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Highly sensitive assay method for gad antibody as marker for early diagnosis of type 1 diabetes mellitus

A measurement method and antibody technology, which is applied in the field of high-sensitivity determination of GAD antibodies, can solve the problems of difficult detection or detection of autoantibodies and low detection sensitivity, and achieve the effects of improving diagnostic accuracy, high sensitivity, and avoiding ignorance of symptoms

Active Publication Date: 2015-10-07
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low detection sensitivity of the conventional enzyme-linked immunoassay (ELISA), it is difficult to detect early detection or detection of low-concentration autoantibodies

Method used

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  • Highly sensitive assay method for gad antibody as marker for early diagnosis of type 1 diabetes mellitus
  • Highly sensitive assay method for gad antibody as marker for early diagnosis of type 1 diabetes mellitus
  • Highly sensitive assay method for gad antibody as marker for early diagnosis of type 1 diabetes mellitus

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0065] The "immune complex transfer enzyme immunoassay method (ICT-EIA method)" of the present invention refers to a method related to a highly sensitive improved method of a non-competitive binding method (sandwich) enzyme immunoassay (EIA) (refer to non-competitive Patent Document 1). Such as figure 1 As shown, the non-specific adsorption (background) of the antibody used can be reduced by the ICT-EIA method, so the measurement of the amol level or less (zmol) of various polymer physiologically active substances becomes possible (see Hashida S, et al., Biotechnology Annual Review Volume 1, (1995) pp403-451, Elsevier Science Publishers B.V., Amsterdam).

[0066] The "GAD antibody" of the present invention refers to an autoantibody against glutamic acid decarboxylase (glutamic acid decarboxylase: GAD). GAD is an enzyme that synthesizes γ-aminobutyric acid (GABA) from L-glutamic acid, and is mostly expressed in brain or pancreatic islet β cells. The role of GABA in pancreati...

Embodiment 1

[0073] [Example 1: ICT-EIA method for GAD antibody]

[0074] 【(1) Reagents】

[0075] (a) General reagents:

[0076] Bovine serum albumin (BSA) was purchased from Nacalai tesque (Kyoto), and streptavidin was purchased from Wako Pure Chemical Industries (Osaka). Other general reagents were purchased from Nacalai tesque and Wako Pure Chemical Industries.

[0077] (b) Antibodies:

[0078] Rabbit anti-2,4-dinitrophenyl (DNP)-bovine serum albumin (BSA) serum was purchased from Goat Company (Gunma).

[0079] (b) Antigen:

[0080] Recombinant-human-GAD65 was purchased from RSR Limited (Cardiff, UK).

[0081] (c) Buffer:

[0082] With 0.1M NaCl, 0.1% BSA, 1mM MgCl 2 and 0.1% NaN3 0.01M sodium phosphate buffer (pH 7.0) as buffer A, containing 0.4M NaCl, 0.1% BSA, 1mM MgCl 2 and 0.1% NaN3 0.01M sodium phosphate buffer (pH 7.0) as buffer B, containing 0.1M NaCl, 0.01% BSA, 1mM MgCl 2 and 0.1% NaN3 0.01M sodium phosphate buffer (pH 7.0) as buffer C, 0.01M sodium phosphate buffer (...

Embodiment 2

[0120] [Example 2: Comparison of the detection sensitivity of ICT-EIA-1 method, ICT-EIA-2 method and ELISA method]

[0121] 【(1) Subjects, etc.】

[0122] (a) Target person:

[0123] From non-diabetic subjects (73 patients), hyperinsulinemia patients (9 patients), Graves' disease patients (30 patients) under administration of antithyroid agent (methimazole), Hashimoto's disease patients (20 patients), 2 Type 2 diabetes patients (30; insulin not treated: 7, insulin therapy: 15, insulin therapy unknown: 8), type 1 diabetes patients (24; insulin therapy: 21, insulin therapy unknown: 3 ) to collect samples.

[0124] (b) Informed Consent:

[0125] The experiment conducted in this study was performed with the approval of the ethics committee of Tokushima Bunri University (approval number No. 4). Informed consent was obtained among the test subjects, and the experiment was carried out after obtaining the consent.

[0126] (c) Statistical processing:

[0127] The cutoff value was...

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Abstract

The present invention addresses the problem of providing an assay method for GAD antibody, in particular, a method for assaying GAD antibody at a higher sensitivity than the conventional ELIZA method. To solve this problem, provided is an assay method for GAD antibody in the blood, said assay method being constructed by using the immune complex transfer enzyme immunoassay (ICT-EIA) method, whereby a sensitivity 33 times or greater as high as in the conventional ELIZA method can be established. As a result, type 1 diabetes mellitus can be detected at the early onset stage so that an appropriate interventional treatment can be carried out.

Description

【Technical field】 [0001] The present invention relates to a high-sensitivity measurement method for GAD antibody, which is an evaluation index for the initial symptoms of type 1 diabetes, for the exact evaluation-diagnosis. 【Background technique】 [0002] Diabetes is defined as "a group of diseases characterized by chronic hyperglycemia with insufficient insulin action as the main symptom, accompanied by various characteristic metabolic abnormalities. Its onset is related to both genetic and environmental factors. Therefore, the metabolic Long-term persistence tends to cause unique complications and also promotes arteriosclerosis. In addition, according to the degree of metabolic abnormality, it shows a wide range of pathological conditions from asymptomatic to ketoacidosis or lethargy." (Non-Patent Document 1). In Japan, a total of about 22.1 million people are identified as those who are strongly suspected of diabetes and those whose possibility cannot be denied (at risk) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/564G01N33/53G01N33/543
CPCG01N33/564G01N2800/042
Inventor 桥田诚一
Owner SYSMEX CORP
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