CN101042399A - Diabetes autoantibody immunoblotting reagent kit - Google Patents
Diabetes autoantibody immunoblotting reagent kit Download PDFInfo
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- CN101042399A CN101042399A CN 200710135677 CN200710135677A CN101042399A CN 101042399 A CN101042399 A CN 101042399A CN 200710135677 CN200710135677 CN 200710135677 CN 200710135677 A CN200710135677 A CN 200710135677A CN 101042399 A CN101042399 A CN 101042399A
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Abstract
This invention discloses one diabetes disease self antigen print agent case, whose islets of Langerhans cell extracts mixture protein composed of Langerhas element, glutamic acid decarboxylase and Langerhas cell; using protein print technique through SDS-polyacrylamide gel electrophoresis ranked by molecule size then into print film; its film bar comprises molecule of each Langerhans self antigen element to be put into reaction tank and serum; if it has antigen, then processing antigen combination and then adding enzyme cross and development agent in the zone; comparing with standard to judge whether serum has the antigen etc.
Description
Technical field
The present invention relates to a kind of diabetes autoantibody immunoblotting reagent kit.
Background technology
Diabetes are general names of one group of hyperglycaemia disease, are divided into type 1 diabetes type (T by the cause of disease and pathogenesis
1DM), diabetes B (T
2DM) and other type.T
1DM claims immune-mediated property diabetes again, is that autoantibody destruction islet cells synthesizes insulin and secretion reduces and causes.T
2DM is that body is to insulin resistance, insulin lowers relatively and causes, so detect diabetes autoantibody is the main foundation of clinical diabetes control to the accurate somatotype of diabetes, the diabetes autoantibody of confirming has insular cellular antibody (ICA), insulin antibody (IAA) and glutamic acid decarboxylase antibody (GADA) at present, enzyme linked immunosorbent assay (ELISA) commonly used and radioimmunology (RIA) are measured, because the restriction of detection method differs greatly in different testing laboratory's testing results.Immunoblot assay is the affirmation test of generally acknowledging in the world, and China Ministry of Public Health is recommended as the confirmation method of acquired immune deficiency syndrome (AIDS) great communicable diseases such as (HIV) to the method.We have invented diabetes autoantibody immunoblotting reagent kit to ICA, IAA and GADA joint-detection, so that clinical in diagnosis of diabetes and somatotype.
Summary of the invention
The purpose of this invention is to provide a kind of diabetes autoantibody immunoblotting reagent kit, it can detect the multiple autoantibodies such as insulin, glutamate decarboxylase and islet cells in diabetic's serum simultaneously, be used for the diabetes somatotype, provide reference frame for clinical diabetes treatment, prediction, prevention simultaneously.
In order to achieve the above object, the present invention is realized by following technical method.
A kind of diabetes autoantibody immunoblotting reagent kit, it is characterized in that: the composition of kit comprises: put blotting membrane bar, elisa reagent, developer A, developer B, stop buffer, concentrated cleaning solution, standard regions band in the reactive tank, the wretch Bai Kangti of islet cells extraction is contained the multiple protein composition of insulin, glutamate decarboxylase and islet cells; Utilize the Western blotting technology, with SDS-polyacrylamide gel electrophoresis PAGE, separate successively by the molecular weight size, be transferred on the blotting membrane by engram technology again, promptly contain the various islet cell autoantigen compositions that vary in size and arrange by molecular weight on its blotting membrane bar, put it into the reaction of reactive tank and test serum, contain autoantibody as test serum, will combine with corresponding antigens respectively, add the enzyme linked immunological chromogenic reagent again, will be at antigen, colour developing district band appears in the antibodies position, can judge to contain which kind of antibody in the test serum with the index zone contrast, the kind and the quantity of the antibody that has that it's too late of its antibody are to the diabetes classification diagnosis, prediction, prevention has the important clinical meaning.
Embodiment
The following examples and example of formulations can illustrate in greater detail the present invention, but do not limit the present invention in any form.
Embodiment 1
The composition of this kit comprises: put 8 of the every plates of blotting membrane bar 5 plates, elisa reagent 0.5ml, developer A25ml, developer B25ml, stop buffer 25ml, concentrated cleaning solution 25ml, standard regions band in the reactive tank.Vein is got blood 2-5ml, puts test tube and waits to solidify back centrifuging serum, and serum specimen is stored in 2-8 ℃, and the sample that can not test in 24 hours should be put-20 ℃ of preservations, and significant hemolysis has floccus or the serum specimen that goes mouldy can influence test.
Determination step:
1, the preparation of liquid is used in washing: have crystallization at the concentrated cleaning solution of 2-8 ℃ of storage and separate out, whole bottle (25ml) thickening and washing is diluted to 500ml with distilled water or deionized water, put into reagent bottle, indicate the preparation time on the label, be stored in 2-8 ℃, the term of validity 6 months, discarded if any floccus or mould allergic effect.
2, in the reactive tank that contains the blotting membrane bar, add washing and use liquid 1ml and serum 10ul to be checked.
3, put on the shaking table that (more than 20 ℃, below 37 ℃) shook 30 minutes under the room temperature.
4, discard reactive tank liquid, pat dry on thieving paper, add washing and use liquid 1ml, wash and discarded reactive tank liquid in 1 minute, cyclic washing 4 times is at last at thieving paper arsis dry liquids.
5, in reactive tank, add washing and use liquid 0.5ml and elisa reagent 10ul.
6, put on the shaking table that (more than 20 ℃, below 37 ℃) shook 30 minutes under the room temperature.
7, discard reactive tank liquid, pat dry on thieving paper, add washing and use liquid 1ml, wash and discarded reactive tank liquid in 1 minute, cyclic washing 4 times is at last at thieving paper arsis dry liquids.
8, in reactive tank, add developer A 0.5ml respectively, developer B 0.5ml.
9, on shaking table, shake 2-10 minute, colour developing.
10, treat that the colour developing of quality control band and hot spot band adds stop buffer 0.5ml after clear, shakes 2 minutes on shaking table.
11, wash away liquid in the groove with tap water, take out the blotting membrane bar, put on the thieving paper, wait to do back and index zone contrast judged result.
The result judges: blotting membrane upper end start line is alignd with the index zone start line, observe positive colour developing district band and can judge that with corresponding index zone position which kind of autoantibody the colour developing band is.According to having or not or what of autoantibody, provide reference frame to diabetes diagnosis, somatotype, treatment, prediction, control.The diabetes autoantibody immunoblotting index zone
Confirm product before using: whether number of registration, lot number, the term of validity, inspection certificate be complete, effective; The blotting membrane aluminium foil bag has or not gas leak phenomenon; Reagent bottle has or not the phenomenon of leakage.The operating environment temperature should be not less than 20 ℃, if environment temperature is low excessively, should place 37 ℃ of incubators to operate.Sample to be measured should as not detecting the same day, should be put 4 ℃ of preservations with fresh serum or whole blood; Surpass more than 24 hours, need put below-20 ℃ and preserve.During judged result, should at first observe quality control band and whether occur, as occurring, please be in time and producer get in touch.The index zone of different lot numbers can not be used with.This kit only is used for in-vitro diagnosis, puts 2-8 ℃ of refrigeration, and is unsuitable freezing.
Claims (3)
1, a kind of diabetes autoantibody immunoblotting reagent kit, it is characterized in that: the composition of kit comprises: put the blotting membrane bar in the reactive tank, elisa reagent, developer A, developer B, stop buffer, concentrated cleaning solution, the standard regions band, mixed protein antigen (insulin with the islet cells extraction, the multiple protein composition of glutamate decarboxylase and islet cells), utilize western blotting technique, by SDS-polyacrylamide gel electrophoresis (PAGE), separate successively by the molecular weight size, transfer on the blotting membrane, promptly contain the various islet cell autoantigen compositions that vary in size and arrange by molecular weight on its blotting membrane bar, put it into the reaction of reactive tank and test serum, contain autoantibody as test serum, will combine with corresponding antigens respectively, add the enzyme linked immunological chromogenic reagent again, will be at antigen, colour developing district band appears in the antibodies position, can judge with the index zone contrast and contain which kind of antibody and antibody type and quantity in the test serum.
2, diabetes autoantibody immunoblotting reagent kit according to claim 1 is characterized in that: kit and determination step:
1., the preparation of liquid is used in washing: have crystallization at the concentrated cleaning solution of 2-8 ℃ of storage and separate out, whole bottle (25ml) thickening and washing is diluted to 500ml with distilled water or deionized water, put into reagent bottle, indicate the preparation time on the label, be stored in 2-8 ℃, the term of validity 6 months, discarded if any floccus or mould allergic effect.
2., in the reactive tank that contains the blotting membrane bar, add washing and use liquid 1ml and serum 10ul to be checked.
3., put on the shaking table that (more than 20 ℃, below 37 ℃) shook 30 minutes under the room temperature.
4., discard reactive tank liquid, on thieving paper, pat dry, add washing and use liquid 1ml, wash and discarded reactive tank liquid in 1 minute, cyclic washing 4 times is at last at thieving paper arsis dry liquids.
5., in reactive tank, add washing and use liquid 0.5ml and elisa reagent 10ul.
6., put on the shaking table that (more than 20 ℃, below 37 ℃) shook 30 minutes under the room temperature.
7., discard reactive tank liquid, on thieving paper, pat dry, add washing and use liquid 1ml, wash and discarded reactive tank liquid in 1 minute, cyclic washing 4 times is at last at thieving paper arsis dry liquids.
8., in reactive tank, add developer A 0.5ml, developer B 0.5ml respectively.
9., on shaking table, shook colour developing 2-10 minute.
10., treat that the colour developing of quality control band and hot spot band adds stop buffer 0.5ml after clear, shakes 2 minutes on shaking table.
(11), wash away liquid in the groove, take out the blotting membrane bar, put on the thieving paper, wait to do back and index zone contrast judged result with tap water.
3, diabetes autoantibody immunoblotting reagent kit according to claim 1, it is characterized in that: blotting membrane upper end start line is alignd with the index zone start line, observe positive colour developing district band and can judge that with corresponding index zone position which kind of autoantibody the colour developing band is, according to having or not or what of autoantibody, provide reference frame to diabetes diagnosis, somatotype, treatment, prediction, control, utilize the contrast of diabetes autoantibody immunoblotting index zone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710135677 CN101042399A (en) | 2007-03-12 | 2007-03-12 | Diabetes autoantibody immunoblotting reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710135677 CN101042399A (en) | 2007-03-12 | 2007-03-12 | Diabetes autoantibody immunoblotting reagent kit |
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| Publication Number | Publication Date |
|---|---|
| CN101042399A true CN101042399A (en) | 2007-09-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN 200710135677 Pending CN101042399A (en) | 2007-03-12 | 2007-03-12 | Diabetes autoantibody immunoblotting reagent kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967704A (en) * | 2012-11-26 | 2013-03-13 | 深圳市伯劳特生物制品有限公司 | Kit for combined detection of 6 diabetic antibodies |
| WO2014067030A1 (en) * | 2012-11-02 | 2014-05-08 | Li Hui | Electronic standard and analysis application software for dna, rna and protein gel |
| CN105353122A (en) * | 2015-11-05 | 2016-02-24 | 厦门敖依生物科技有限公司 | Sjogren syndrome specific autoantibody immunoblotting kit |
| CN107449904A (en) * | 2017-09-06 | 2017-12-08 | 深圳市亚辉龙生物科技股份有限公司 | A kind of autoimmune diabetes detection reaction film bar, preparation and application |
| CN107643397A (en) * | 2017-07-26 | 2018-01-30 | 中国人民解放军总医院 | A kind of film bar for autoantibody parting of being caused a disease for neuromyelitis optica |
-
2007
- 2007-03-12 CN CN 200710135677 patent/CN101042399A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014067030A1 (en) * | 2012-11-02 | 2014-05-08 | Li Hui | Electronic standard and analysis application software for dna, rna and protein gel |
| CN102967704A (en) * | 2012-11-26 | 2013-03-13 | 深圳市伯劳特生物制品有限公司 | Kit for combined detection of 6 diabetic antibodies |
| CN102967704B (en) * | 2012-11-26 | 2014-05-14 | 深圳市伯劳特生物制品有限公司 | Kit for combined detection of 6 diabetic antibodies |
| CN105353122A (en) * | 2015-11-05 | 2016-02-24 | 厦门敖依生物科技有限公司 | Sjogren syndrome specific autoantibody immunoblotting kit |
| CN107643397A (en) * | 2017-07-26 | 2018-01-30 | 中国人民解放军总医院 | A kind of film bar for autoantibody parting of being caused a disease for neuromyelitis optica |
| CN107449904A (en) * | 2017-09-06 | 2017-12-08 | 深圳市亚辉龙生物科技股份有限公司 | A kind of autoimmune diabetes detection reaction film bar, preparation and application |
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Open date: 20070926 |