207 results about "Immunoblottings" patented technology

The invention is drawn to composite agarose / acrylamide compositions and gels. In particular it relates to gels for the separation of molecules, particularly macromolecules such as proteins. The invention is also directed to the preparation of composite gels, the separation of molecules by techniques such as electrophoresis using such gels, and the transfer of proteins from such gels to a transfer membrane using an immunoblot transfer gel.

The invention relates to the technical field of biology and in particular discloses an anti-silk fibroin polyclonal antibody and a preparation method thereof. The anti-silk fibroin polyclonal antibody is produced by an animal under the condtion that polypeptide with an amino acid sequence shown in SEQ ID NO:1 is taken as an immunogen. The anti-silk fibroin polyclonal antibody has the characteristics of good specificity and high purity, can be specifically conjugated with silk fibroin and is widely applicable to immunohistochemistry, immunoblotting and in vitro immunoassay. The preparation method of the anti-silk fibroin polyclonal antibody is simple in operation and is applicable to preparation of a large quantity of anti-silk fibroin polyclonal antibodies.

The invention discloses preparation of novel duck reovirus recombinant sigma B protein antigen, and application of the novel duck reovirus recombinant sigma B protein antigen in detection of a novel duck reovirus antibody. With RNA (ribonucleic acid) of the novel duck reovirus (NDRV) as a template, a nucleotide complete sequence of an NDRV S3 gene coding region can be amplified through a reverse-translation polymerase-chain reaction, is directionally subcloned to a fusion expression vector of the prokaryotic expression vector pET-30a-C(+) of escherichia coli, and transformed into the host cell of the Escherichia coli BL21 (DE3), and is subjected to inducible expression with IPTG (isopropyl-beta-d-thiogalactoside), purification of Ni-NTA Agarose, inclusion body refolding to obtain the expressed NDRV alpha B recombinant protein antigen. The expression mode of the protein is fusion protein (His-alpha B) with molecular weight being about 44kU; and immunoblotting assays prove that the protein has immunoreactivity similar to natural protein..

The invention discloses an anti-PD-L1 antibody as well as application, a preparation method, a kit and a drug, and relates to the technical fields of biological detection and treatment. The anti-PD-L1 antibody provided by the invention contains a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6. The anti-PD-L1 antibody can be combined with PD-L1 recombinant proteins and inflammation tissue cells or tumor tissue cells expressing PD-L1 molecules via specific recognition, so that the anti-PD-L1 antibody has a relatively high specificity and avidity, can be used for immunoblotting, enzyme-linked immuno sorbent assay, immunohistochemistry and flow cytometry and can also be used for treating diseases of tumors, immunological diseases, chronic infectious diseases and the like.

The invention discloses a chip for diagnosing the proteins. The antigens corresponding to multiple relevant indexes are printed on the different positions of the membrane of cellulose nitrate. The multi-tape immunoblotting method is utilzied through the coloration of gold mark or the precipitation enzyme without coloration to detect the antibody in the specimen of the sufferer aiming at the antibodies all antigens. The invention raises the detecting sensitivity and the specificity of the immunizing method so as to avoid the false negative or the false positive, providing the results for detecting multiple antigens in one one-stroke detection. Since the chip possesses more information than the information in the testing sheet so as to reduce the collecting quantity and shorten the testing period.

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

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 aims at providing chimeric protein of enterovirus EV71 neutralization epitope and a human norovirus P structure domain and an establishing method of a chimeric vector. By means of immunoblotting, it is verified that the chimeric protein of the enterovirus EV71 neutralization epitope and the norovirus P structure domain can be used for research and development of detection kits of EV71 and norovirus and novel bivalent subunit vaccines.

Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. A method to detect proteins which contain nitrosothiols involves several steps. Nitrosylated cysteines are converted to tagged cysteines. Tagged proteins can then be detected, for example, by immunoblotting and / or can be purified by affinity chromatography. The method is applicable to the detection of S-nitrosylated proteins in cell lysates following in vitro S-nitrosylation, as well as to the detection of endogenous S-nitrosothiols in selected protein substrates.

The present invention relates to a classical swine fever antibody PPA-ELISA detection kit and a preparation method thereof. The kit preparation comprises: adopting RT-PCR to amplify a 550 bp gene fragment of E2 gene, wherein the fragment contains 4 neutralizing antigen regions of A, B, C and D; cloning the amplified 550 bp gene fragment into a pMD18-T vector; carrying out identification, wherein the identification result is correct; further directionally cloning the fragment into a PGEX-6P-1 prokaryotic expression vector; transforming BL21 expression bacterial; carrying out IPTG induced expression to obtain recombinant protein expressed in a form of inclusion body; adopting an affinity chromatography method to separate and purify the recombinant protein; adopting immunoblotting to detect antigenicity and specificity of the purified protein; and adopting the recombinant protein as coating antigen, adopting horseradish peroxidase-labeled staphylococcal protein A (SPA) as second antibody, optimizing reaction conditions of indirect ELISA, establishing an indirect ELISA detection method for detection of classical swine fever virus antibody, and assembling the classical swine fever antibody PPA-ELISA detection kit.

The invention provides a multiphase microfluidic immunoblotting chip, and a preparation method and applications thereof. The chip comprises a substrate fixed with a separate protein band, and a chip body, wherein at least one micro fluid concave groove is arranged on one surface of the chip body, the micro fluid concave groove and the a polymer film together form at least one microflow passage for antibody circulation, and a via hole is respectively arranged on each end port, corresponding the microflow passage, on another surface, to form a passage inlet and a passage outlet of the microflow passage. The chip preparation method comprises the following steps of: preparing the substrate fixed with the separate protein band; and assembling the prepared substrate and the clip body. The chip is used for protein detection and for preparing a kit for protein detection. Single-time immunoblotting reaction of the invention can determine existence of multiple target proteins, and the chip and the preparation method has advantages of high detection sensitivity, simple operation, high detection efficiency and low cost.

The invention discloses a detection method of a self immune antibody. The method comprises the following steps of performing cotransfection cells by expression vectors of allergen protein of an antibody to be detected and a chaperone protein expression vector; after the transfected cells are fixed and after washing, adding serum to be tested for incubation; adding second antibodies for incubation; then, performing analysis, wherein the antigen protein of the antibody to be detected is MOG protein. The detection method has the characteristics of high detection efficiency, high specificity and high sensitivity. The common problem of false positive or false negative detection result in the immunoblotting and enzyme linked immunosorbent assay are successfully solved; very important study and practical values are realized.

The invention relates to an extraction method of protein from animal tissues, in particular to an extraction method of total protein from a rumen epithelial tissue of a milk cow. The method comprises the steps of putting a rumen epithelial tissue sample of the milk cow in a precooled mortar, adding liquid nitrogen, grinding into powder, adding an acetone solution of precooled trichloroacetic acid, oscillating, eddying, uniformly mixing for overnight aging, centrifugally collecting sediment, freeze-drying the tissue sediment, adding lysate, incubating and eddying at intervals, and centrifugally collecting supernate, namely the total protein of the rumen epithelial tissue of the milk cow. For the total protein of the rumen epithelial tissue of the milk cow, extracted by the method, a total protein mixture can be separated by a two-dimensional gel electrophoresis technology according to an isoelectric point and molecular weight of the protein, the relative abundance of the protein is acquired, the isoelectric point, the molecular weight and a relative transcript level of each protein can be clearly and visually shown in a gel atlas, and the method has the advantages of more obvious integrity and visualization in comparison with the prior arts such as a high efficiency liquid chromatography separation method, an enzyme-linked immunosorbent assay and immunoblotting.

The invention provides an establishing method of a Chinese tongue squamous cancer cell line (CTSC-1), and provides a material base for researching the characteristics of tongue squamous cancer and therapeutics thereof. The establishing method comprises the steps of: carrying out primary culture on the tongue squamous cancer cells by adopting a tissue explants adherent method; subculturing when the cell density reaches about 80 percent; carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on stably subcultured cells to indicate that the cells express tongue squamous cancer cell symbolic molecules CK 8, 18 and 19; and detecting adherent cells through a western blot test to express proteins of the tongue squamous cancer cell symbolic molecules CK 8 and 18, therefore, proving on a molecular level the successful establishment of the CTSC-1.

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

The invention discloses a theileria equi immunoblotting detecting method and a method for preparing a kit, wherein the theileria equi immunoblotting detecting method in the invention is established on the basis of acquiring diagnostic antigen and positive control serum via Cochlearia officinalis hyperoxide enzyme label and chemical light emitting prime. The solvent for the method in the inventionis little in amount, low in cost, high in specificity, high in sensitivity, high and fast in operation, free of toxicity and harm, low in required conditions, easy for judging the test result, long-time for storage, and stronger in specificity than ELISA. The method in the invention detects no false positive and false negative result.

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 western blotting luminescence and grey level combined foot and mouth disease virus antigen detection method. The method comprises the following steps: (1) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis); (2) blotting transfer; (3) immune reaction; (4) developing or luminous reaction; (5) judgment of the property of an antigen to be detected according to a specific antigen band; (6) calculation of the content according to the luminous grey level of the antigen. The method can not only qualitatively but also quantitatively detect the foot and mouth disease virus antigen, has the characteristics of being safe, quick, simple, convenient, accurate and specific, realizes specific quantitative detection of various foot and mouth disease virus antigens, originates the mode of adopting western blotting luminescence to quantitatively detect the foot and mouth disease virus antigen, and has an important guiding meaning for vaccine production.

The invention discloses a kit for detecting an IgM antibody for resisting a novel coronavirus SARS-CoV-2 in a sample. The invention further discloses an application of the kit in preparation of a product for diagnosing novel coronavirus SARS-CoV-2 infection. The kit provided by the invention has the advantages of short operation time, low cost, low dependence on a machine, capability of simultaneously carrying out detection by utilizing S protein and N protein, low detection cost, high detection efficiency, high detection accuracy rate and the like.

The invention relates to the biology field, in particular to a monoclonal antibody of influenza virus matrix protein M1 and application of the monoclonal antibody. The monoclonal antibody of influenzavirus matrix protein comprises a heavy chain variable region and a light chain variable region, amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ IDNO.1-3 separately; and amino acid sequences of the CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO.4-6 separately. The monoclonal antibody can specifically recognize andbind multiple subtypes of influenza virus matrix protein M1, has broad spectrum applicability among influenza virus subtypes, has higher specificity and sensitivity, can be well applied to western blotting experiments, indirect immunofluorescence experiments, immunoprecipitation technologies and the like and has higher application values.

The invention discloses a monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins. The monoclonal antibody is produced by secreting rock bream iridovirus ORF049L recombinant protein hybridoma of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.7304. A preparation method of the monoclonal antibody comprises the following steps of: obtaining the rock bream iridovirus ORF049L recombinant proteins with the molecular weight of 16.9 kDa through gene cloning and prokaryotic expression; taking the obtained recombinant proteins as antigens to immune mice; performing cell fusion on mice spleens and myeloma cells, and screening and secreting the hybridoma of the rock bream iridovirus ORF049L recombinant protein monoclonal antibody by an immunoblotting method, a dot immunobinding assay and an indirect immunofluorescence; and cloning positive hybridoma by a finite dilution method.

The invention belongs to the field of life science research, and specifically relates to a method for detecting the expression quantity of nicotine acetylcholine receptors (nAChRs) in peripheral bloodlymphocytes of rats, which is characterized by comprising the following steps: 1) separating peripheral blood lymphocytes of rats; 2) acquiring cell total protein, concentration determination and sample preparation; and 3) detecting the expression quantity of the nAChRs in the lymphocytes by a western blot (WB) experiment. According to the invention, the detection of the protein expression in theanimal peripheral blood in real time can be realized by determining the protein in the peripheral blood lymphocytes, and the kit has important significance for the research of nicotine addiction biomarkers.

The invention provides a reaction membrane strip for detecting autoimmune diabetes, a preparation method and a using method, and belongs to the field of biomedical science. Aiming at the lack of an immunoblotting product special for various immune diabetes experimental examinations and physical examination at present and some defects of an existing immunoblotting agent, the invention provides the reaction membrane strip for detecting the autoimmune diabetes. The reaction membrane strip comprises a bottom lining plate, not less than one detecting zone and not less than one indicating zone, wherein the detecting zones and the indicating zones are arranged on the bottom lining plate; the detecting zones, the indicating zones as well as the detecting zones and the indicating zones are parallel to each other. Compared with the prior art, the reaction membrane strip provided by the invention has the following advantages that various autoantibodies can be detected through one clinic sample and once detection reaction, insufficient sensitivity and specificity of single autoantibody detection and operation complexity of independently detecting autoantibodies related to various diseases one by one are overcome, and the detecting efficiency and the result judgment accuracy are greatly increased.

The invention belongs to the technical field of immunoassay and in particular relates to a method for detecting trace protein by fluorescent immunoblotting of quantum dot nanospheres. The method comprises the following steps: firstly, carrying out gel electrophoresis separation on a protein mixture to be detected; transferring gel subjected to the electrophoresis separation into a solid-phase carrier film; then sealing a protein-loaded solid-phase film; after adding a specific primary antibody of protein to be detected, washing; then reacting with a fluorescent secondary antibody of the quantum dot nanosphere marker and washing; finally, developing and photographing through a protein blot film under the illumination of exciting light. Compared with chemiluminescence, the method for detecting the protein through the fluorescent immunoblotting, provided by the invention, has the characteristics that luminescent substrates are not needed, a detection signal is more stable and can be stored for a long period, results have consistency and repeatability and the like; compared with near-infrared fluorescent immunoblotting, the method has the characteristics that an additional signal amplification step is not needed, a fluorescent signal is more stable, light-shielding operation is not needed, an expensive near-infrared laser scanner does not need to be used for detecting and the like.

The invention discloses an anti-HPV16 E6 protein monoclonal antibody, which is characterized by having heavy chain variable regions with the amino acid sequences shown as SEQ ID NO:4, SEQ ID NO:6 andSEQ ID NO:8 and light chain variable regions with the amino acid sequences shown as SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14. The immunoblot experiments show that the antibody can be used for specifically recognizing the recombinant HPV16 E6 protein and cervical cancer tissues expressing HPV16 E6 protein. The antibody can be used for detecting the expression level of the HPV16 E6 protein in thecells in artificial or automatic modes through immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA) or western blot manners; the HPV16 viruses can be specifically diagnosed;the novel measure is provided for detecting HPV16 viruses on the protein level; the antibody can be used or the study of human reproductive system infectious diseases and tumors, and can also be usedfor the study of relevant malignant tumor such as esophagus cancer, breast cancer and anal carcinoma.

The invention relates to polypeptide biological technical field, which discloses a spatial conformation mimic antigen epitope of H3-type influenza virus hemagglutinin, and relative application. The sequence of the spatial conformation mimic antigen epitope is SEQ ID No. 1. The invention utilizes anti-recombination HA1 rabbit serum IgG selection phage 12 peptide library, uses phage ELISA, DNA sequencing, western bolt, and software analysis to analyze selected colony, and selects the small peptide from random phage 12 peptide library, which can be specially combined with anti-recombination HA1 rabbit serum IgG. The inventive spatial conformation mimic antigen epitope can prepare diagnostic reagent box of H3-type influenza virus.

The invention belongs to the technical field of liquid chromatographic separation, and particularly relates to a rapid and high-purity separation method of human plasma exosome. The method comprises the steps of firstly, removing apoptotic bodies and microvesicles of human plasma through high-speed centrifugation, then loading a sample to an agarose gel small column for pre-separation, and removing most of abundance proteins with relatively small sizes, such as human serum albumin and immune globulin; sequentially carrying out merging and ultrafiltration concentration on the collected fractions, and finally carrying out high performance liquid volume exclusion chromatography separation to realize separation of exosome and interference lipoprotein, thereby finally obtaining the high-purity human plasma exosome fraction. The whole process from plasma pretreatment to exosome fraction obtaining does not exceed two hours. Characterization of immunoblotting proves that the removal rate of high-density lipoprotein (HDL) can reach 95%, and the removal rate of low-density lipoprotein or very low-density lipoprotein (VHDL) can reach 85%.

The invention discloses a marker for cancer detection and an application thereof. The marker is mRNA of a Nurim gene or an expression product thereof; and a reagent comprises a reagent for specifically detecting the mRNA of the Nurim gene or a reagent for specifically detecting the expression product of the mRNA of the Nurim gene. Through RT-PCR detection of different transcripts or immunoblots of the Nurim or immunofluorescence assay of protein expression of the Nurim, whether a patient suffers a cancer or not can be diagnosed. The marker for the cancer detection, disclosed by the invention, is general and capable of detecting a plurality of different malignant tumors; meanwhile, according to the quantity of the mRNAs or the expression products, the tumor progression degree can be reflected; through the adoption of the molecular marker or the detecting reagent disclosed by the invention, the cancer can be conveniently and rapidly diagnosed with high accuracy; and the marker for the cancer detection, disclosed by the invention, has higher reference values to clinical diagnosis.

The invention belongs to the technical field of biomedicine, and particularly relates to the application of a molecular target in the prognosis prediction and treatment of esophageal squamous cell carcinoma. The molecular target is a long-chain non-coded RNA AGPG. The expression level of an AGPG gene in esophageal squamous cell carcinoma tissues is detected through RT-PCR, RNA scope in-situ hybridization, in-situ hybridization, a chip or a high-throughput sequencing platform. The expression level of a target protein PFKFB3 corresponding to the AGPG gene in the esophageal squamous cell carcinoma tissues is detected through immunoblot and an immunohistochemical technology. A biologically active drug is successfully developed, the biologically active drug comprises an inhibitor of the long-chain non-coded RNA AGPG and an inhibitor PFK15 of a PFKFB3 protein, and the biologically active drug can be effectively used for the prognosis prediction and treatment of the esophageal squamous cell carcinoma. A novel molecular target is provided for the prognosis prediction and treatment of the esophageal squamous cell carcinoma by a researching outcome, and a novel treatment strategy is providedfor the clinical treatment of the esophageal squamous cell carcinoma.