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; each polypeptide in the seneca valley virus structural protein epitope polypeptide composition is obtained by chemical artificial synthesis.
3. The enzyme-linked immunoassay kit of claim 1, characterized in that: dissolving the seneca valley virus structural protein antigen epitope polypeptide composition in 100 mu l of carbonate solution with pH of 9.6, adding the solution into a 96-hole polystyrene enzyme linked reaction plate, placing 200ng of the seneca valley virus structural protein antigen epitope polypeptide composition in each hole for 8-12 hours at 2-8 ℃ to ensure that the seneca valley virus structural protein antigen epitope polypeptide composition is fully combined with the enzyme linked reaction plate, then adding PBS buffer solution containing 0.01g/ml bovine serum albumin with pH of 7.4 according to 300 mu l/hole, sealing for 2-3 hours at 37 ℃, drying, and sealing and storing at 4 ℃ after the enzyme linked reaction plate is dried.
4. The enzyme-linked immunoassay kit of claim 1, characterized in that: the kit also comprises a substrate solution A, a substrate solution B and a stop solution, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.0006g/ml of urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use; the stop solution is a 2mol/L sulfuric acid solution; the kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent is a phosphate buffer solution with a value of 7.4 and a concentration of 0.01mol/L, pH containing 0.00005g/ml casein; the concentrated washing solution is 0.01mol/L phosphate buffer solution containing 0.8-1.2% of Tween-20 by volume percentage and with the pH value of 7.4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910028355.4A CN109627293B (en) | 2017-08-13 | 2017-08-13 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710688944.6A CN107253978B (en) | 2017-08-13 | 2017-08-13 | Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus |
| CN201910028355.4A CN109627293B (en) | 2017-08-13 | 2017-08-13 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710688944.6A Division CN107253978B (en) | 2017-08-13 | 2017-08-13 | Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109627293A CN109627293A (en) | 2019-04-16 |
| CN109627293B true CN109627293B (en) | 2021-02-09 |
Family
ID=60026733
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910028355.4A Active CN109627293B (en) | 2017-08-13 | 2017-08-13 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
| CN201710688944.6A Active CN107253978B (en) | 2017-08-13 | 2017-08-13 | Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710688944.6A Active CN107253978B (en) | 2017-08-13 | 2017-08-13 | Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109627293B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627293B (en) * | 2017-08-13 | 2021-02-09 | 中牧实业股份有限公司 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
| CN109627292B (en) * | 2018-01-03 | 2021-02-09 | 中牧实业股份有限公司 | Porcine circovirus type 3 epitope polypeptide and application thereof |
| CN108329378B (en) * | 2018-03-12 | 2020-09-25 | 华中农业大学 | Senakava virus VP1 protein, coding gene, hybridoma cell strain and monoclonal antibody and application thereof |
| CN108761074A (en) * | 2018-05-23 | 2018-11-06 | 中国农业科学院兰州兽医研究所 | Senecan virus ELISA antibody assay kit and preparation method, application |
| CN110542755A (en) * | 2018-05-28 | 2019-12-06 | 金宇保灵生物药品有限公司 | kit for detecting Selenecar valley virus by using immune labeling method and detection method thereof |
| CN108872575A (en) * | 2018-07-12 | 2018-11-23 | 中国农业科学院兰州兽医研究所 | A kind of Seneca Valley virus nonstructural protein 3A BC antibody ELISA detection kit |
| CN108872574A (en) * | 2018-07-12 | 2018-11-23 | 中国农业科学院兰州兽医研究所 | A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test |
| CN108872576A (en) * | 2018-07-12 | 2018-11-23 | 中国农业科学院兰州兽医研究所 | A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus nonstructural protein 3A BC antibody test |
| CN110196325B (en) * | 2019-06-24 | 2021-10-15 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | In-plug virus diagnosis kit and test paper |
| CN112225780B (en) * | 2019-06-24 | 2022-02-11 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Polypeptide for detecting Seneca virus antibody and application thereof |
| CN112745387B (en) * | 2019-10-31 | 2022-11-08 | 洛阳普泰生物技术有限公司 | Anti-porcine Seneca valley virus monoclonal antibody and application thereof |
| CN112964871A (en) * | 2021-02-07 | 2021-06-15 | 中国农业科学院北京畜牧兽医研究所 | Pig encephalomyocarditis indirect ELISA diagnostic kit and preparation method thereof |
| CN114675024B (en) * | 2022-03-03 | 2023-08-15 | 中国农业科学院兰州兽医研究所 | Pig type-A sai virus neutralizing antibody, competitive ELISA detection kit and detection method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MXPA06003318A (en) * | 2003-09-26 | 2006-06-08 | Novartis Ag | Seneca valley virus based compositions and methods for treating disease. |
| CN101448526A (en) * | 2005-03-23 | 2009-06-03 | 纽特罗佩克斯公司 | Seneca valley virus based compositions and methods for treating disease |
| WO2008112891A2 (en) * | 2007-03-13 | 2008-09-18 | Neotropix, Inc. | Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof |
| CN106432434B (en) * | 2016-08-29 | 2019-11-12 | 中牧实业股份有限公司 | Foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent |
| CN109627293B (en) * | 2017-08-13 | 2021-02-09 | 中牧实业股份有限公司 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
-
2017
- 2017-08-13 CN CN201910028355.4A patent/CN109627293B/en active Active
- 2017-08-13 CN CN201710688944.6A patent/CN107253978B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109627293A (en) | 2019-04-16 |
| CN107253978B (en) | 2020-11-20 |
| CN107253978A (en) | 2017-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109627293B (en) | Seikaga valley virus structural protein epitope polypeptide and application thereof | |
| CN110618279B (en) | African swine fever virus epitope antigen polypeptide and application thereof | |
| CN110642925B (en) | African swine fever virus synthetic peptide ELISA antibody detection kit | |
| CN109485703B (en) | Foot-and-mouth disease A-type structural protein VP1 antigen epitope polypeptide and application thereof | |
| CN103864906B (en) | Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent | |
| CN111961120B (en) | African swine fever virus MGFs and CD2v ELISA antibody detection kit | |
| NO166466B (en) | PROCEDURE FOR DETECTING OR DETERMINING A PEPTIDE CONTAINING AN ANTIGENIC ACTIVE SEQUENCE OF AMINO ACIDS. | |
| CN108152511B (en) | Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit | |
| CN104987368B (en) | Foot-and-mouth disease antibody ELISA immunity detection reagent | |
| CN109239355B (en) | Enzyme-linked immunoassay kit for avian influenza virus H9 subtype antibody | |
| CN108196065B (en) | Porcine circovirus type 3 ELISA antibody detection kit | |
| CN104478998B (en) | Swine foot-and-mouth disease virus nonstructural protein 3A BC antibody ELISA immunity detection reagents | |
| CN107513101B (en) | Pig foot-and-mouth disease virus non-structural protein antibody enzyme-linked immunoassay kit | |
| CN104961809B (en) | The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa | |
| CN108802384B (en) | ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide | |
| CN109900903B (en) | Porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof | |
| CN107991499B (en) | Enzyme-linked immunoassay kit for porcine parvovirus structural protein antibody | |
| CN110669112B (en) | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus | |
| CN110229218B (en) | Reagent for detecting Seneca virus antibody and polypeptide used by same | |
| CN117209569A (en) | African swine fever virus p72 protein ELISA antibody detection kit | |
| CN117783523A (en) | Rabbit hemorrhagic disease virus 2 type structural protein VP60 antibody enzyme-linked immunosorbent assay kit | |
| CN115993449A (en) | Niu Jiejie skin disease virus ELISA antibody detection kit | |
| CN116482356A (en) | ELISA antibody detection kit for porcine reproductive and respiratory syndrome | |
| CN117143205A (en) | ELISA antibody detection kit and application thereof | |
| CN117777256A (en) | ELISA kit for clostridium perfringens toxin antibody of cattle A |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |