52 results about "ELISPOT" patented technology

The invention relates to an ELISpot tuberculosis infection diagnostic kit and the application, which adopts the genetic engineering technology to obtain a CFP10-ESAT6 fusion protein antigen from a mycobacterium tuberculosis by cloning, expression and purification, a tuberculosis specific cell stimulator is used for stimulating the peripheral blood T lymphocyte cell of the detected person to secrete a specific IFN-Gamma, then the invention is detected by ELISpot, and the whole process needs to take two days. The usage of the invention can be used for detecting whether the detected person is infected by mycobacterium tuberculosis by using the naked eye or instrument to determine the result according to the number of the generated purple blue spots, so as to assist the diagnosis of tuberculosis and differential diagnosis.

The invention discloses a kit for assisted diagnosis of tuberculosis, which comprises a specific antibody composition, wherein the specific antibody composition comprises the following five antibodies: (1) IFN-gamma antibody, (2) TNF-alpha antibody, (3) IL-2 antibody, (4) MIG antibody and (5) IP-10 antibody. The kit disclosed by the invention can distinguish a Mycobacterium tuberculosis infected person from a BCG vaccinee. Compared with the ELISPOT tuberculosis diagnosis method, the tuberculosis diagnosis kit based on multi-molecular marker detection has obviously enhanced sensitivity to active tuberculosis (from 72% to 89%), and the positive rate for normal healthy persons is obviously reduced (from 27% to 16%).

Here, we describe a sensitive and specific assay and kit for the detection of chemokines having activity that is upregulated by Th-1 cytokines (such IFN-gamma) and chemokines that upregulate the activity of Th-1 cytokines (such as IFN-gamma). In a typical embodiment, detection of the chemokine monokine induced by gamma interferon (MIG) provides a measure of the biological effect of IFN-gamma rather than direct quantitation of IFN-gamma or IFN-gamma secreting cells per se. Upregulation of MIG expression was observed following in vitro activation of PBMC with defined CD8+ T cell epitopes derived from influenza virus, CMV, or EBV, and in all cases this was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Responses as assessed by the MIG assay paralleled those detected by conventional IFN-gamma ELISPOT, but the magnitude of response and sensitivity of the MIG assay were superior. Our data validate this novel method for the detection of high as well as low levels of antigen-specific and genetically restricted IFN-gamma activity or MIG.

The invention discloses a method for simultaneously detecting neoantigen immunogenicity and a neoantigen specificity TCR (T cell receptor). A neoantigen polypeptide is added to peripheral blood mononuclear cells (PBMCs) to obtain antigen-stimulated cells, and compared with a traditional method, the method has the advantages that the time required for obtaining antigen-specific cells is shortened,the whole process of verifying the neoantigen immunogenicity and obtaining a TCR CDR3 sequence only needs 13 days, and compared with the 40-day treatment process of the traditional method, the methodsaves the time cost greatly. Compared with a traditional MHC-peptide tetramer technology, the method is low in cost, a special tetramer needs to be prepared for one antigen polypeptide in the MHC-peptide tetramer technology, the cost of the sequencing method is remarkably reduced, and compared with a traditional ELISPOT method with the test flux only reaching 10 pieces per batch, the sequencing method is not limited in test flux, higher in efficiency, high in test result accuracy and wide in applicability.

The invention discloses a CTL (Cytotoxic T Lymphocyte) epitope peptide of a foot-and-mouth disease virus type O as well as a screening method and application of the CTL epitope peptide. The CTL epitope peptide is composed of nine amino acid residues, and the amino acid sequence of the CTL epitope peptide is as follows: Ala-Thr-Arg-Val-Thr-Glu-Leu-Leu-Tyr. The epitope peptide has relatively strong combining capacity with SLA (Swineleukocyteantigen)-I proteins from various strains of swine and can induce cytotoxic immune response so as to be suitable for preparing vaccines for preventing and controlling foot-and-mouth disease viruses of various strains of swine and wide in application range. According to the invention, a CTL simulated epitope peptide of a foot-and-mouth disease virus is combined with a single-chain molecule of SLA-I of six strains of constructed swine in vitro, thus a polypeptide which can be combined with a complex can be screened through mass spectrum measurement; in addition, a simulated epitope peptide which can be induced to generate the immune response capacity of T cells is determined through ELISPOT (Enzyme-Linked Immunospot Assay) detection. The invention provides a method for screening and authenticating the CTL epitope of the foot-and-mouth disease virus in a large scale, and lays the foundation for researching and preparing a multi-epitope vaccine of a foot-and-mouth disease.

The invention provides universal CD8+T cell epitope polypeptide of a chicken IBV (Infectious Bronchitis Virus) S1 protein, and belongs to the field of gene and protein engineering. The epitope polypeptides are prepared by the following steps: screening 21 epitope polypeptides in accordance with binding motif sequences in amino acid sequences of the S1 genes of the IBV virus according to the binding motif sequences of haplotype chicken major histocompatibility complex (MHC) I-type molecules; then, taking lymphocytes of three constructed SPF (Specific Pathogen Free) chicken immunized by DNA (Deoxyribonucleic Acid) recombinant plasmids of the S1 genes containing different subtype IBVs, determining the capacity of the 21 polypeptides which induce chicken splenic lymphocytes to secrete interferon-gamma by using an ELIspot (Enzyme-Linked Immunospot Assay) method, and finally, screening to obtain four universal functional T cell epitope polypeptides of IBV, wherein the sequences are respectively shown in SEQ ID NO. 1-4. The four epitope polypeptides provided by the invention are combined in use to prepare a universal vaccine for IBV. The invention further provides a method for screening the epitope of the functional T cell of the S1 protein of the IBV.

The invention discloses a multi-epitope vaccine YL66, the pronucleus expression carrier pGEX-4T-2-YL66 and the eukaryon expression carrier pCDNA3.1-YL66 are built on the basis of the multi-epitope vaccine YL66, and the fusion protein GST-YL66 is obtained through the pronucleus expression of the carrier pGEX-4T-2-YL66. Through the in-vitro and in-vivo ELISPOT experiment and the LDH experiment on the peripheral blood of a healthy supplier HLA-A2 and the transgenosis mouse HLA-A2.1/Kb, a research is performed on immunological competence of the fusion protein GST-YL66 and the DNA vaccine pCDNA3.1-YL66. The research finds that a certain kill rate for the tumour cell EC-9706 by the specificity CTL induced in-vivo mouse and in-vitro human body is indicated according to the tumour-resistant multi-epitome vaccine, meanwhile the induced CTL has a certain specificity of antigen, and can be applied in a further research on the strategy and the application of building tumour multi-epitome vaccine.

The invention belongs to the field of biological engineering and diagnostic medicine, in particular to a mycobacterium tuberculosis ESAT-6 recombinant dipolymer, a preparation method thereof, an antigenicity analysis and an application thereof in the serodiagnosis of tuberculosis. The invention uses the DNA of a chimeric gene nucleic acid vaccine HG856A plasmid containing 2 * esat-6 copies as the template, obtains 2 * esat-6 gene segments by PCR amplification, and clones, expresses and purifies an ESAT-6 recombinant dipolymer protein in E.coli. The protein is connected with the two ESAT-6 monomers through amino acid Tyr and Val which are coded by the AccI restriction enzyme cutting site, i.e. GTC TAC, carries six* His tags at the N end, and can be purified by nickel-chelate affinity chromatography. Experiments confirm that the purified recombinant dipolymer has favorable reactogenicity and antigenic specificity. The recombinant dipolymer can be used as an antigen for the early diagnosis of the tuberculosis, including the serodiagnosis of ELISA, ELISPOT or the like, or can be used as an antigen for a skin test.

The invention discloses a detection method for identifying mycobacterium tuberculosis and non-mycobacterium tuberculosis infections. The detection method comprises the following steps: respectively contacting the specific antigen of mycobacterium tuberculosis and the specific antigen of mycobacterium with a to-be-detected in-vitro T cell, and detecting a cell factor released by the T cell; comparing the detection results of the specific antigen of mycobacterium tuberculosis and the specific antigen of mycobacterium; judging that the to-be-detected in-vitro T cell is suffered from mycobacterium tuberculosis infection or non-mycobacterium tuberculosis infection according to a comparison result. According to the detection method provided by the invention, the specific antigen of mycobacterium and the ELISPOT detection method for detecting the specificity of MTB can be combined and utilized to simultaneously identify MTB and NTM infections. The detection method provided by the invention has important guidance significance on clinical diagnosis and drug usage of a doctor.

The invention belongs to the technical field of biology, and particularly relates to a method for preparing tetramer from O type foot and mouth disease virus polypeptide. Aiming at designed foot and mouth disease virus polypeptide, two methods of ELISPOT and tetramer construction are adopted for screening and identifying the foot and mouth disease virus polypeptide, a Hebao pig SLA-2 heavy chain tetramer is constructed, expression is carried out on a prokaryotic expression system pET-21a(+)/BL21, and an inclusion body is extracted; in the foot and mouth disease virus polypeptide, polypeptide causing a specific CTL reaction is screened out through an ELISPOT method, then the polypeptide, the SLA-2 heavy chain, and existing light chains beta 2m are subjected to renaturation, biotinylation and FITC label streptavidin reactions to generate the SLA-2-BSP/peptide tetramer, and then the function of the tetramer is detected through flow cytometry. Through an ELISPOT method, a specificity polypeptide epitope of O type foot and mouth disease viruses VP1 is obtained through identification, the peptide tetramer is successfully constructed, a pig tetramer technology platform is preliminarily built, and a foundation is laid for study of pig specificity CTL immune responses and T epitope screening.

This document provides methods and materials related to identifying mammals having a LTBI. For example, methods and materials for using ELISpot assays to identify mammals (e.g., humans) having a LTBI are provided.

The present invention discloses a polypeptide with tubercle bacillus immunogenicity, which contains a sequence shown by Seq ID No.2 in the sequence table. The present invention also discloses an ELISPOT method, which utilizes the polypeptide to detect if a biological sample is infected with tubercle bacillus, and an agent box containing the polypeptide. When the polypeptide, the method and the agent box are adopted to detect the infectivity of tubercle bacillus, the false positive rate is greatly decreased, while the true positive rate can be kept unchanged.

The present invention relates to Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof, wherein the three Mycobacterium tuberculosis latent infection related proteins are LTBIPr1, LTBIPr2 and LTBIPr3, and the nucleotide sequences of the protein epitope are represented by the sequences 1-3 in the sequence table. According to the present invention, the genetic engineering technology is adopted to clone, express and purify Mycobacterium tuberculosis to obtain the three protein antigens related with Mycobacterium tuberculosis latent infection; the protein antigen is adopted as the tuberculosis specific cell stimulating agent to stimulate the peripheral blood mononuclear T-lymphocyte of a subject so as to make the T-lymphocyte secrete specific IFN-gamma, and then the spot number of the T-lymphocyte secreting the IFN-gamma is detected through the ELISpot technology, wherein the whole process takes two days; and according to the generated blue-purple spot number, the result is read with the naked eye or instrument, such that the Mycobacterium tuberculosis latent infection related proteins can be used for detection of whether a subject is infected with tubercle bacillus, and active tuberculosis assisted diagnosis and differential diagnosis.

Disclosed are compositions and methods for detecting, isolating, and/or characterizing a T cell or autoantibody associated with type I diabetes. The composition and methods comprise the use of a hybrid insulin peptide having an N-terminal amino acid sequence taken from the human insulin peptide and a C-terminal amino acid sequence taken from a secretory granule protein that are joined through a peptide bond to form an autoimmune antigen. The detecting, isolating and characterization step further includes performing an immunoassay and/or T cell proliferation assay with the disclosed hybrid insulin peptides, where preferably, the immunoassay is an ELISPOT assay.

The invention discloses a tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL (Cytotoxic T Lymphocyte) epitope peptide which is a nonapeptide, wherein the amino acid sequence of the nonapeptide is P10: MLIAGLPCL. According to the tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL epitope peptide, immunological informatics means are adopted according to a primary structure of an antigen, an HLA-A*0201-restrictive CTL epitope of a tuberculosis-resisting-drug-related protein antigen is subjected to predictive analysis by using databases, namely SYFPEITHI, BIMAS and NetCTL1.2, the epitope peptide is obtained through screening, and assayed nonapeptides are not reported in documents. The epitope peptide is assayed through in-vitro ELISPOT experiments, and the results provide a theoretical basis for the development of tuberculosis vaccines based on resisting-drug-associated protein antigens and provide more information for designing tuberculosis multi-epitope peptide vaccines based on mixed T cell epitopes.

An ELISpot diagnosis kit for NMO and its application are characterized in that, the polypeptide fragment specific to NMO effector T-cell is obtained through topological conformation analysis of aquaporin-4 (AQP-4), followed by the structural analyses of the related polypeptides after combination and rearrangement so as to screen out the brand-new polypeptide fragment suitable for NMO disease diagnosis. In the ELISpot experiment, by utilizing the obtained polypeptide fragment, stimulate the effector T-cell in the NMO disease to secrete IL-4, proving the feasibility and scientific value of that polypeptide fragment in the NMO diagnosis. This method is with strong specificity, high sensitivity and easy operations, and it can be developed into the diagnosis kit for the diagnosis and differential diagnosis of NMO in the clinical examination, laying the foundation for the early discovery and treatment of NMO.

The invention relates to a method for screening point mutation BIRC5 antigen epitope peptides, and the method comprises the following specific steps: 1), comprehensively predicting CTL epitope peptides of BIRC5 antigen 9 amino acid residues by using NetMHCpan 4.1 and SYFPEITHI on-line prediction systems, and changing the types of corresponding amino acid residues through a point mutation technology to improve the score of on-line prediction; 2) according to the amino acid sequence obtained in the step 1), artificially synthesizing a corresponding epitope peptide, co-incubating the epitope peptide and T2 cells, and detecting the actual binding capacity of the epitope peptide and HLA-A2 molecules; 3) establishing specific CTLs cloning in vitro by utilizing the mutant peptide obtained by screening in the step (2), and identifying the immunological activity of the mutant peptide induced CTLs for specifically recognizing and killing target cells by utilizing ELISPOT experiment, calcein killing and other methods. The invention lays a foundation for further preparation of tumor peptide vaccines.