51 results about "African swine fever virus Antibody" patented technology

The invention discloses a hybridoma cell line of a monoclonal antibody against African swine fever virus and the secreted monoclonal antibody thereof. The preparation method of the invention comprises the following steps: preparing a recombined P30 soluble antigen by prokaryotic expression; immunizing a BALB/c mouse; and finally fusing, screening and cloning by a hybridoma technology to obtain the hybridoma cell line which can stably secrete the monoclonal antibody against African swine fever virus P30 protein. The invention further discloses a method for preparing the monoclonal antibody with the cell line, an antibody purification method and a labeling method for horseradish peroxidase of the antibody. The monoclonal antibody can be used in detecting the African swine fever viral antibody in pig serum.

The invention provides a multi-antigen enzyme linked immunosorbent assay (ELISA) kit for detecting an African swine fever virus (ASFV) antibody, belonging to the field of a biotechnology and diagnosis and research of animal-borne diseases. The multi-antigen enzyme linked immunosorbent assay kit comprises expression and purification of three types of ASFV recombined antigens, preparation of positive and negative control blood serum of an ASFV antibody, optimal envelope antigen combination and concentration determination, optimization of multi-antigen ELISA (MA-ELISA (Microalbumin-Enzyme Linked Immunosorbent Assay)) reaction parameters, determination of an ASFV antibody negative blood serum critical value, MA-ELISA detection artificial infection and determination of sensitivity, specificity and repeatability of field blood serum samples. By detecting and testifying a large quantity of known blood serum samples, the sensitivity, the specificity and the repeatability of detecting the ASFV antibody by the MA-ELISA are obviously higher than those of an ELISA method recommended by World Organization for Animal Health and oversea similar kits; and the multi-antigen enzyme linked immunosorbent assay kit can be used for ASFV serological diagnosis, epidemiological investigation and live pig import and export quarantine inspection.

The invention discloses an indirect ELISA kit for detecting an African swine fever virus antibody and an application thereof, and belongs to the technical field of biology. The kit adopts prokaryotic expression recombinant P30 protein as a coating antigen, and detects the antibody of African swine fever virus in porcine serum based on the indirect ELISA principle. The coating antigen in a 96-well plate of the kit is prokaryotic expression recombinant P30 protein which has good antigenicity. The enzyme-linked immunoassay kit provided by the invention comprises a 96-well plate coated with P30 protein, a positive control, a negative control, a horseradish peroxidase-labeled rabbit anti-porcine IgG polyclonal antibody, a concentrated washing liquid, a serum diluent, a TMB substrate, and a terminating liquid. The kit of the invention is applicable to the screening of large quantities of samples, and main reagents in the kit are provided in a form of operating fluid which is convenient for use.

The invention relates to a preparation method for an African swine fever virus (ASFV) antibody detection colloidal gold immunochromatography test paper strip, belonging to the field of bioengineering and animal epidemic disease diagnosis. The preparation method comprises the following steps of: expressing an ASFV recombinant antigen p54, purifying, preparing an antibody, preparing a colloidal gold immunochromatography test paper strip, and detecting the sensitivity and specificity of the ASFV antibody. Compared with the other conventional serological methods, the colloidal gold immunochromatography test paper strip prepared by the method has the advantages of convenience, quickness, sensitivity, specificity and the like, can be used for obtaining a definite diagnosis result within 5 minutes and directly detecting a suspicious swine serum (or anticoagulation) virus antibody, and is particularly suitable for field ASFV serological diagnosis, epidemiological survey and swine international trade quarantine and inspection.

The invention discloses an epitope antigen polypeptide of an African swine fever virus and an application of the epitope antigen polypeptide of the African swine fever virus. The epitope antigen polypeptide of the African swine fever virus is a polypeptide as shown in a sequence 2 or a polypeptide as shown in a sequence 3 in a sequence table. A composition of the epitope antigen polypeptide is thecomposition formed by the polypeptide as shown in the sequence 2 in the sequence table and the polypeptide as shown in the sequence 3 in the sequence table. According to the epitope antigen polypeptide or the composition thereof disclosed by the invention, a detection kit is prepared by coating an elisa plate with a chemically synthesized antigen peptide, so that the epitope antigen polypeptide is small in amount of antigen, high in sensitivity and high in specificity; whether an African swine fever virus antibody exists or not can be efficiently detected; and the kit has the advantages of being high in sensitivity, good in specificity and convenient to operate, and has a good market prospect

The invention discloses an indirect ELISA kit for detecting African swine fever virus (ASFV) antibody, and a use thereof, and belongs to the technical field of biology. The kit is characterized by: adopting prokaryotic expressed recombined P54 protein as coating antigen; detecting antibody against to the ASFV in swine serum according to an indirect ELISA principle. The coating antigen in 96-well plates of the kit is the prokaryotic expressed recombined P54 protein which has good antigenicity. The enzyme-linked immunospecific assay kit provided by the present invention comprises the 96-well plates coated by the P54 protein, positive control, negative control, horseradish peroxidase-labeled rabbit anti-porcine IgG polyclonal antibody, a concentrated cleaning solution, a serum dilution, a TMB substrate indicator and a stop buffer. The kit provided by the present invention can be provided for screening mass samples. In addition, the main reagents in the kit are provided in a working fluid manner such that the reagents are used conveniently.

The invention discloses a competitive ELISA kit for detecting african swine fever virus antibody and the purposes thereof, which belong to the biological technical field. The kit adopts prokaryotic expression recombinant P30 protein as coating antigen, and detects the african swine fever virus antibody in pig serum according to a competitive ELISA principle. The coating antigen in a 96 pore platein the kit is the prokaryotic expression recombinant P30 protein, and has good antigenicity. The ELISA kit comprises the 96 pore plate coated by the P30 protein, and monoclonal antibody, concentratedwashing liquid, serum dilution, TMB substrate and stopping solution of positive control, negative control and horseradish peroxidase labeling. The kit can be used for screening a large batch of samples, and the main reagent in the kit is provided in the form of working solution, so that the use is convenient.

The invention relates to a method for preparing a genetic engineering product, in particular to a prokaryotic expression protein of VP73 gene from African swine fever virus (ASFV) and a preparation method thereof. The preparation method comprises: artificially synthesizing the whole-length sequence of VP73 gene according to the sequence of the VP73 gene from ASFV in GenBank, constructing a recombinant expression vector pET32a-VP73, sequencing, verifying, transforming prokaryotic expression recipient bacteria E.coli BL21(DE3), and inducing expression by isopropyl-1-thio-beta-d-galactopyranoside (IPTG), wherein the molecular weight of the recombinant fusion protein is about 65KD. Protein purified by nickel column affinity chromatography can undergo a specific immune imprinting reaction with ASFV positive serum and avoid cross reaction with viruses such as swine fever virus, hog cholera virus, porcine circovirus, porcine reproductive and respiratory syndrome virus, swine influenza virus and pseudorabiesvirus. Experiments show the expressed protein has high detection sensitivity, and high specificity. When the antigen is used for detection, risk of spreading poison is avoided. And the antigen can be used as a detection antigen for use in an enzyme-linked immuno sorbent assay (ELISA) method for identifying an ASFV antibody.

The invention provides African swine fever virus test paper, a kit and a preparation method of the test paper. The African swine fever virus test paper comprises a back plate; a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere pad is coated with a labeled antibody labeled by latex microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one sideof the latex microsphere pad; the quality control line is coated with a goat-anti-mouse anti-antibody, and the quality control line is arranged close to one side of the water absorption pad; and thelabeled antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with a preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with a preservation number of CGMCC No.18539, and the capture antibody is an African swine fever virus antibody. The African swine fever virus test paper can realize rapid, high-specificity and high-sensitivity detection of the African swine fever virus.

The invention discloses a specific IgY antibody for resisting African swine fever virus p54 protein and a preparation method thereof, and application of the IgY antibody prepared by the method to specific detection of an African swine fever virus antibody. The preparation method comprises the following steps: preparing p54 protein antigen by utilizing a genetic recombination expression method, immunizing Hy-Line laying hens aged 23 weeks for 4 times, collecting eggs, and extracting and purifying immunoglobulin (IgY antibody) of egg yolk by utilizing a saturated ammonium sulfate precipitation method. A competitive ELISA method for detecting African swine fever serum antibodies established based on the specific IgY antibody is good in effect, only the positive serum detection result of African swine fever is positive, and the detection result is consistent with the parallel detection result of a commercialized ASFV antibody detection ELISA kit. The competitive ELISA method can be used for generally surveying and monitoring the serum epidemiology of African swine fever so as to prevent and control the disease from being spread to China from entry and exit ports.

The invention belongs to the technical field of immune protein preparation and applications thereof, and particularly relates to a test paper for detecting African swine fever virus antibody. The invention provides a gene sequence E183L-1 for encoding an extracellular region of an African swine fever virus p54 protein. Based on a general inventive concept, the invention also proposes a primer pairfor amplifying the gene sequence and a synthetic protein encoded by the gene sequence. In order to solve the problems existing in the detection of the African swine fever virus, the invention prepares the test paper for detecting the antibody of the African swine fever virus by utilizing the synthetic protein encoded by the gene. The test paper can rapidly and accurately detect the antibody of the African swine fever virus, and is very suitable for basic-level and on-site rapid detection and diagnosis.

The invention belongs to the technical field of biology, particularly relates to an African swine fever virus CD2v extracellular domain recombinant protein, and further discloses the application of the African swine fever virus CD2v extracellular domain recombinant protein to construction of ELISA (enzyme-linked immuno sorbent assay) and an ELISA detection kit prepared from the African swine fever virus CD2v extracellular domain recombinant protein and used for detecting the African swine fever virus. According to the scheme, prokaryotic expression CD2v extracellular domain recombinant protein is constructed based on the characteristics of strains popular in China at present, the recombinant protein has high acquisition amount, high purity and good reactogenicity and can induce an organism to generate a neutralizing antibody, and an ELISA kit for detecting the African swine fever virus can be further constructed based on the recombinant protein. The kit has high specificity, sensitivity and repeatability, and can be used as a reliable detection method for monitoring the African swine fever virus antibody or recognizing the virus.

The invention provides an African swine fever virus antibody detection kit, relates to the field of animal infectious disease detection, and specifically relates to an African swine fever virus antibody detection kit. The African swine fever virus antibody detection kit comprises an elisa plate coated with a recombinant polypeptide tandem protein R10 and a recombinant protein PS273R, wherein the amino acid sequence of the recombinant polypeptide tandem protein R10 is shown as SEQ ID NO: 2, and the amino acid sequence of the recombinant protein PS273R is shown as SEQ ID NO: 4. The African swinefever virus antibody detection kit provided by the invention can be used for rapidly and accurately detecting the African swine fever virus antibody with high specificity.

The invention relates to an African swine fever virus CD2v protein as well as a kit and an antibody prepared from the African swine fever virus CD2v protein. The gene sequence of the African swine fever virus CD2v protein provided by the invention comprises a nucleotide sequence as shown in SEQ ID No.1 or a nucleotide sequence which is at least 90% homologous with the sequence as shown in SEQ ID No.1. According to the invention, codon optimization is carried out on an African swine fever virus CD2v protein gene sequence according to preferred codons of a CHO expression system, and then fusion protein mFc-CD2v and fusion protein gD-CD2v are prepared through the CHO expression system. An ELISA antibody detection kit or a monoclonal antibody prepared from the fusion protein mFc-CD2v can be used for detecting the African swine fever virus antibody, and the detection is rapid and sensitive and has good specificity.

The invention belongs to the field of virus and epidemic disease diagnosis and animal quarantine, particularly relates to an African swine fever virus p72 recombinant protein, and further discloses colloidal gold immunochromatography test paper constructed based on the recombinant protein and a method for detecting an African swine fever virus antibody. According to the colloidal gold test paper based on African swine fever virus p72 protein double-antigen sandwich, a recombinant antigen of ASFV strong immunogenicity main structural protein p72 is marked by colloidal gold, and ASFV antibody in porcine serum is detected in a double-antigen sandwich mode. According to the double-antigen sandwich colloidal gold immunochromatography test paper, after a sample is collected, the sample is mixed with a sample diluent according to a certain proportion and then dropwise added into a sample hole, a detection result can be obtained within 5 minutes, the result is accurate, the double-antigen sandwich colloidal gold immunochromatography test paper can be directly used for detecting a virus antibody in suspicious porcine serum, and the double-antigen sandwich colloidal gold immunochromatography test paper is especially suitable for on-site ASFV serological diagnosis, epidemiological investigation and swine international trade quarantine inspection.

The invention discloses a colloidal gold duplex detection test strip for whole-course monitoring of African swine fever virus antibodies and a preparation method thereof. The test strip comprises a PVC base plate, wherein the PVC base plate is sequentially provided with a sample pad, a combination pad, a nitrocellulose film and a water absorption pad from left to right, the nitrocellulose film is provided with a detection line and a quality control line, the combination pad is coated with ASFV p30 protein and ASFV p72 protein which are labeled by colloidal gold, the detection line is coated with ASFV p72 and ASFV p30 protein, and the quality control line C is coated with an anti-ASFV p30 protein monoclonal antibody and/or an anti-ASFV p72 protein monoclonal antibody; by optimizing the preparation of the combination pad, the whole-process monitoring of the African swine fever virus antibody is realized, compared with a single p30 or p72 test strip, the infection period of an organism can be more accurately obtained, and meanwhile, the combination is good in specificity and relatively high in sensitivity and is expected to become a technical means for on-site or laboratory detection of the African swine fever virus antibody; and a new idea is provided for accurate and rapid diagnosis of African swine fever.

The invention discloses an African swine fever virus (ASFV) P54 antibody detection method and kit based on bluetongue virus core-like particles with a chimeric ASFV P54 protein antigen epitope. The chimeric core-like particle is used as a coating antigen, the antigen epitope is better presented in a form similar to that of an original virus, and meanwhile, the monoclonal antibody with the specificity of the antigen epitope is used as a blocking enzyme-labeled antibody, so that better specificity and better sensitivity are achieved. The ASFV P54 antibody detection kit is high in safety, good in sensitivity, high in specificity, high in accuracy and convenient to operate, and compared with an imported kit, the detection coincidence rate reaches 100%.

The invention relates to an indirect ELISA antibody identification and detection kit for the African swine fever virus. The kit comprises a coating plate, a sample diluent, an enzyme conjugate, a positive control, a negative control, a color developing solution A, a color developing solution B, a concentrated washing solution, a solid washing solution, a stop solution, a sample adding groove for sample adding, a plate sealing film and a serum diluting plate for serum dilution. According to the kit of the invention, the indirect ELISA technology is applied; on the basis of an indirect method principle, through detecting an African swine fever virus antibody, an African swine fever virus wild virus infection antibody and a vaccination generated antibody are further identified and detected, so that the African swine fever virus wild virus infection antibody and the vaccine immune antibody are distinguished; and the sensitivity and the specificity of the kit are good, high-throughput detection is convenient to realize, and the detection efficiency and the detection speed are greatly improved.

The invention relates to the technical field of immunodetection, and discloses a colloidal gold immunochromatography test paper card for detecting an African swine fever virus antibody. According to the immunochromatography test paper card for detecting the African swine fever virus antibody, an African swine fever virus capsid protein P72 monomer is sprayed on a marker pad, a P72 trimer is sprayed on a T line, and the prepared test paper card has extremely high sensitivity; meanwhile, the test paper card further has extremely high specificity and accuracy, the result is visual and can be judged by naked eye, and the test paper card is capable of rapidly and specifically detecting the African swine fever virus antibody in serum, high in practical value and convenient to popularize and use in grassroots.

The invention designs an African swine fever virus antibody detection colloidal gold immunochromatography test paper established based on capsid protein p72 and a preparation method thereof. The test paper comprises a supporting bottom plate, and a sample pad, a gold-labeled protein pad, a chromatography detection membrane and a water absorption pad are sequentially fixed on the supporting bottom plate; the gold-labeled protein pad is coated by glass fiber cotton and is fixed with African swine fever virus p72 recombinant protein labeled by colloidal gold; the chromatographic detection membrane is a nitrocellulose membrane, a detection line and a quality control line are arranged on the nitrocellulose membrane, staphylococcus protein A is fixed on the detection line, and a monoclonal antibody or a polyclonal antibody of anti-African swine fever virus p72 recombinant protein is fixed on the quality control line. The test paper is specific, sensitive, rapid, simple and convenient, can be used for ASFV serological diagnosis and epidemiological investigation, and is easy to popularize and apply in production practice.

The invention provides an anti-African swine fever virus (ASFV) IgY antibody, an anti-ASFV and CD composite IgY antibody, an anti-ASFV and CD polyclonal micromolecular antibody and composite antibodyoptimal combination application. Aiming to solve the problems that the ASFV takes porcine immune cells as target cells, destroys immune tissue of a host and causes vaccine failure, antigen proteins causing 'immunosuppression' and 'immune escape' and antigen proteins playing an important role in virus diffusion are used as target antigens, and the antigen proteins are inhibited and killed by adopting a composite antibody through humoral immunity, cellular immunity and ADCC action; the two bottlenecks of 'immunosuppression' and 'immune escape' are broken through, immune cells are protected and activated, and ASFVs and infected cells are removed. The composite antibody can be prepared into a biological medicine or a disinfectant for preventing and treating African swine fever, and can also beused as feed or a feed additive or an oral preparation component for blocking propagation of the ASFVs through oral cavities and digestive tracts.