26 results about "ELISPOT" patented technology

The invention provides an applicable population screening method and application of a glioma dry-like cell sensitized DC vaccine, peripheral blood of a patient before and after inoculation of the glioma dry-like cell sensitized DC vaccine is obtained, mononuclear cells are extracted, CD8 + T cells are respectively separated from the mononuclear cells through a magnetic bead sorting method, and the CD8 + T cells are separated from the mononuclear cells. The method comprises the following steps: uniformly inoculating CD8 < + > T cells into an ELISPOT cell culture plate coated with an IFN-gamma antibody for culture, respectively setting an experimental group and a positive control group for culture, adding A2B5 < + > glioma cell lysis buffer into the experimental group and adding phytohemagglutinin into the positive control group, and carrying out ELISPOT staining on the cultured cells to obtain the secretion amount of FN-gamma before and after treatment of a patient. According to the secretion amount of the FN-gamma, patients with specific immune response to the glioma dry-like cell sensitization DC vaccine are screened, so that the glioma dry-like cell sensitization DC vaccine is conveniently popularized and applied, and the immune mechanism of the glioma dry-like cell sensitization DC vaccine is researched.

The invention discloses an African swine fever virus T cell epitope peptide for inducing swine T cell immune response and a composition of the African swine fever virus T cell epitope peptide. The amino acid sequence of the African swine fever virus T cell epitope peptide is shown as any one of SEQ ID NO.1-19. Or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1-19 and maintains the protein function shown in SEQ ID NO.1-19 through substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1-19; the composition comprises amino acids of which the sequences are SEQ ID NO.1-19. A group of ASFV key T cell antigens and epitope peptides are obtained by combining IFN-gamma ELISpot and flow cytometry screening, the ASFV key T cell antigens and epitope peptides can be used as vaccine components to induce strong T cell immune response, and T cells activated by the antigen peptides have the maximum immune protection potential and are essential key antigens for ASFV vaccine research and development.

The invention provides a polypeptide for activating cellular immunity in chronic hepatitis B and application of the polypeptide, and belongs to the technical field of biological medicine. A polypeptide library is designed and synthesized on the basis of a preS1 structural domain for coding HBV large HBsAg, a full-length core protein Core, a polymerase protein fragment rich in T cell epitopes and an mRNA-PreS1CPX holoantigen sequence (as shown in SEQ ID NO.1) of full-length X protein HBX, and peptide fragments capable of activating T cell immunity are screened by utilizing ELISPOT and flow cytometry. Experimental results show that the polypeptide sequences as shown in SEQ ID NO.2-15 can promote HBV antigen specific immune response by stimulating CD8 + T lymphocytes to secrete IFN-gamma, so that immune activation treatment of hepatitis B is realized.

The invention relates to the technical field of biology, and discloses a restrictive epitope peptide of which the amino acid sequence is shown as SEQ ID NO.1. According to the invention, chicken MHC I-BF2 * 1901 restrictive epitopes are screened through an in-vivo capture elution experiment, an epitope polypeptide of H9N2AIV is further determined by combining with a BF2 * 1901 binding motif and a binding force test, and then the immunogenicity of candidate polypeptides is detected through an in-vitro ELISpot test, so that the immunogenicity of the H9N2AIV is detected. Finally, the restrictive epitope peptide is determined to be a polypeptide epitope with remarkable immunogenicity, and in addition, the invention further discloses a screening method and application of the restrictive epitope peptide and a vaccine.

The invention discloses an influenza B virus T3 peptide fragment capable of inducing cellular immune response and application of the influenza B virus T3 peptide fragment, and belongs to the technical field of immunotherapy and immunodetection. According to the invention, detection is carried out through ELISpot, and the immunogenicity of the long peptide (SEQ ID NO.1, named as T3) is verified in 21 volunteers. The result shows that the T3 is detected to be positive in four persons, and the positive rate is 19.05%; the IFN-gamma secretory cells of the human PBMCs are detected through ELISpot, and the immune response induced by the T3 in people can reach up to 2870 SFC / 106 PBMC. Therefore, the polypeptide has good immunogenicity and crowd universality, can be used as a specific detection peptide for monitoring the cellular immune level, can effectively activate the T cell immune response of the crowd, and has the potential of being used as a candidate vaccine epitope peptide to be applied to novel vaccine design.

This invention relates to the field of genetic engineering technology, and in particular to a low-immunogenic SaCas9 protein mutant and its applications. The SaCas9 protein mutant of this invention involves one or more mutations among Q433H, K426W, and K297D in the wild-type SaCas9 protein. The SaCas9 protein mutant of this invention significantly reduces the number of IFN-γ spots induced in ELISpot experiments compared to the wild-type, effectively reducing humoral and cellular immune responses, lowering the immunogenicity of SaCas9, making it more suitable for gene editing, and providing technical support for developing safer CRISPR-based gene editing. The gene editing efficiency of the SaCas9 protein mutant provided by this invention is comparable to that of the wild-type, and it does not affect the nucleic acid recognition and cleavage function of SaCas9.

A method, apparatus and computer program product to provide machine learning-assisted detection of objects of interest in live cell-based assays, where the objects of interest are larger than cells used in the assay. The technique herein comprises receiving image data that has been captured from a set of imaging devices, such as immune monitoring analyzer machines. Representative image data is an enzyme-linked immune absorbent spot (ELISPOT) assay captured from an ELISPOT analyzer. For each set of image data, the image data is then processed using, for example, one of: (a) a first pre-trained model; and (b) a set of one or more detection algorithms, to generate training data comprising a set of labels for the image data. A model, e.g., a deep neural network (DNN), is then trained using the set of labels. Following training, the model is provided for detection of the objects of interest.

The invention provides an ELISPOT (Enzyme-Linked Immunospot Assay) analysis method for detecting IFN-gamma and IL-2 secretion, which comprises the following steps: activating an ELISPOT plate, washing, adding IFN-gamma and IL-2 capture antibody coating with specific concentration, and staying overnight at 4 DEG C; recovering and counting target sample cells, resuspending the target sample cells according to a certain concentration, and adding the target sample cells into coated plate holes; incubating for more than 16 hours in an environment of 37 DEG C and 5% COC; washing the incubated plate, adding IFN-HRP and IL-2-ALP detection antibodies, and incubating at room temperature for 2 hours; after washing, adding a blue developing solution for developing for 10-15 minutes; adding a red developing solution, incubating for 10-15 minutes, and stopping developing; according to the method, T cell subset identification and multifunctional analysis can be carried out, in the early stage of vaccine research and development, due to the fact that target positive population is small, the number of obtained PBMC cells is small, two-factor detection can detect different cell factor reaction frequencies and locate different cell populations and subtypes at the same time, and comparison of two-factor data is beneficial to finding out population immune differences; and scientific research and vaccine development are promoted.

A rapid, comprehensive and sensitive method for neoantigen screening from recurrent cancer mutations comprising: (A) STEP 1: selecting recurrent cancer mutations dividing into three tiers based on ranking features; (B) STEP 2: Generating a library of artificial antigen presenting cells (aAPC) co-expressing HLA types (including a list of 13 most frequent HLA class I types covering 66% of Asian population or 67% of Vietnamese population, and a list of 04 most frequent HLA class II types covering 41% Asian population or 50% of Vietnamese population) and a co-stimulatory molecule; and (C) STEP 3: Using the library of aAPC at step (B) for rapid and comprehensive screening for selecting of immunogenic peptides (neoantigens) from the off-the-shelf peptides at step (A) activate T cells via immunological assays (ELISpot or intracellular staining flow cytometry).

The present invention discloses a method for detecting mRNA-specific immune activation, which comprises the following steps: preparing mRNA, including preparing a linearized template, in vitro transcription, capping, removing the linearized template, and purifying mRNA; preparing iDC cells, transfecting the iDC cells, and loading the mRNA onto the DC cells by electroporation or chemical transfection; inducing mDC from iDC, culturing, and plating for the Elispot assay; performing Elispot staining and detection. The present invention mainly designs a method for directly detecting mRNA-specific immune activation by Elispot. The method of the present invention can directly detect mRNA-specific immune activation, and has the advantages and characteristics of short detection time, high convenience, less consumption of experimental cells, and low detection cost, and can be well applied to detecting the effect of mRNA vaccine-specific immune activation.

A rapid, comprehensive and sensitive method for neoantigen screening from recurrent cancer mutations comprising: (A) STEP 1: selecting recurrent cancer mutations dividing into three tiers based on ranking features; (B) STEP 2: Generating a library of artificial antigen presenting cells (aAPC) co-expressing HLA types (including a list of 13 most frequent HLA class I types covering 66% of Asian population or 67% of Vietnamese population, and a list of 04 most frequent HLA class II types covering 41% Asian population or 50% of Vietnamese population) and a co-stimulatory molecule; and (C) STEP 3: Using the library of aAPC at step (B) for rapid and comprehensive screening for selecting of immunogenic peptides (neoantigens) from the off-the-shelf peptides at step (A) activate T cells via immunological assays (ELISpot or intracellular staining flow cytometry).

This invention discloses a type B influenza virus T4 peptide that can induce cellular immune responses and its applications, belonging to the fields of immunotherapy and immunoassay technology. The immunogenicity of this long peptide (SEQ ID NO.1, named T4) was verified in 21 volunteers using ELISpot assay. Results showed that T4 was positive in 5 individuals, with a positive rate of 23.81%. ELISpot assay of human PBMCs (IFN-γ secretory cells) showed that T4 induced an immune response of up to 3060 SFCs / 10 in the population. 6 PBMC. Therefore, this peptide has good immunogenicity and universality in the population, and can be used as a specific detection peptide for monitoring cellular immune levels. At the same time, this peptide can effectively activate the T cell immune response in the population, and has the potential to be used as a candidate vaccine epitope peptide for the design of novel vaccines.

The present application relates to a kind of detection method and kit of cancer cell specific T cell, T cell in peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte is co-incubated with the particle activated antigen presenting cell prepared by the particle loaded whole cell component to activate the cancer cell specific T cell that can recognize and kill cancer cell, then using ELISPOT, flow cytometry, ELISA and other techniques detect and analyze the number and proportion of activated cancer cell specific T cell, further evaluate the strength of cancer cell specific immunity in patient body.The present application overcomes the difficulty that currently cannot effectively screen specific T cell of broad spectrum and polyclonal in peripheral blood, peripheral immune organ or tumor infiltrating lymphocyte, can detect broad spectrum of specific T cell of effector cell specific T cell with specific killing function from cell, and with the characteristics of easy separation and high specificity, can be used as effective biomarker.

The present application relates to the field of immunodetection technology, and particularly relates to a method for enriching and detecting antigen-specific T cells. The method for enriching antigen-specific T cells comprises the following steps: stimulating a whole blood sample with a specific antigen, and then sorting T cells in the whole blood sample stimulated by the antigen by using an immunomagnetic bead sorting technology to obtain a T cell sample. The method is based on the strategy of stimulating the antigen first and then sorting the cells, and can quickly and accurately separate T cells from a complex cell system of whole blood, effectively avoiding the complicated operation of traditional sorting methods, realizing automatic sorting, simplifying the operation steps, shortening the sorting time, improving the sorting throughput, and better ensuring the accuracy of subsequent cell detection. The T cells obtained by the above enrichment method can be detected by using an ELISPOT method, and the antigen-specific T cells can be accurately detected.

The present invention relates to a method for assaying antigen-specific T-cell responses through boosted flow cytometry and boosted ELISPOT technologies that are based on the use of specific combinations of monoclonal antibodies to stain simultaneously cytokines that in their combination identify specific T-cell polarization profiles. The method allows the identification of Tfc CD8+ T cell responses unrecognized so far.

The invention belongs to the field of biological medicine, and relates to immunology and virology. Specifically, the invention relates to a method for screening antiviral drugs, in particular to a method for screening anti-influenza virus drugs. Specifically, the invention relates to a method for screening antiviral drugs, which comprises the following steps: (1) culturing host cells; (2) infecting host cells with the virus, and culturing under conditions suitable for virus and cell growth; (3) adding a covering solution and a candidate drug with a proper concentration, and continuing to culture, the covering solution comprising microcrystalline cellulose, a cell culture medium and TPCK pancreatin; (4) detecting virus antigens through ELISA (enzyme-linked immuno sorbent assay), and reading original data by using an ELISPOT instrument; (5) calculating IC50 according to the original data; and (6) selecting the candidate drugs according to the IC50 value. The method is rapid, simple, accurate and reliable, and has a good application prospect.

The invention discloses an influenza B virus T2 peptide fragment capable of inducing cellular immune response and application of the influenza B virus T2 peptide fragment, and belongs to the technical field of immunotherapy and immunodetection. Verification is carried out through ELISpot, T2 is detected to be positive in six persons, the positive rate is 28.57%, S-T2 is detected to be positive in four persons, and the positive rate is 19.05%; the IFN-gamma secretory cells of the human PBMCs are detected through ELISpot, the highest immune response induced by T2 in the HLA-A2 population can reach 890 SFC / 106 PBMC, and the highest immune response induced by S-T2 in the HLA-A2 population can reach 650 SFC / 106 PBMC. Therefore, T2 and S-T2 can stably induce human T cells to generate immune response, can be used for monitoring the cellular immune level, can effectively activate the T cell immune response of people, and has the potential of being used as candidate vaccine epitope peptide to be applied to novel vaccine design.

The invention discloses a method for detecting the infectivity of oncolytic viruses in urine and swab preserving fluid based on an ELISpot technology, and relates to the technical field of detection of the infectivity of the oncolytic viruses in the urine and swab preserving fluid, and the specific method comprises the following steps: firstly, placing a proper amount of cells in a transparent cell culture plate for overnight culture, then transferring a sample into a cell plate, and carrying out ELISA detection on the cell plate; culturing for a period of time. An indirect immunofluorescence method is established by adopting an immunofluorescence technology and taking a rabbit anti-Vaccinia Virus Polyclonal Antibody polyclonal antibody as a primary antibody and an Alexa FluorR 488 labeled goat anti-rabbit antibody as a secondary antibody, and the virus infectivity is judged according to the quantity of fluorescent cells infected by the virus. The invention fills the technical blank in the field of detecting the infectivity of the oncolytic virus by using the ELISpot technology, is accurate in detection and high in sensitivity, and has important significance for new drug research and development of oncolytic virus drugs.

The invention discloses an influenza B virus T1 peptide fragment capable of inducing cellular immune response and application of the influenza B virus T1 peptide fragment, and belongs to the technical field of immunotherapy and immunodetection. Immunogenicity verification is carried out through ELISpot, T1 is detected to be positive in seven persons, the positive rate is 33.33%, S-T1 is detected to be positive in four persons, and the positive rate is 19.05%; the IFN-gamma secretory cells of the human PBMCs are detected through ELISpot, the immune response induced by the T1 in the HLA-A2 population can be up to 4160 SFC / 106 PBMC at most, and the S-T1 can be up to 990 SFC / 106 PBMC at most. Therefore, T1 and S-T1 can stably induce human T cells to generate immune response, can be used for monitoring the cellular immune level, can effectively activate the T cell immune response of people, and have the potential of being used as candidate vaccine epitope peptides to be applied to novel vaccine design.

The invention discloses a method and a kit for evaluating treatment response of a circular mRNA (messenger Ribonucleic Acid) drug. The method and the kit are used for solving the problem that individualized administration of the drug cannot be realized through blood concentration monitoring. According to the method, the traditional thought is abandoned, and the curative effect is evaluated by detecting the functional immune response induced by the medicine. The method specifically comprises the step of quantitatively detecting the level of T cells capable of recognizing the antigen in peripheral blood by using an MHC polymer or ELISpot detection plate specifically bound with the antigen peptide 7-14 days after a patient is administered with the annular mRNA encoding the specific antigen SEQ ID NO: 1. By comparing the detection result with a preset threshold value, a basis is provided for clinically adjusting a subsequent administration scheme. According to the invention, paradigm conversion from pharmacokinetics to pharmacokinetics monitoring is realized, and a key tool is provided for precise treatment of novel drugs such as annular mRNA. The corresponding kit comprises the specific detection component and is convenient for clinical standardized application.

The present disclosure describes methods and devices for detecting SARS-CoV-2 specific T cell interferon gamma activation. By using a microfluidic chip to allow ELISpot Interferon Gamma Release Assay (IGRA) to be performed using a small amount of a whole blood sample collected from a fingertip, results can be rapidly provided in a small amount of a blood sample.

This invention discloses a tetanus vaccine and its enzyme-linked immunosorbent assay (ELISpot) analysis method. The tetanus vaccine of this invention is designed based on screened tetanus antigen peptides, which contain any of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:61. Addressing the technical problem of the lack of specific T-cell immune response detection methods for existing tetanus vaccines, this invention designs an overlapping peptide library covering the entire sequence of tetanus toxoid, screening out dominant peptides that can effectively stimulate T-cell responses, significantly improving the sensitivity and specificity of detection. This method optimizes the processing flow of PBMCs in the test samples, establishes a quantitative analysis standard based on IFN-γ secretion, and achieves accurate assessment of the T-cell immune response induced by the tetanus vaccine.

The application relates to the technical field of biology, and discloses a restrictive epitope peptide, the amino acid sequence of which is shown as SEQ ID NO:1. The application screens a chicken MHC I-BF2*1901 restrictive epitope through in-vivo capture elution experiment, combines BF2*1901 binding motifs and binding force test, further determines an epitope polypeptide of H9N2 AIV, and then detects immunogenicity of a candidate polypeptide through in-vitro ELISpot test, so that the polypeptide epitope with significant immunogenicity is finally determined. In addition, the application also discloses a screening method and use of the restrictive epitope peptide and a vaccine.

Screening methods are provided that are based on using ELISpot assays to measure the distribution profile of maximum spot intensity or mean spot intensity of spontaneous cytokine or chemokine or the number of "low intensity" spontaneous cytokine or chemokine producing cells as a clinically relevant measure of immune function for diagnosing patients with a particular disorder based on one or more of these measurements and for treating the particular disorder in the patient. In some embodiments, the cytokine is IFN-gamma or TNF-alpha. Patients can optionally be further evaluated to determine whether they are of an immunosuppressed or hyperinflammatory immune phenotype by quantifying cytokine and / or chemokine levels in a biological sample. Representative disorders include sepsis, autoimmune disease, autoimmunity, cancer, and lymphopenia.

This invention discloses an African swine fever virus (ASFV) T-cell epitope peptide and its composition for inducing porcine T-cell immune responses. The amino acid sequence is as shown in any of SEQ ID NO. 1-19, or an amino acid sequence derived from SEQ ID NO. 1-19 but retaining the protein function shown in SEQ ID NO. 1-19, through substitution and / or deletion and / or addition of one or more amino acid residues. The composition comprises amino acids with the sequences of SEQ ID NO. 1-19. This invention, combined with IFN-γ ELISpot and flow cytometry screening, yielded a group of key ASFV T-cell antigens and epitope peptides, which can be used as vaccine components to induce strong T-cell immune responses. The T cells activated by these antigenic peptides have the greatest immunoprotective potential and are essential key antigens for ASFV vaccine development.

This invention provides an ELISpot kit for detecting T-cell immunity in porcine epidemic diarrhea (PEDD) and its applications, belonging to the field of in vitro diagnostic technology. The hybridoma cell line 6E8-5G5, which secretes anti-porcine interferon-gamma monoclonal antibodies, can be used to prepare high-purity anti-porcine interferon-gamma monoclonal antibodies stably and in large quantities over a long period. Furthermore, an enzyme-linked immunospot (ELISpot) method based on PEDV N protein-specific IFN-γ is established. By detecting the level of specific T-cell immune responses against PEDV N protein after antigen stimulation, this provides technical support for the assessment of cellular immunity after PEDV infection or vaccine immunization.