52 results about "ELISPOT" patented technology

With the goal of creating a combination vaccine against Shigella and other diarrheal pathogens we have constructed a prototype vaccine strain of Shigella flexneri 2a (SC608) that can serve as a vector for the expression and delivery of heterologous antigens to the mucosal immune system. SC608 is an asd derivative of SC602, a well-characterized vaccine strain, which has recently undergone several phase 1 and 2 trials for safety and immunogenicity. Using non-antibiotic asd-based plasmids, we have created novel constructs for the expression of antigens from enterotoxigenic E. coli (ETEC), including CFA / I (CfaB and CfaE) and the B-subunit from heat-labile enterotoxin (LTB) in Shigella vaccine strain SC608. Heterologous protein expression levels and cellular localization are critical to immune recognition and have been verified by immunoblot analysis. Following intranasal immunization (SC608(CFAI) and SC608(CFAI / LTB) of guinea pigs, serum IgG and IgA immune responses to both the Shigella LPS and ETEC antigens can be detected by ELISA. In addition, ELISPOT analysis for ASCs from cervical lymph nodes and spleen showed similar responses. All vaccine strains conferred high levels of protection against challenge with wild-type S. flexneri 2a using the Sereny test. Furthermore, serum from guinea pigs immunized with SC608 expressing CfaB and LTB contained antibodies capable of neutralizing the cytological affects of heat-labile toxin (HLT) on Chinese Hamster Ovary (CHO) cells. These initial experiments demonstrate the validity of a multivalent invasive Shigella strain that can serve as a vector for the delivery of pathogen-derived antigens.

The invention discloses a tuberculosis medicament resistance related tuberculosis-resisting cytotoxic T lymphocyte (CTL) epitope peptide derived from refflux protein, namely, nonapeptide. The amino acid sequence of the nonapeptide is as follows: P5: YLGGTTGPV, or P6: YIVGFCLLV, or P7: TLTWLFAFV, or P8: GLVAGLSAV, or P9: ALGMLIAGL, or P10: MLIAGLPCL, or P11: LLCAIFAEV, or P12: RLWPTVGCL. Accordingto the invention, the HLA-A*0201 restrictive CTL epitope of a tuberculosis medicament resistance related protein antigen is predicted and analyzed by applying SYFPEITHI, BIMAS and NetCTL1.2 databasesand using an immunoinformatics mean according to a primary structure of the antigen so that the epitope peptide is obtained by virtue of selection, and the identified nonapeptide is not reported in documents. The epitope peptide is identified through an in-vitro enzyme linked immunospot (ELISPOT) experiment; and according to the result, a theoretical basis is provided for developing tuberculosis vaccine based on the medicament resistance related protein antigen and more information is provided for designing a tuberculosis polyepitope peptide vaccine based on mixed T cell epitope.

The invention belongs to the technical field of biology, and particularly relates to screening and identification of antigen epitope polypeptides. The invention discloses screening, identification and application of a series of rabies virus glycoprotein and nucleoprotein antigen epitope polypeptides. The rabies virus glycoprotein and nucleoprotein are predicted by biological information means to obtain the candidate epitope polypeptides; and a lymphopoiesis experiment, ELISPOT experiment and a stream-type cell method are utilized to carry out in-vitro experimental verification on the subsequent epitope polypeptides to obtain the four rabies virus protein antigen epitope polypeptides. The invention is characterized in that the antigen epitope polypeptides respectively comprise a Th epitope and a CTL epitope, can stimulate the lymphopoiesis of the vaccine-immunized mouse in vitro and induce the cells to secrete related cell factors, and have the functions of killing virus-infected cells and stimulating the generation of the antibody. The invention can be used for developing rabies virus epitope vaccines and detecting the vaccine effect, and has important value for developing and producing immunologic function detection kits for rabies virus vaccines.

With the goal of creating a combination vaccine against Shigella and other diarrheal pathogens we have constructed a prototype vaccine strain of Shigella flexneri 2a (SC608) that can serve as a vector for the expression and delivery of heterologous antigens to the mucosal immune system. SC608 is an asd derivative of SC602, a well-characterized vaccine strain, which has recently undergone several phase 1 and 2 trials for safety and immunogenicity. Using non-antibiotic asd-based plasmids, we have created novel constructs for the expression of antigens from enterotoxigenic E. coli (ETEC), including CFA / I (CfaB and CfaE) and the B-subunit from heat-labile enterotoxin (LTB) in Shigella vaccine strain SC608. Heterologous protein expression levels and cellular localization are critical to immune recognition and have been verified by immunoblot analysis. Following intranasal immunization (SC608(CFAI) and SC608(CFAI / LTB) of guinea pigs, serum IgG and IgA immune responses to both the Shigella LPS and ETEC antigens can be detected by ELISA. In addition, ELISPOT analysis for ASCs from cervical lymph nodes and spleen showed similar responses. All vaccine strains conferred high levels of protection against challenge with wild-type S. flexneri 2a using the Sereny test. Furthermore, serum from guinea pigs immunized with SC608 expressing CfaB and LTB contained antibodies capable of neutralizing the cytological affects of heat-labile toxin (HLT) on Chinese Hamster Ovary (CHO) cells. These initial experiments demonstrate the validity of a multivalent invasive Shigella strain that can serve as a vector for the delivery of pathogen-derived antigens.

The invention discloses a precise localization method for analysis of Elispot by using image identification. The method mainly comprises the steps of transforming grey level value of an original micro-well plate single-pore coloured image; detecting the flange of the grey level image; searching for pore bottom circle of the flange detection image; carrying out pore bottom trimming for an original whole origin image. According to the method disclosed by the invention, the image characteristic of an Elispot analysis instrument is combined, the pore bottom circumference searching is conducted based on the micro-well plate based on flange detected image, the problem on precise trimming of the micro-well plate pore bottom image is solved, and the demands on positioning of a motor and manufacture precision of the micro-well plates are lowered. The method of the invention can be used for image analysis of an Elispot reader and also for similar target area positioning based on the micro-well plate image analysis.

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-γ) and chemokines that upregulate the activity of Th-1 cytokines (such as IFN-γ). In a typical embodiment, detection of the chemokine monokine induced by gamma interferon (MIG) provides a measure of the biological effect of IFN-γ rather than direct quantitation of IFN-γ or IFN-γ 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-γ. Responses as assessed by the MIG assay paralleled those detected by conventional IFN-γ 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-γ activity or MIG.

The invention belongs to the technical field of biology and particularly relates to a method for preparing tetramer from Asia I foot and mouth disease virus polypeptide. According to the designed foot and mouth disease virus polypeptide, the foot and mouth disease virus polypeptide is screened and identified by the ELISPOT method and the tetramer construction method, pouch pig SLA-2 heavy-chain tetramer is constructed and expressed in a prokaryotic expression system pET-21a(+) / BL21, and an inclusion body is extracted. Polypeptide capable of causing specificity CTL reaction is screened from the foot and mouth disease virus polypeptide through the ELISPOT method, the polypeptide and SLA-2 heavy-chain and known light-chain beta2m are subjected to renaturation and biotinylation, the polypeptide and FITC labeled-streptavidin react to generate SLA-2-BSP / tetramer, and then the function of tetramer is detected through flow cytometry. A specificity peptide epitope of a pig Asia I foot and mouth disease virus VP1 is obtained through identification through the ELISPOT method, the tetramer is successfully constructed, the specificity CTL of the tetramer is detected, a pig tetramer technical platform is primarily built, and a foundation is laid for researching pig specificity CTL immune response and T epitope screening.

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 P8: GLVAGLSAV. 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.

A method of quantifying analyte secreted by a cell or released from a drug delivery vehicle, typically by ELISpot or fluorispot assay is described. Quantification is possible through the use of an analyte-releasing reagent that includes a bead and the analyte releasably bound to the bead, or a container pre-spotted with analyte released from the reagent. The reagent or pre-spotted containers can be used to provide a standard curve for release of the analyte. By detecting analyte secreted by one or more cells or drug released by a drug delivery vehicle, and comparing the detected analyte to the standard curve, it is possible to quantify the amount of analyte released by the one or more cells or drug released by the drug delivery vehicle. Kits and reagents for practicing the methods of the present invention are also disclosed.

The invention relates to hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof. An HLA-A33 restrictive cell level epitope peptide screening platform is constructed; and 5 important HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) are determined by combining computer simulation analysis, detecting on the cell level epitope peptide screening platform and using three detection technologies of trimerical protein level renaturation, tetrameric patient's blood sample detection and Elispot detection. The invention also discloses the application of the screened HBV specific HLA-A33 restrictive epitope peptides in preparation of a medicament for treating hepatitis B in examinees, and specifically, the hepatitis B of the examinees is of HLA-A33 type. The invention also provides a medicament for treating the HLA-A33 hepatitis B in the examinees, and the medicament contains one or more of the 5 HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) and a medicinal carrier.

The invention belongs to the technical field of microbial immunity, and particularly relates to dominant epitope peptide of Ebola virus envelope glycoprotein as well as a coding gene and application thereof. The epitope peptide is composed of sequences as shown in SEQ ID NO.1-25. According to the dominant epitope peptide of Ebola virus envelope glycoprotein as well as the coding gene and application thereof, potential epitopes derived from EBOV GP are predicted and verified through an ELISpot test, protection properties of the potential epitopes in different isolates around the world are subjected to a variety of deep analyses, so that screened dominant isolates are verified. The binding affinity and immunogenicity of the epitopes and proper sites of respective MHC class I molecules are further predicted, which is finally helpful for understanding the immunobiology of the EBOV GP and perfecting the epitope vaccine design for virus in the future.

The invention discloses a polypeptide. An amino acid sequence of the polypeptide is shown as SEQ ID No. (sequence identifier number) 1 or 2. The invention further provides a drug composition, food ora health care product containing the polypeptide and a corresponding application of the polypeptide. An epitope peptide is obtained through screening and identified by in-vitro ELISPOT (enzyme-linkedimmunospot assay), a cell killing experiment and the like; a theoretical basis is provided for subsequent development of a tuberculosis vaccine and a diagnostic agent based on a drug-resistant mutation antigen; and more options are provided for a design of a multi-epitope tuberculosis vaccine based on a mixed T cell epitope.

The invention discloses an O-type foot-and-mouth disease CTL epitope peptide, a screening method and application thereof. The CTL epitope peptide is composed of nine amino acid residues, and its amino acid sequence is: Ala-Thr-Arg-Val-Thr-Glu-? Leu-Leu-Tyr. The epitope peptide can have strong binding ability with SLA-I proteins derived from different strains of pigs and can cause cytotoxic immune response, and is suitable for the preparation of vaccines for the prevention and treatment of various strains of porcine foot-and-mouth disease viruses, and has a wide range of applications. The invention uses the constructed six-strain pig SLA-I single-chain molecule to bind the CTL mimetic epitope peptide of foot-and-mouth disease virus in vitro, mass spectrometry to screen the polypeptide that can bind to the complex, and through ELISPOT detection, it is determined that it can induce T cell immune response Capable mimotope peptides. The invention provides a method for screening and identifying a large number of CTL epitopes of foot-and-mouth disease virus in the future, and lays a foundation for developing a porcine foot-and-mouth disease multi-epitope vaccine.