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Soluble liver antigen T-cell antigen epitope and detection kit prepared therefrom

A cell antigen, soluble technology, applied in the field of immunology, can solve the problems of no research, no environmental protection, increased experimental costs, etc., and achieve the effect of reducing misdiagnosis and mistreatment

Inactive Publication Date: 2011-12-28
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] This scheme has the following disadvantages: 1. The test time is long, which increases the probability of false positive contamination; 2. The amount of cells used is large, which increases the cost of the experiment; 3. The repeatability of the experiment is not reflected in the experiment process; 4. The experiment cannot simultaneously prompt the cells Cytokine secretion after stimulation; 5. Radioactive isotopes are required in the experiment, which is not environmentally friendly; 6. Poor sensitivity
[0014] There is no domestic research on T cell immunity against the target antigen of SLA disease
[0015] 4. The sensitivity of the method used to observe the function of T cells is poor

Method used

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  • Soluble liver antigen T-cell antigen epitope and detection kit prepared therefrom
  • Soluble liver antigen T-cell antigen epitope and detection kit prepared therefrom
  • Soluble liver antigen T-cell antigen epitope and detection kit prepared therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Preparation of SLA Synthetic Peptides

[0030] The SLA sequence peptide was synthesized by overlapping peptide technology, and the SLA protein gene sequence and its corresponding SLA protein amino acid sequence (NP722547, 441 amino acids) were retrieved from the human gene library. Design of overlapping peptides: each peptide is 20 amino acids long and overlaps 12 amino acids. A total of 54 SLA sequence peptides were synthesized, and the purity of the synthesized peptides was >75%. The amino acid sequences of each peptide are shown in Table 1.

[0031] Table 1

[0032] No.

amino acid position

peptide sequence

1

1-20

MDSNNFLGNCGVGEREGRVA

2

9-28

NCGVGEREGRVASALVARRH

3

17-36

GRVASALVARRHYRFIHGIG

4

25-44

ARRHYRFIHGIGRSGDISAV

5

33-52

HGIGRSGDISAVQPKAAGSS

6

41-60

ISAVQPKAAGSSLLNKITNS

7

49-68

AGSSLLNKITNSLVLDIIKL

8

...

Embodiment 2

[0056] Example 2 Preparation of a kit for detecting autoimmune hepatitis

[0057] Materials required for kit preparation:

[0058] 96-well PVDF membrane-covered ELISPOT plate

[0059] 1. IFN-γ coated antibody

[0060] 2. Biotin-labeled IFN-γ detection antibody

[0061] 3. SLA peptide combination (final concentration 10μg / ml)

[0062] Consists of SEQ ID No.1 to SEQ ID No.7. The specific embodiment of the present invention is the best embodiment of the present invention, the kit is composed of 7 peptide segments (SEQ ID No.1 to SEQ ID No.7), one or more of the 7 peptide segments of the present invention ( The combination of non-seven peptides can also achieve the final detection result, but compared with the combination of seven peptides, the experimental reagents and the amount of isolated whole blood need to be increased. Therefore, this embodiment is the best implementation mode, and the present invention does not subject to this limitation.

[0063] 4. Avidin-labeled a...

Embodiment 3

[0068] Embodiment 3 detection experiment

[0069] (1) Using the mixed seven peptides (SEQ ID No.1 to SEQ ID No.7) as a stimulus to stimulate peripheral blood PBMCs to secrete IFN-Γ;

[0070] (2) Detection by ELISPOT method. The specific steps of the ELISPOT detection here are exactly the same as those in Example 1.

[0071] Detection object:

[0072] 41 Cases of Autoimmune Hepatitis

[0073] 20 patients with primary biliary cirrhosis

[0074] Patients with viral hepatitis, including 10 cases of hepatitis C and 20 cases of hepatitis B

[0075] 20 cases of normal control

[0076] Test results:

[0077] Among the 41 AIH patients, 22 (53.66%) were positive for SLA epitope mixed peptide stimulation;

[0078] One of the 20 PBC patients (5%) showed a weak positive reaction to the mixed peptide of the SLA epitope;

[0079] None of the patients with hepatitis B or C responded to the stimulation of the SLA epitope pool;

[0080] None of the normal controls responded to stimulat...

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Abstract

The invention discloses a soluble liver antigen T cell antigen epitope of autoimmune hepatitis, the amino acid sequence of which is as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No. Shown in ID No.5, SEQ ID No.6 and SEQ ID No.7. The recognition and determination of the T cell antigen epitope expands the cognition angle of this disease; one or more combinations of the antigen epitope can be used for the application of preparing the kit for detecting autoimmune hepatitis. The detection sensitivity and specificity of the kit for preparing the soluble liver antigen T cell antigen epitope of the present invention are higher than the level of the existing detection kits. The method of the soluble liver antigen T cell antigen epitope obtained in the present invention is simple and accurate, and the kit prepared by using the epitope is low in cost and remarkable in effect, and has good application value and market value.

Description

technical field [0001] The invention relates to a T cell antigen epitope, in particular to an autoimmune hepatitis soluble liver antigen T cell epitope and a detection kit prepared therefrom, belonging to the field of immunology. Background technique [0002] Antigenic epitope: It is usually difficult for immune cells to recognize the entire antigen molecule, but only a specific part of the antigen macromolecule, called epitope or antigenic determinant. Thus epitope represents an immunologically active region on the antigen molecule, which is responsible for binding to the antigen receptor on the surface of immune cells or free antibody molecules. Strictly speaking, the specificity of an antibody is for an epitope rather than for a complete antigen molecule. T and B cells often recognize different epitopes on antigen molecules. [0003] Determining T cell epitopes is of great significance for studying the occurrence process and mechanism of diseases, improving disease diag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08G01N33/68
Inventor 闫惠平赵艳马筠冯霞张永宏刘妍檀玉芬
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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