Autoantibody assay method for immunological mediated I type diabetes diagnosis

A type 1 diabetes and autoantibody technology, applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of low diagnosis rate, long time, and many operation steps, so as to improve the detection rate and reduce the number of reaction steps. , the effect of reducing operating errors

Inactive Publication Date: 2006-05-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantage is that it can only target GAD in patients with type 1 diabetes 65 、IA 2 Although a single determination of multiple autoantibodies can predict and diagnose the disease to a certain extent, each antibody determination costs 100-150 yuan, and the diagnostic rate is relatively low. The cost of joint detection of two or more antibodies Expensive, with many operation steps and long time, it is not a small burden for the tester and the testee

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Anti-GAD 65 , IA 2, Phogrin, ICA69 antibody determination

[0024] 1. Preparation of four antigens:

[0025] (1) Extract mRNA from 1 gram of fresh pancreatic tissue for IA 2 , Phogrin, ICA69 protein expression; and used in GAD 65 The protein expressed mRNA is a commercial human brain hippocampus mRNA (Clontech company Cat No 6578-1).

[0026] (2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit is used.

[0027] (3) PCR amplification of each target gene: the primers used for amplification of each target gene

[0028] GAD 65 : P1: ACAGGATTGGCATCTCCG,

[0029] P2: GGTCGACTTGGTGAGCAAGGTTA

[0030] ICA69: P1: GAAGGATTCTTGTCAGGACACA,

[0031] P2: GGTGTCGACTCATGCATTGAGCA

[0032] IA2: P1: GGATCCCATGGTGACACTAC,

[0033] P2: AAGCTTAACACAGAGATGCTCACACAG

[0034] Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

[0035] P2: GATCAAGCTTCCTGACAACATC

[0036] A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High Fide...

Embodiment 2

[0055] Example 2: Anti-GAD 65 , IA2, Phogrin, ICA69 antibody determination

[0056] 1. Preparation of four antigens:

[0057] (1) Extract mRNA from 1 gram of fresh pancreatic tissue, the mRNA is a commercial human brain mRNA (Clontech Company Cat No 6578-1).

[0058] (2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit is used.

[0059] (3) PCR amplification of each target gene: the primers used for each target gene

[0060] GAD 65 : P1: ACAGGATTGGCATCTCCG,

[0061] P2: GGTCGACTTGGTGAGCAAGGTTA

[0062] ICA69: P1: GAAGGATTCTTGTCAGGACACA,

[0063] P2: GGTGTCGACTCATGCATTGAGCA

[0064] IA2: P1: GGATCCCATGGTGACACTAC,

[0065] P2: AAGCTTAACACAGAGATGCTCACACAG

[0066] Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

[0067] P2: GATCAAGCTTCCTGACAACATC

[0068] A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High FidelityGIBCO) was used to amplify the DNA of each reverse transcription product to synthesize the corresponding cDN...

Embodiment 3

[0087] Example 3: Anti-GAD 65 , IA2, Phogrin, ICA69 antibody determination

[0088] 1. Preparation of four antigens:

[0089] (1) Take 1 gram of fresh pancreatic tissue and extract mRNA with a strong denaturant. The mRNA is a commercial human brain mRNA (Clontech Company Cat No 6578-1).

[0090] (2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit is used.

[0091] (3) PCR amplification of each target gene: the primers used for each target gene

[0092] GAD 65 : P1: ACAGGATTGGCATCTCCG,

[0093] P2: GGTCGACTTGGTGAGCAAGGTTA

[0094] ICA69: P1: GAAGGATTCTTGTCAGGACACA,

[0095] P2: GGTGTCGACTCATGCATTGAGCA

[0096] IA2: P1: GGATCCCATGGTGACACTAC,

[0097] P2: AAGCTTAACACAGAGATGCTCACACAG

[0098] Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

[0099] P2: GATCAAGCTTCCTGACAACATC

[0100] A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High FidelityGIBCO) was used to amplify the DNA of each reverse transcription product to syn...

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Abstract

The present invention relates to a self-body antibody detection method for immune mediated diabetes type A diagnosis, belonging to the field of biological medicine technology. Said invention includes the following steps: (1), using reverse transcriptive enzyme to make mRNA undergo the process of reverse transcription. Then using high fidelity DNA polymerase to respectively synthesize target DNA, using ligase and protein expression plasmid to make connection, making it be transformed into colibacillus to respectively express target protein; (2), using maltose-binding protein affinity chromatographic column and GST affinity chromotographic column to purify and separate out expression protein, dividing every purified expression protein into two portions; (3), coating antigen: making four antigens be mixed in the coating buffer solution, adding coating buffer solution in every enzyme-labeled well, standing still; (4), closing; and (5), using double-antigen sandwich method to determine antibody. Said invention can once detect any one or several kinds of antibodies of GAD65, IA2, Phogrin and ICA 69.

Description

Technical field [0001] The present invention relates to a detection method used in the field of biomedical technology, specifically, an autoantibody detection method used in immune-mediated diagnosis of type 1 diabetes. Background technique [0002] Diabetes type diagnosis is based on the cause of diabetes. Diabetes is divided into 4 types, namely type 1 diabetes, type 2 diabetes, gestational diabetes and other special types of diabetes. Their harm to patients and treatment methods are completely different. Diagnosis before treatment is very important for patients. Type 1 diabetes is also called youth-onset diabetes, because it often develops before the age of 35, accounting for less than 10% of diabetes. The insulin-producing cells of the pancreas in the patient's body have been damaged, and insulin is absolutely lacking. The patient needs to use insulin from the onset of the disease. Treatment and lifelong use. Because type 1 and type 2 diabetes not only have different causes, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N1/34G01N33/531
Inventor 潘晓平项坤三贾伟平
Owner SHANGHAI JIAO TONG UNIV
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