CN104034904B - 一种糖尿病肾病检测试纸 - Google Patents
一种糖尿病肾病检测试纸 Download PDFInfo
- Publication number
- CN104034904B CN104034904B CN201410317187.8A CN201410317187A CN104034904B CN 104034904 B CN104034904 B CN 104034904B CN 201410317187 A CN201410317187 A CN 201410317187A CN 104034904 B CN104034904 B CN 104034904B
- Authority
- CN
- China
- Prior art keywords
- urine
- hoptoglobin
- solution
- protein
- detection zone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 208000007342 Diabetic Nephropathies Diseases 0.000 title claims abstract description 41
- 208000033679 diabetic kidney disease Diseases 0.000 title claims abstract description 41
- 238000012360 testing method Methods 0.000 title claims abstract description 39
- 210000002700 urine Anatomy 0.000 claims abstract description 143
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 64
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 52
- 238000001514 detection method Methods 0.000 claims abstract description 43
- 239000000084 colloidal system Substances 0.000 claims abstract description 33
- 239000010931 gold Substances 0.000 claims abstract description 33
- 229910052737 gold Inorganic materials 0.000 claims abstract description 33
- 239000003365 glass fiber Substances 0.000 claims abstract description 12
- 230000002745 absorbent Effects 0.000 claims abstract description 8
- 239000002250 absorbent Substances 0.000 claims abstract description 8
- 241001529936 Murinae Species 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 48
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 27
- 238000010790 dilution Methods 0.000 claims description 21
- 239000012895 dilution Substances 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 238000009835 boiling Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 11
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 239000010419 fine particle Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 239000012460 protein solution Substances 0.000 claims description 9
- 238000013207 serial dilution Methods 0.000 claims description 9
- 229910052708 sodium Inorganic materials 0.000 claims description 9
- 239000011734 sodium Substances 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 238000005199 ultracentrifugation Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 abstract description 19
- 238000012631 diagnostic technique Methods 0.000 abstract description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 52
- 229940109239 creatinine Drugs 0.000 description 27
- 239000008280 blood Substances 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 12
- 206010012601 diabetes mellitus Diseases 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 238000002405 diagnostic procedure Methods 0.000 description 8
- 102000009027 Albumins Human genes 0.000 description 6
- 108010088751 Albumins Proteins 0.000 description 6
- 230000002485 urinary effect Effects 0.000 description 6
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 5
- 230000005856 abnormality Effects 0.000 description 4
- 238000002306 biochemical method Methods 0.000 description 4
- 201000001421 hyperglycemia Diseases 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 208000002249 Diabetes Complications Diseases 0.000 description 2
- 206010012655 Diabetic complications Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 208000037157 Azotemia Diseases 0.000 description 1
- 102100025255 Haptoglobin Human genes 0.000 description 1
- 108050005077 Haptoglobin Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 208000009852 uremia Diseases 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N2021/752—Devices comprising reaction zones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N2021/757—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated using immobilised reagents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7763—Sample through flow
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00108—Test strips, e.g. paper
- G01N2035/00118—Test strips, e.g. paper for multiple tests
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4713—Plasma globulins, lactoglobulin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及一种糖尿病肾病检测试纸,属于医学体外诊断技术领域。该试纸包括从上到下包括依次层叠的吸水滤纸、玻璃纤维素膜、NC膜,所述的试纸横向方向设有T1检测区、T2检测区和T3参照区;所述的NC膜在T1检测区包被有尿微量白蛋白抗体,在T2检测区包被有尿结合珠蛋白抗体,在T3参照区包被有抗鼠抗人IgG的抗体;所述的玻璃纤维素膜在T1检测区包被有胶体金标记的尿微量白蛋白,在T2检测区包被有胶体金标记的尿结合珠蛋白,在T3参照区包被有胶体金标记的鼠抗人IgG蛋白。该试纸采用尿微量蛋白和尿结合珠蛋白作为生物靶标配,可以检测出尿液中尿微量白蛋和尿结合珠蛋白的含量,根据尿微量白蛋和尿结合珠蛋白的含量比作为诊断依据,操作简单,快速准确。
Description
技术领域
本发明属于医学体外诊断技术领域,涉及一种糖尿病肾病检测试纸。
背景技术
糖尿病是一种以高血糖为特征的代谢性疾病。高血糖则是由于胰岛素分泌缺陷或其生物作用受损,或两者兼有引起。糖尿病时长期存在的高血糖,导致各种组织,特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍,这些称为糖尿病并发症。在众多糖尿病并发症中,由于长期高血压和没有很好控制的高血糖导致糖尿病肾病,引起糖尿病患者的慢性肾衰竭或尿毒症,是晚期糖尿病患者死亡的主要原因。因此,对于中国1亿多的糖尿病患者来说,对于糖尿病肾病的早期诊断显得尤为重要。
传统的糖尿病肾病早期诊断方法在各方面都存在一些不足之处,如传统的血肌氨酸酐判断肾脏疾病的方法,肾脏疾病的漏诊率达到62%;传统的尿白蛋白的诊断方法,受很多药物等因素的影响,很高的假阳性和假阴性;传统的尿微量蛋白的诊断方法,虽然可以作为绝大多数肾脏疾病的诊断标准,但是不适用于II糖尿病引起的糖尿病肾病,漏诊率50-60%;国际上几个顶级实验室结果表明尿结合珠蛋白可以通过计算尿肌氨酸酐的浓度,来判断II型糖尿病引起的糖尿病肾病,但是由于需要计算尿肌氨酸酐的浓度,操作过于复杂,很难广泛应用于临床上糖尿病肾病的诊断,现今针对“糖尿病肾病的准确诊断”,主要通过诊断肾脏疾病以及糖尿病肾病的“金标准”诊断指标——肌氨酸酐清除率做参照,该项指标可以准确的反应肾小球滤过率功能,从而直接判断肾脏功能的好坏。但是,由于该项指标诊断复杂,一般很少用于临床诊断,不仅仅需要复杂的计算公式,还需要准确测量尿流量(毫升/小时)、血肌氨酸酐的浓度和尿肌氨酸酐的浓度,因此,这项“金标准”主要用于医学研究中。研究表明,尿结合珠蛋白(haptoglobin)是糖尿病肾病的新型靶标,并且该生物靶标通过计算尿肌氨酸酐的浓度,可以早期发现II型糖尿病肾病。但是,这些研究过于学术化,其诊断标准操作很复杂,无法快速诊断糖尿病肾病,很难应用于实际的临床中。
糖尿病肾病检测试纸作为一种检测糖尿病肾病的手段,具有快速、简便、廉价的特点,全部检测过程仅需3-10分钟,不需其它任何仪器设备,操作也极其简单,无需专业人员,携带方便,可随时随地进行,对标本既能成批检测,又可单份检测。但是,现有的糖尿病肾病检测试纸通过胶体金免疫层析法对尿液进行检测,只能针对I型糖尿病或II型糖尿病进行检测,而且检测的准确性不高,无法作为准确的检测诊断依据。
发明内容
糖尿病肾病的时候,尿微量白蛋白如果异常,尿液中尿微量白蛋白表现为增加;相反地,糖尿病肾病患者的尿结合珠蛋白如果异常,尿液中尿结合珠蛋白表现为下降。但是,很多情况下,即使是患有糖尿病肾病,二者仍然可能表现为单个指标正常,从而造成漏诊。针对上述问题,本发明提供一种快速准确的糖尿病肾病检测试纸,具体方案如下:
一种糖尿病肾病检测试纸,从上到下包括依次层叠的吸水滤纸、玻璃纤维素膜、NC膜,所述的试纸横向方向设有T1检测区、T2检测区和T3参照区;所述的NC膜在T1检测区包被有尿微量白蛋白抗体,在T2检测区包被有尿结合珠蛋白抗体,在T3参照区包被有抗鼠抗人IgG的抗体;所述的玻璃纤维素膜在T1检测区包被有胶体金标记的尿微量白蛋白,在T2检测区包被有胶体金标记的尿结合珠蛋白,在T3参照区包被有胶体金标记的鼠抗人IgG蛋白。所述的尿微量白蛋白抗体是根据尿微量白蛋白特异C端结构产生的多种抗体;所述的尿结合珠蛋白抗体是根据尿结合珠蛋白特异C端结构产生的多种抗体。
所述的胶体金标记的尿微量白蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液2.5ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液。
尿微量白蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿微量白蛋白储存液,分别取尿微量白蛋白储存液,稀释为15μg/ml、20μg/ml、25μg/ml、30μg/ml、35μg/ml、40μg/ml、45μg/ml、50μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿微量白蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿微量白蛋白量再加10%即为胶体金标记的尿微量白蛋白量。
所述的胶体金标记的尿结合珠蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液1ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液。
尿结合珠蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿结合珠蛋白储存液,分别取尿结合珠蛋白储存液,稀释为10μg/ml、15μg/ml、20μg/ml、25μg/ml、30μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿结合珠蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿结合珠蛋白量再加10%即为胶体金标记的尿结合珠蛋白。
所述的胶体金标记的鼠抗人IgG蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液4ml,继续沸腾加热直至溶液出现橙红色,得到胶体金溶液。
鼠抗人IgG蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到鼠抗人IgG蛋白储存液,分别取鼠抗人IgG蛋白储存液,稀释为15μg/ml、25μg/ml、35μg/ml、45μg/ml、55μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加鼠抗人IgG蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适鼠抗人IgG蛋白量再加10%即为胶体金标记的鼠抗人IgG蛋白。
上述的糖尿病肾病检测试纸的使用方法为:将尿液滴加在试纸一端的吸水滤纸上,借助毛细作用泳动至T1检测区、T2检测区和T3参照区,T1检测区、T2检测区和T3参照区发生颜色、宽度变化,测量T1、T2和T3的颜色密度来决定各测试区的物质量,得出T1检测区、T2检测区分别对应检测的尿液中尿微量白蛋和尿结合珠蛋白的物质量含量。
本发明益效果在于:该试纸采用尿微量蛋白和尿结合珠蛋白作为生物靶标配,尿液检测时,可以同时快速、准确检测出尿液中尿微量白蛋和尿结合珠蛋白的含量,根据尿微量白蛋和尿结合珠蛋白的含量比作为诊断依据,操作简单,快速准确,漏诊率底,便于广泛应用于临床上糖尿病肾病的快速准确诊断,给糖尿病患者预防病情恶化提供科学的诊断依据。
附图说明
图1为实施例中糖尿病肾病检测试纸的横向剖视图。
具体实施方式
为了更充分理解发明的技术内容,下面结合具体实施例对本发明的技术方案进一步介绍和说明。
实施例
如图1所示的糖尿病肾病检测试纸,包括吸水滤纸1、玻璃纤维素膜2、NC膜3,吸水滤纸1、玻璃纤维素膜2、NC膜3从上到下依次层叠,T1检测区、T2检测区和T3参照区位于试纸的中间区域;NC膜3在T1检测区包被有尿微量白蛋白抗体31,在T2检测区包被有尿结合珠蛋白抗体32,在T3参照区包被有抗鼠抗人IgG的抗体33;所述的玻璃纤维素膜2在T1检测区包被有胶体金标记的尿微量白蛋白21,在T2检测区包被有胶体金标记的尿结合珠蛋白22,在T3参照区包被有胶体金标记的鼠抗人IgG蛋白23。所述的尿微量白蛋白抗体是根据尿微量白蛋白特异C端结构产生的多种抗体;所述的尿结合珠蛋白抗体是根据尿结合珠蛋白特异C端结构产生的多种抗体。
所述的胶体金标记的尿微量白蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液2.5ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液。
尿微量白蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿微量白蛋白储存液,分别取尿微量白蛋白储存液,稀释为15μg/ml、20μg/ml、25μg/ml、30μg/ml、35μg/ml、40μg/ml、45μg/ml、50μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿微量白蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿微量白蛋白量再加10%即为胶体金标记的尿微量白蛋白量。
所述的胶体金标记的尿结合珠蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液1ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液。
尿结合珠蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿结合珠蛋白储存液,分别取尿结合珠蛋白储存液,稀释为10μg/ml、15μg/ml、20μg/ml、25μg/ml、30μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿结合珠蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿结合珠蛋白量再加10%即为胶体金标记的尿结合珠蛋白。
所述的胶体金标记的鼠抗人IgG蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液4ml,继续沸腾加热直至溶液出现橙红色,得到胶体金溶液。
鼠抗人IgG蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到鼠抗人IgG蛋白储存液,分别取鼠抗人IgG蛋白储存液,稀释为15μg/ml、25μg/ml、35μg/ml、45μg/ml、55μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加鼠抗人IgG蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适鼠抗人IgG蛋白量再加10%即为胶体金标记的鼠抗人IgG蛋白。
胶体金蛋白结合物玻璃纤维素膜制备:将胶体金标记的尿微量白蛋白、尿结合珠蛋白、鼠抗人IgG蛋白用喷点仪分别喷在玻璃纤维素膜的T1检测区、T2检测区、T3参照区,37℃真空干燥2h。
抗体包被固相NC膜的制备:
取尿微量白蛋白抗体用PBS进行梯度稀释2倍、4倍、8倍、16倍,用标准尿液与各稀释浓度后的抗体溶液进行反应,选择抗体-抗原反应适宜的稀释浓度用1μl移液器点在NC膜上的T1检测区。
取尿结合珠蛋白抗体用PBS进行梯度稀释2倍、4倍、8倍、16倍,用标准尿液与各稀释浓度后的抗体溶液进行反应,选择抗体-抗原反应适宜的稀释浓度用1μl移液器点在NC膜上的T2检测区。
取抗鼠抗人IgG的抗体用PBS进行梯度稀释2倍、4倍、8倍、16倍,用标准尿液与各稀释浓度后的抗体溶液进行反应,选择抗体-抗原反应适宜的稀释浓度用1μl移液器点在NC膜上的T3参照区。
将抗体包被的NC膜37℃干燥1h。
将吸水滤纸1、玻璃纤维素膜2、NC膜3组装为试纸。
上述的糖尿病肾病检测试纸的使用方法为:将尿液滴加在试纸一端的吸水滤纸上,借助毛细作用泳动至T1检测区、T2检测区和T3参照区,T1检测区、T2检测区和T3参照区发生颜色、宽度变化,测量T1、T2和T3的颜色密度来决定各测试区的物质量,得出T1检测区、T2检测区分别对应检测的尿液中尿微量白蛋和尿结合珠蛋白的物质量含量。
本发明的试纸采用尿微量白蛋白和尿结合珠蛋白作为生物靶标配,可以同时快速、准确检测出尿液中尿微量白蛋和尿结合珠蛋白的含量,通过计算尿液中尿微量白蛋白÷尿结合珠蛋白的物质量比值对糖尿病肾病进行诊断,漏诊率极低(约1-2%),并且由于两者在糖尿病肾病指标变化的差异性,通过上述计算公式,将两个指标合并为一个数值,增加了该数值的诊断灵敏性,从而使该方法容易在临床上应用,尿液中尿微量白蛋白÷尿结合珠蛋白的物质量比值判断标准:正常范围为小于(包含等于)0.8;异常范围为大于0.8。
利用本发明试纸的诊断方法与测定尿白蛋白诊断方法、胶体金试纸测定尿微量白蛋白诊断方法、胶体金试纸测定尿结合珠蛋白诊断方法对糖尿病肾病实验体进行诊断,试验方法如下:
(1)成年wistar大鼠150~200g,高脂喂养1个月,然后持续高脂喂养,并腹腔注射链尿菌素(STZ)2周,测定成年wistar大鼠的(血肌氨酸酐÷尿肌氨酸酐)*尿流量。其中尿流量用毫升每小时表示,具体操作是记录两次尿液产生的间隔时间,收集间隔时间后的尿液,通过尿量÷时间来计算尿流量;血肌氨酸酐和尿肌氨酸酐通过生物化学试剂盒测定。
选取该指标异常的大鼠300只,用生物化学的方法测定其尿液中尿白蛋白,本发明的糖尿病肾病检测试纸测定尿微量白蛋白和尿结合珠蛋白,并进一步计算尿微量白蛋白÷尿结合珠蛋白的比值,统计各方法诊断出大鼠患糖尿病的比例。
(2)选择20~30g左右db/db小鼠,饲养2周后,测定小鼠的(血肌氨酸酐÷尿肌氨酸酐)*尿流量。其中尿流量用毫升每小时表示,具体操作是记录两次尿液产生的间隔时间,收集间隔时间后的尿液,通过尿量÷时间,来计算尿流量;血肌氨酸酐和尿肌氨酸酐通过生物化学试剂盒测定。
选取该指标异常的小鼠300只,用生物化学的方法测定其尿液中尿白蛋白,本发明的糖尿病肾病检测试纸测定尿微量白蛋白和尿结合珠蛋白,并进一步计算尿微量白蛋白÷尿结合珠蛋白的比值,统计各方法诊断出小鼠患糖尿病的比例。
(3)选择I型糖尿病患者,测定(血肌氨酸酐÷尿肌氨酸酐)*尿流量。其中尿流量用毫升每小时表示,具体操作是记录两次尿液产生的间隔时间,收集间隔时间后的尿液,通过尿量÷时间,来计算尿流量;血肌氨酸酐和尿肌氨酸酐通过生物化学试剂盒测定。
选取该指标异常的患者220例,用生物化学的方法测定尿白蛋白,本发明的糖尿病肾病检测试纸测定尿微量白蛋白和尿结合珠蛋白,并进一步计算尿微量白蛋白÷尿结合珠蛋白的比值,统计各方法诊断出I型糖尿病患者的比例。
(4)选择II型糖尿病患者,测定(血肌氨酸酐÷尿肌氨酸酐)*尿流量。其中尿流量用毫升每小时表示,具体操作是记录两次尿液产生的间隔时间,收集间隔时间后的尿液,通过尿量÷时间,来计算尿流量;血肌氨酸酐和尿肌氨酸酐通过生物化学试剂盒测定。
选取该指标异常的患者235例,用生物化学的方法测定尿白蛋白,本发明的糖尿病肾病检测试纸测定尿微量白蛋白和尿结合珠蛋白,并进一步计算尿微量白蛋白÷尿结合珠蛋白的比值,统计各方法诊断出II型糖尿病患者的比例。
上述各方法诊断出糖尿病的比例统计结果见表1。
表1:
由于上述每个样本都通过测定(血肌氨酸酐÷尿肌氨酸酐)*尿流量做比对,该指标是实验研究中公认的判断糖尿病肾病的金标准,各试验方案选取的(血肌氨酸酐÷尿肌氨酸酐)*尿流量指标异常的测试个体均为糖尿病病患个体。上述统计的诊断结果表明,测定尿微量白蛋白÷尿结合珠蛋白是诊断准确率最高的诊断方法,是一种有效的、可用于临床的判断糖尿病肾病的诊断指标。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也视为本发明的保护范围。
Claims (4)
1.一种糖尿病肾病检测试纸,其特征在于:所述的糖尿病肾病检测试纸从上到下包括依次层叠的吸水滤纸、玻璃纤维素膜、NC膜,所述的试纸横向方向设有T1检测区、T2检测区和T3参照区;所述的NC膜在T1检测区包被有尿微量白蛋白抗体,在T2检测区包被有尿结合珠蛋白抗体,在T3参照区包被有抗鼠抗人IgG的抗体;所述的玻璃纤维素膜在T1检测区包被有胶体金标记的尿微量白蛋白,在T2检测区包被有胶体金标记的尿结合珠蛋白,在T3参照区包被有胶体金标记的鼠抗人IgG蛋白。
2.根据权利要求1所述的糖尿病肾病检测试纸,其特征在于,所述的胶体金标记的尿微量白蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液2.5ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液;
尿微量白蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿微量白蛋白储存液,分别取尿微量白蛋白储存液,稀释为15μg/ml、20μg/ml、25μg/ml、30μg/ml、35μg/ml、40μg/ml、45μg/ml、50μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿微量白蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿微量白蛋白量再加10%即为胶体金标记的尿微量白蛋白量。
3.根据权利要求1所述的糖尿病肾病检测试纸,其特征在于,所述的胶体金标记的尿结合珠蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液1ml,继续沸腾加热至溶液出现橙红色,得到胶体金溶液;
尿结合珠蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到尿结合珠蛋白储存液,分别取尿结合珠蛋白储存液,稀释为10μg/ml、15μg/ml、20μg/ml、25μg/ml、30μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加尿结合珠蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适尿结合珠蛋白量再加10%即为胶体金标记的尿结合珠蛋白。
4.根据权利要求1所述的糖尿病肾病检测试纸,其特征在于,所述的胶体金标记的鼠抗人IgG蛋白的制备方法为:
取0.01%HAuCl4水溶液100ml,加热至沸腾,迅速加入1%枸橼酸三钠水溶液4ml,继续沸腾加热直至溶液出现橙红色,得到胶体金溶液;
鼠抗人IgG蛋白对低离子强度的水透析,用微孔滤膜或超速离心除去蛋白质溶液中的细小微粒,得到鼠抗人IgG蛋白储存液,分别取鼠抗人IgG蛋白储存液,稀释为15μg/ml、25μg/ml、35μg/ml、45μg/ml、55μg/ml的系列稀释液,分别取0.1ml的稀释液加到1ml胶体金溶液中,另设一不加鼠抗人IgG蛋白质的对照组,5min后加入0.1ml 10%NaCl溶液,混匀后静置2小时,能使胶体金稳定的最适鼠抗人IgG蛋白量再加10%即为胶体金标记的鼠抗人IgG蛋白。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410317187.8A CN104034904B (zh) | 2014-07-03 | 2014-07-03 | 一种糖尿病肾病检测试纸 |
US14/655,748 US20160202271A1 (en) | 2014-07-03 | 2014-08-20 | Test paper for detection of diabetic nephropathy |
DE112014002343.4T DE112014002343T5 (de) | 2014-07-03 | 2014-08-20 | Testpapier zum Nachweis diabetischer Nephropathie |
PCT/CN2014/084800 WO2016000299A1 (zh) | 2014-07-03 | 2014-08-20 | 一种糖尿病肾病检测试纸 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410317187.8A CN104034904B (zh) | 2014-07-03 | 2014-07-03 | 一种糖尿病肾病检测试纸 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104034904A CN104034904A (zh) | 2014-09-10 |
CN104034904B true CN104034904B (zh) | 2015-11-04 |
Family
ID=51465750
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201410317187.8A Expired - Fee Related CN104034904B (zh) | 2014-07-03 | 2014-07-03 | 一种糖尿病肾病检测试纸 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20160202271A1 (zh) |
CN (1) | CN104034904B (zh) |
DE (1) | DE112014002343T5 (zh) |
WO (1) | WO2016000299A1 (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106353511A (zh) * | 2016-08-19 | 2017-01-25 | 基蛋生物科技股份有限公司 | 一种尿微量白蛋白/尿肌酐一体化检测二联条及其制备方法 |
CN107091927B (zh) * | 2017-06-28 | 2018-11-06 | 安徽惠邦生物工程有限公司 | 一种用于糖尿病肾病早期诊断的试剂盒 |
US20210389307A1 (en) * | 2018-09-28 | 2021-12-16 | Siemens Healthcare Diagnostics Inc. | Methods for detecting hook effect(s) associated with anaylte(s) of interest during or resulting from the conductance of diagnostic assay(s) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6251608B1 (en) * | 2000-04-20 | 2001-06-26 | Technion Research & Development Foundation, Ltd. | Method of determining a potential of a hyperglycemic patients of developing vascular complications |
RU2348038C1 (ru) * | 2007-06-26 | 2009-02-27 | Государственное образовательное учреждение высшего профессионального образования "Воронежская государственная медицинская академия им. Н.Н. Бурденко Федерального агентства по здравоохранению и социальному развитию" | Способ диагностики диабетической нефропатии на доклинической стадии |
CN102539786A (zh) * | 2011-12-31 | 2012-07-04 | 上海凯创生物技术有限公司 | 微量尿白蛋白胶体金检测试剂盒及其制备工艺 |
CN102707067A (zh) * | 2012-05-24 | 2012-10-03 | 蓝十字生物药业(北京)有限公司 | 半定量检测尿微量白蛋白的试纸条 |
CN203432976U (zh) * | 2013-08-07 | 2014-02-12 | 天津中新科炬生物制药有限公司 | 一种半定量微量尿白蛋白快速检测装置 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005002416A2 (en) * | 2003-06-04 | 2005-01-13 | Joslin Diabetes Center, Inc. | Predictors of renal disease |
CN1854738A (zh) * | 2005-04-20 | 2006-11-01 | 北京怡成生物电子技术有限公司 | 一种尿微量白蛋白测试片及其测试装置 |
-
2014
- 2014-07-03 CN CN201410317187.8A patent/CN104034904B/zh not_active Expired - Fee Related
- 2014-08-20 DE DE112014002343.4T patent/DE112014002343T5/de not_active Withdrawn
- 2014-08-20 US US14/655,748 patent/US20160202271A1/en not_active Abandoned
- 2014-08-20 WO PCT/CN2014/084800 patent/WO2016000299A1/zh active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6251608B1 (en) * | 2000-04-20 | 2001-06-26 | Technion Research & Development Foundation, Ltd. | Method of determining a potential of a hyperglycemic patients of developing vascular complications |
RU2348038C1 (ru) * | 2007-06-26 | 2009-02-27 | Государственное образовательное учреждение высшего профессионального образования "Воронежская государственная медицинская академия им. Н.Н. Бурденко Федерального агентства по здравоохранению и социальному развитию" | Способ диагностики диабетической нефропатии на доклинической стадии |
CN102539786A (zh) * | 2011-12-31 | 2012-07-04 | 上海凯创生物技术有限公司 | 微量尿白蛋白胶体金检测试剂盒及其制备工艺 |
CN102707067A (zh) * | 2012-05-24 | 2012-10-03 | 蓝十字生物药业(北京)有限公司 | 半定量检测尿微量白蛋白的试纸条 |
CN203432976U (zh) * | 2013-08-07 | 2014-02-12 | 天津中新科炬生物制药有限公司 | 一种半定量微量尿白蛋白快速检测装置 |
Non-Patent Citations (3)
Title |
---|
2型糖尿病并发代谢综合征与糖尿病肾损害的关系;阮园等;《浙江实用医学》;20120831;第7卷(第4期);237-239、242 * |
2型糖尿病肾病尿液蛋白组学研究及早期诊断标志物的筛选;姜洪娟;《中国博士学位论文全文数据库医药卫生科技辑》;20100515(第05期);E065-3 * |
Urine haptoglobin levels predict early renal functional decline in patients with type 2 diabetes;NM Bhensdadia et al;《Kidney International》;20130327;第83卷;1136-1143 * |
Also Published As
Publication number | Publication date |
---|---|
DE112014002343T5 (de) | 2016-04-07 |
US20160202271A1 (en) | 2016-07-14 |
CN104034904A (zh) | 2014-09-10 |
WO2016000299A1 (zh) | 2016-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Smith et al. | The measurement of the tubular excretory mass, effective blood flow and filtration rate in the normal human kidney | |
CN101221189B (zh) | 一种用于测定活化部分凝血活酶时间aptt的体外诊断试剂盒 | |
CN104034904B (zh) | 一种糖尿病肾病检测试纸 | |
CN106353511A (zh) | 一种尿微量白蛋白/尿肌酐一体化检测二联条及其制备方法 | |
Bahlmann et al. | Micropuncture study of isolated perfused rat kidney | |
Bartoli et al. | Effect of intraluminal flow on proximal tubular reabsorption | |
CN204422546U (zh) | 肾损伤五合一检测卡 | |
Haque et al. | Association of HbA1c with urinary ACR & eGFR in Type-2 diabetes mellitus | |
CN204882450U (zh) | 一种糖化血红蛋白-血糖联合检测装置 | |
Sano et al. | Anaesthesia and circulating blood volume | |
CN209059682U (zh) | 一种具有尿液检测功能的纸尿裤 | |
CN105051533A (zh) | 基于脂肪酸燃烧的测定胰岛素抵抗性的方法及用于该方法的组合物 | |
CN206772984U (zh) | 一种用于急性肾损伤快速定量检测的多指标胶体金试剂盒 | |
Balaky et al. | Indications of Liver and Kidney Functions in Non-Insulin Dependent Diabetic Patients | |
WO2011104710A1 (en) | Device, system and method for in-flow analyte concentration detection | |
Flasar | What is urine specific gravity? | |
CN206772987U (zh) | 一种用于急性肾损伤快速定量检测的多指标时间分辨荧光免疫层析试剂盒 | |
CN208808525U (zh) | 一种医疗检验科方便采集尿液的装置 | |
Weinstein et al. | Early postglomerular plasma concentrations of chloride, sodium, and inulin in the rat kidney | |
CN201642489U (zh) | 一种精确计量尿袋 | |
CN211206532U (zh) | 一种家用克罗恩病检测组件 | |
CN207270342U (zh) | 一种真空采血管 | |
CN207384271U (zh) | 一种用于医疗护理的便捷集液器 | |
Peduruhewa et al. | Pit falls in interpretation of electrolyte results | |
CN205493848U (zh) | 一种尿量仪校准装置 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151104 Termination date: 20210703 |
|
CF01 | Termination of patent right due to non-payment of annual fee |