165 results about "Indirect immunofluorescence" patented technology

The invention discloses a preparation method for a PCV-II Cap protein monoclonal antibody, an antibody and application. The invention adopts ultracentrifuged and purified PCV-II as an immunogen to immunize a BALB/c mouse by the conventional method, takes spleen cells of the immunized BALB/c mouse to fuse with SP2/0 cells, obtains two strains of hybridoma cells secreting the PCV2-Cap protein monoclonal antibodies by indirect ELISA screening, respectively names the two strains of hybridoma cells as 8-60 and 10-48, identifies biological characteristics of the two strains 8-60 and 10-48, and usesthe two strains 8-60 and 10-48 as the first antibodies to establish an indirect immunofluorescence diagnostic method. The result of the indirect immunofluorescence diagnostic method is basically consistent with that of the PCR diagnostic method, and the positive and negative coincidence rates are respectively 93.75 percent and 100 percent so as to provide reference for preventing and treating theporcine circovirus disease.

The invention discloses a HCMVPP65 antigenemia indirect immune fluorescence method detection kit, which uses cytomegalovirus-AD169 virus strain pp65 protein as the immunogen to immune a Balb/c mouse. The spleen cells of the immunized mouse and the myeloma cells of the mouse which belongs to the same type with the immune mouse are conventionally integrated, by indirect ELISA screening and finite dilution cloning, the hybridoma cell lines of the mouse cytomegalovirus pp65 protein cloning antibody are obtained, and the characteristics of the hybridoma cell lines are identified by ELISA, immune fluorescence experiment and other methods; two monoclonal antibodies that stably secrete the pp65 protein are established successfully and named respectively as 1A6 and 4A8. A monoclonal antibody which differs from the former report and aims at the pp65 protein of the cytomegalovirus (CMV) is prepared and a method used for preparing erythrocyte fast pyrolysis is established; compared with other detection kits which belongs to the same kind, the detection kit of the invention is faster, simpler and more convenient and has higher specificity and sensitivity.

This invention discloses monoclonal antibody against anti-LCDV immunoglobulin of Paralichthys olivaceus, which is excreted by hybridoma JF-lgM-H (CCTCC-C200631). The method comprises: immunizing Paralichthys olivaceus with LCDV inactivated by formalin to prepare antiserum, purifying Paralichthys olivaceus immunoglobulin, immunizing Balb/c mice as antigen, preparing hybridoma cells by cell engineering method, and screening the monoclonal antibody by immunoassay. Indirect ELISA and indirect immunofluorescent antibody assay show that this monoclonal antibody is located on the heavy chain (7-80 kDa) of the anti-LCDV immunoglobulin. The monoclonal antibody can be used for preparing reagents for detecting LCDV infection in early stage, and evaluating the immune effects of LCDV vaccine inactivated by formalin.

The present invention provides a kit for quantitative detection of homogeneous or speckled antinuclear antibody, and a use method thereof. The kit comprises: (1) a Hep-2 cell antigen sheet; (2) a fluorescence labeled secondary antibody; (3) fluorescence intensity calibration microspheres; (4) a buffer; (5) a sheet sealing agent; and (6) an experimental operation and quantitative fluorescence intensity analysis instruction. According to the present invention, the (1) Hep-2 cell antigen sheet provided by the kit, the (2) fluorescence labeled secondary antibody provided by the kit, and a serum sample to be detected are subjected to an indirect immunofluorescence reaction, the (3) fluorescence intensity calibration microspheres are added to the (1) Hep-2 cell antigen sheet to be detected, observing and microscopic image shooting are performed through a fluorescence microscope, a microscopic image analysis software is used to carry out fluorescence intensity analysis, and the fluorescence intensity value is converted into the corresponding serum antinuclear antibody titer value according to the (6) experimental operation and quantitative fluorescence intensity analysis instruction provided by the kit, such that the quantitative detection of the homogeneous or speckled antinuclear antibody in the serum sample is achieved.

The invention discloses a staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use. The staphylococcal enterotoxin gene engineering reshaped antibody has an amino acid sequence shown in the formula of SEQ ID No.1 in the sequence table. Through construction of light and heavy chain eukaryotic co-expression vectors of the staphylococcal enterotoxin monoclonal antibody, a high-efficiency expression and stable-secretion mammalian cell line and the gene engineering reshaped antibody having high singularity and strong affinity are obtained. The staphylococcal enterotoxin gene engineering reshaped antibody can be used in staphylococcal enterotoxin detection, cell indirect immunofluorescence detection and flow cytometry detection.

The invention discloses a staphylococcal enterotoxin micromolecule antibody and its preparation method and use. The staphylococcal enterotoxin micromolecule antibody has an amino acid sequence shown in the formula of SEQ ID No.1 in the sequence table. The preparation method comprises the following steps of designing and constructing eukaryotic mono-promoter co-expression vectors of staphylococcal enterotoxin monoclonal antibody light chain and heavy chain variable region genes, introducing cysteine residues to C- ends of the light chain and the heavy chain to form interchain disulfide bonds by an intracellular peptide fragment self-assembling principle, and simulating antigen binding domain spatial conformation of natural antibodies to obtain high-efficiency expression stable-secretion mammal cell line and the high-singularity strong-affinity micromolecule antibody. The staphylococcal enterotoxin micromolecule antibody can be used for staphylococcal enterotoxin detection, cell indirect immunofluorescence detection and flow cytometry detection.

The invention belongs to the technical field of new biological veterinary medicaments, and in particular relates to a method for testing the efficacy of a swine fever live vaccine. The efficacy of the live vaccine is evaluated by detecting the virus content of the live vaccine through indirect immunofluorescence (IFA). The method for testing the efficacy of the swine fever live vaccine has the advantages of short test time, simple operation, low cost, accurate obtained result, high repeatability, excellent application value and wide market prospects.

The invention relates to a method for preparing an allophycocyanin(APC)-marked fluorescent antinuclear antibody, which comprises: separating and purifying spirulina APC by anion exchange chromatography; crosslinking APC and antinuclear antibody derivatives in a liquid phase in a proper molar ratio; and preparing the APC-marked fluorescent antinuclear antibody by purification by high-pressure liquid chromatogram. The fluorescent antinuclear antibody prepared by the method has high crosslinking efficiency, high purity, bright red fluorescence, high stability and high sensibility, and can be used as a common fluorescent probe for indirect immunofluorescent detection of infectious diseases in animals such as chickens and pigs.

The invention belongs to the technical field of biological quarantine and particularly relates to a method for detecting the virus content of a swine fever live vaccine through indirect immunofluorescence. The method for detecting the virus content of the swine fever live vaccine through indirect immunofluorescence comprises the following steps: (1) preparing subculture cells; (2) measuring the virus content of the swine fever live vaccine in the subculture cells; (3) fluorescently dyeing virus-inoculated cells. According to the method, the viruses of the live vaccine in the subculture cells are detected through indirect immunofluorescence so as to achieve the purpose of rapidly, accurately, simply and effectively detecting the virus content of the swine fever live vaccine.

The invention discloses a recombinant expression protein of 1301 ORF136 genes of herpesvirus cyprinid type 3, a polyclonal antibody and an application of the polyclonal antibody. A full-length amino acid sequence of the recombinant expression protein of 1301 ORF136 genes of the herpesvirus cyprinid type 3 is as shown in SEQ ID NO:2, and a full-length nucleotide sequence encoding the protein is as shown in SEQ ID NO:1. A pET32a-ORF136 recombinant prokaryotic expression vector is constructed by selecting one part of gene sequence of ORF136; the recombinant expression protein is obtained through IPTG-induced expression; the obtained polyclonal antibody is subjected to western blot analysis by adopting a purified CyHV-3 virus and a KS cell infected by the CyHV-3; and the detection application of the antibody is further verified by adopting an indirect immunofluorescence assay. An important material is provided for construction of an ORF136 protein function research and CyHV-3 serological diagnosis method through preparation of the polyclonal antibody of the recombinant expression protein of the ORF136 genes.

The invention discloses a method for detecting a bovine viral diarrhea virus by virtue of an indirect immunofluorescence, belonging to the technical field of biological quarantine. According to the method, the BVDV (bovine viral diarrhea virus) is bred by virtue of MDBK cells and is detected by virtue of the indirect immunofluorescence. The method specifically comprises the following steps: (S1) culturing the MDBK cells; (S2) inoculating the BVDV; and (S3) detecting the bovine viral diarrhea virus by virtue of the indirect immunofluorescence, namely carrying out washing, acetone fixation, primary antibody incubation, secondary antibody incubation and result determination. According to the method, the activities of BVDV particles can be rapidly and accurately detected, and polluted BVDV in swine fever live vaccines can be stably detected; the method is applied to the detection on the BVDV polluting bovine-origin raw materials in the vaccines, and a relatively efficient detection method for preparing pure swine fever live attenuated live vaccines is provided.

The invention discloses a method for determining baculovirus titer. The method comprises the following steps: paving a cell culture plate with insect cells in the logarithmic phase at the cell density of 2*104-4*104/0.1mL/hole, placing the cell culture plate into an incubator for culture and sticking the insect cells to hole bottom; inoculating baculovirus to be detected into the insect cell plate and culturing in the incubator; removing a medium in the cell plate, fixing, washing the cell plate, adding gp64 McAb for incubation, washing the cell plate, adding goat-anti-mouse fluorescent antibody for incubation, washing the cell plate, and observing under a fluorescence microscope; and calculating TCID50 of the virus. The method for determining baculovirus titer is combined with indirect immunofluorescence, is a TCID50 determination method for directly judging whether each hole cell is infected and has good repeatability. Obtained detection results are more accurate. It is not necessary to combine cytopathic effect for determination, and there is no influence from aspects of subjective and experience factors.

The invention relates to an antibody indirect immunofluorescence test method for distinguishing whether an immune animal is infected with influenza A virus or not. The method comprises the following steps of: expressing NA protein of influenza A virus as antigen with insect baculovirus; establishing the antibody indirect immunofluorescence test method; and detecting the serum antibody of the NA protein of the influenza A virus. The expressed protein can be directly used as the antigen without purification. The method established by the invention is a detection method matched with the marking vaccine for the influenza A virus, which is used for distinguishing whether the animal vaccinated with the influenza A virus marking vaccine, such as birds, pigs and the like, are infected with corresponding subtype influenza virus or not; as a screening method, the method can be used for detecting the clinical serum sample and determining whether the animal vaccinated with the influenza A virus marking vaccine, such as the birds, pigs and the like, are infected with the corresponding subtype influenza virus or not; and therefore, corresponding preventative measures can be taken as soon as possible so as to eliminate and kill the corresponding epidemic clinical influenza A virus.

The invention discloses an immunofluorescence reagent for detecting an E-type enterovirus and a detection kit thereof. A direct immunofluorescence detection method set up by applying the immunofluorescence reagent for detecting the E-type enterovirus can be used for clinically fast diagnosing E-type enterovirus infection and used for positioning and authenticating an E-type enterovirus antigen in tissue. The time for direct immunofluorescence detection is short, and a result can be reported within 40 min; specificity is high, and the immunofluorescence reagent only reacts with the E-type enterovirus; the immunofluorescence reagent is high in repeatability and accuracy, the coincidence rate between results of immunofluorescence reagent and results of an indirect immunofluorescence method is 90% or more, and the immunofluorescence reagent can be used for fast diagnosing E-type enterovirus infection.

The invention provides a chicken C-type acian metapneumovirus strain(aMPV-JCX) and application thereof. The preservation number of the aMPV/C-JCX strain is CGMCC No.10900. The virus is a cell culture adaptive strain, a preparation method of the cell culture adaptive strain comprises the steps that tissue grinding fluid of a sick chicken infected by aMPV/C-JCX which is firstly obtained through separation domestically is collected to be inoculated into single-layer Vero cells which grow being adherent to the wall, a cell culture serves as an inoculum for the next round of passage, sequentially continuous passage is conducted, and the cell culture adaptive strain is obtained. In the adaptive process, the early-generation culture cycle is 7-9 days, the culture cycle is shortened to 3-5 days when the 60th generation is cultured, and an obvious cytopathic effect can be observed. It is verified through a transmission electron microscope, indirect immunofluorescence and a western-blotting method that the aMPV/C-JCX can be effectively cultured. The titer 1 g TCID50 of the virus is equal to 10<-4.2>/0.1 ml. It is verified through SPF chickens that the virus virulence is weak, good immunogenicity and protectiveness are achieved, and the cell-adapted virus is an ideal candidate strain for researching and producing viral vaccine.

The invention discloses a method for detecting autoantibodies of a systemic lupus erythematosus patient. The method includes steps of: detecting antinuclear antibodies and double-strand deoxyribonucleic acid (DNA) of the systemic lupus erythematosus patient respectively in an indirect immunofluorescence method, measuring antinuclear antibodies spectrums in a euroimmun western blotting method, and finally detecting single-strand DNA of the systemic lupus erythematosus patient through an enzyme-linked immunosorbent assay. The method for detecting autoantibodies of the systemic lupus erythematosus patient simplifies the existing detection method, improves detection efficiency and accurate diagnosis rate, and is beneficial to clinical diagnosis and treatment of systemic lupus erythematosus.

The invention provides a construction method of porcine reproductive and respiratory syndrome virus (PRRSV) recombinant plasmid expressing African swine fever virus (ASFV) p30 protein, and a genetically engineering vaccine constructed based on the recombinant plasmid and a construction method thereof. Indirect immunofluorescence is carried out on rPRRSV-p30 infected wells, it is found that the specific fluorescence of anti-PRRSV N protein and anti-ASFV p30 protein appears in the field of vision. Protein samples of rPRRSV-p30 infected wells are collected and analyzed by Western blot, results show that p30 bands of the expected size are found. It is suggested that the recombinant PRRSV expressing African swine fever virus p30 protein can ensure good, efficient and stable expression of foreign protein ASFV p30 protein. However, some studies show that p30 is an important antigen protein, and can be used in the development of ASF vaccine.

The invention relates to monoclonal antibody of N-terminal protein of chlamydia abortus POMP18D as well as a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell strain 1F10D4 with the collection number of CGMCC No.4658. The invention also provides application of the monoclonal antibody in direct fluorescence immune or indirect fluorescence immune detection of chlamydia abortus. The monoclonal antibody of N-terminal protein of chlamydia abortus POMP18D has strong specificity and fluorescent characteristic, and is suitable for being used as a fluorescence antibody to establish a direct fluorescence detection method or indirect immune fluorescence detection method.

The invention provides a hybridoma cell strain 2D5 capable of secreting an anti-cattle-interferon inducing protein-10(IP-10) monoclonal antibody and a secreted monoclonal antibody MAb 2D5. The monoclonal antibody MAb 2D5 has the advantages of high titer, excellent specificity and strong affinity with natural cattle IP-10 antigen. A blocking ELISA detection kit, a Western-Blot detection kit, an indirect immunofluorescence (IFA) detection kit and a flow cytometry (FCM) detection kit which are established on the basis of the monoclonal antibody MAb 2D5 have higher sensitivity and specificity and can be applied to the detection for recombination expression cattle IP-10, the detection for in vitro tissues and the non-diagnosis purpose research, such as epitope identification.