12 results about "ELISPOT" patented technology

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 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.

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.

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 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 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 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.

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 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.