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Composition for immunologically diagnosing type I diabetes

A technology for type 1 diabetes and immunodiagnosis, applied in biological testing, material inspection products, etc., can solve the problems that patients with delayed type 1 diabetes should not use it

Inactive Publication Date: 2010-09-08
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, patients with late-onset type 1 diabetes should not use this indicator for detection

Method used

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  • Composition for immunologically diagnosing type I diabetes
  • Composition for immunologically diagnosing type I diabetes
  • Composition for immunologically diagnosing type I diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Preparation of four kinds of antigens of IGRP, ZnT8, GAD65 and IA-2

[0044] 1. Cloning of four antigenic peptide genes

[0045] The upstream and downstream primers were designed and synthesized according to the nucleotide sequence of the antigenic peptide, and the restriction enzymes used were Xho I and Xba I respectively.

[0046] The primer sequences used to amplify the IGRP antigen are as follows:

[0047] IGRP-F: 5'-GCCTCGAGGATTTCCTTCACAGGAATGGAGTGCTCATAATTCAGCATTTGCA-3'

[0048] IGRP-R: 5'-GCTCTAGAAAAAGTGTAGTAAGCTCGGTAGTCCTTCTGCAAATGCTGAATTA-3'

[0049] The primer sequences used to amplify the ZnT8 antigen are as follows:

[0050] ZnT8-F: 5'-GCCTCGAGAAGGACTTCTCCATCCT-3'

[0051] ZnT8-R: 5'-GCTCTAGATTACTAGTCACAGGGGTCTTCACA-3'

[0052] The primer sequences used to amplify the GAD65 antigen are as follows:

[0053] GAD65-F: 5'-GCCTCGAGTGGATGCATGTGGATGCA-3'

[0054] GAD65-R: 5'-GCTCTAGATTAAACGTGGCGTCCGCACTGT-3'

[0055] The primer sequences used ...

Embodiment 2

[0063] Embodiment 2: Preparation of IGRP-ZnT8 fusion antigen

[0064] 1. Primer Design

[0065] The upstream and downstream primers were designed according to the sequences of the two antigenic peptide genes and linkers. The restriction enzymes used were Xba I and Xho I respectively. All primers were synthesized by Shanghai Handsome Biotechnology Co., Ltd., and their sequences are as follows:

[0066] IGRP-FL: 5'-GCCTCGAGGATTTCCTTCACAGGAATGGAGTGCTCATAATTCAGCATTTGCA-3'

[0067] IGRP-RL: 5'-TCCACCACCACTAGAACCTCCACCAAAAAGTGTAGTA-3'

[0068] ZnT8-FL: 5'-GGTGGAGGTTCTAGTGGTGGTGGAAAGGACTTCTCC-3'

[0069] ZnT8-RL: 5'-GCTCTAGATTACTAGTCACAGGGGTCTTCACA-3'

[0070] 2. Vector Construction

[0071] Using the antigen gene plasmid constructed in Example 1 as a template, the gene fragments of IGRP and ZnT8 antigens with linker gene sequences were respectively amplified. Then, the gene fragments with complementary adapters are used as templates to amplify each other, and the IGRP and ZnT8 ...

Embodiment 3

[0074] Example 3: Indirect ELISA method to evaluate the antigenicity of 4 individual antigens

[0075] Using the selected IGRP, ZnT8, GAD65 and IA-2 antigens as antigens, 50 healthy blood donors’ serum samples were preliminarily detected by indirect ELISA method. The cutoff values ​​of ZnT8, GAD65 and IA-2 were 0.19, 0.18, 0.17 and 0.19, respectively. Then, these four antigens were used to detect the type 1 diabetes autoimmune antibodies in the sera of 20 autoimmune type 1 diabetics and 20 healthy blood donors. Table 1 shows the OD value and cutoff value of the tested samples The ratio (S / co=sample OD value / cutoff value), a ratio greater than 1 was considered positive, a ratio greater than 2 was determined to be strongly positive, and a ratio less than 1 was determined to be negative. The sensitivity and specificity of the four antigens of IGRP, ZnT8, GAD65 and IA-2 in the preliminary detection were as follows: 80% and 95% for IGRP, 65% and 100% for ZnT8, 50% and 85% for GAD6...

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Abstract

The invention relates to a composition for immunologically diagnosing type I diabetes, which contains specific IGRP antigen and ZnT8 antigen of an islet beta cell or is selected from one of common antigens GAD65 and IA-2 or the combination of the antigens GAD65 and IA-2.

Description

technical field [0001] The invention relates to the field of diagnosis of autoimmune type 1 diabetes. More specifically it relates to the field of diagnosis of autoimmune type 1 diabetes using compositions derived from multiple autoantigens. Background of the invention [0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by absolute or relative insufficiency of insulin secretion, which is affected by a variety of genetic and environmental factors, which can lead to the inability of the body to effectively utilize carbohydrates, fats and proteins . Diabetes is harmful, mainly because of its serious complications and high mortality. With the prolongation of diabetes, if the metabolic disorder in the body is not well controlled, it can lead to chronic complications in the eyes, kidneys, nerves, blood vessels, heart and other tissues and organs, so that blindness, lower extremity gangrene, and uremia will eventually occur. , stroke or myocar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/66
Inventor 冯晓燕张贺秋宋晓国王国华陈坤朱翠侠方平葛海鹏
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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