CN111796103A - 一种检测棘球蚴病的胶体金试纸条及其制备方法 - Google Patents
一种检测棘球蚴病的胶体金试纸条及其制备方法 Download PDFInfo
- Publication number
- CN111796103A CN111796103A CN202010690018.4A CN202010690018A CN111796103A CN 111796103 A CN111796103 A CN 111796103A CN 202010690018 A CN202010690018 A CN 202010690018A CN 111796103 A CN111796103 A CN 111796103A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- test strip
- solution
- gold
- drying
- 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.)
- Pending
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 238000012360 testing method Methods 0.000 title claims abstract description 49
- 206010014096 Echinococciasis Diseases 0.000 title claims abstract description 13
- 208000009366 Echinococcosis Diseases 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 9
- 239000000427 antigen Substances 0.000 claims abstract description 6
- 102000036639 antigens Human genes 0.000 claims abstract description 6
- 108091007433 antigens Proteins 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 31
- 238000001035 drying Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 241000283707 Capra Species 0.000 claims description 11
- 239000010931 gold Substances 0.000 claims description 11
- 229910052737 gold Inorganic materials 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 238000005507 spraying Methods 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 2
- 238000006748 scratching Methods 0.000 claims 1
- 230000002393 scratching effect Effects 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 19
- 238000001514 detection method Methods 0.000 abstract description 15
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 2
- 230000000405 serological effect Effects 0.000 abstract description 2
- 230000000007 visual effect Effects 0.000 abstract description 2
- 238000003756 stirring Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 206010033794 paragonimiasis Diseases 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 206010009344 Clonorchiasis Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 208000007316 Neurocysticercosis Diseases 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 201000000539 sparganosis Diseases 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 208000003322 Coinfection Diseases 0.000 description 2
- 241001126309 Fasciolopsis Species 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 208000009434 Schistosomiasis japonica Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- ZBKIUFWVEIBQRT-UHFFFAOYSA-N gold(1+) Chemical compound [Au+] ZBKIUFWVEIBQRT-UHFFFAOYSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 201000006675 intestinal schistosomiasis Diseases 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000244163 Echinococcus multilocularis Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 238000012752 Hepatectomy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 241000242677 Schistosoma japonicum Species 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000023402 cell communication Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000006275 fascioliasis Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
Images
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
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- 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
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
- G01N2333/43539—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes
- G01N2333/43543—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes from Taenia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供了一种检测棘球蚴病的胶体金试纸条及其制备方法,本方法以EmAgB3‑GGGS‑Em18蛋白为抗原,制备了用于AE早期诊断的胶体金免疫试纸条。本试纸条具有良好的敏感性和特异性,对血清的检测下限可达到10‑6,检测准确率高,且操作简便、迅速,结果判断直观,便于基层现场使用,为该病的的大规模筛查提供了血清学诊断工具。
Description
技术领域
本发明涉及生物检测领域,具体为一种检测棘球蚴病的胶体金试纸条及其制备方法。
背景技术
泡型棘球蚴病(Alveolar echinococcosis, AE)是由多房棘球绦虫(Echinococcus multilocularis, Em)的续绦期幼虫感染引起的一种人畜共患寄生性蠕虫疾病。该病在中国、日本、俄罗斯、中亚和北美部分地区流行严重。根治性肝切除手术是目前唯一比较可靠的治疗方法。因此,AE早期免疫诊断是降低本病死亡率的关键要求。对AE进行早期诊断并采取及时措施,可显著降低因AE造成的低死亡率。目前,对于该病的诊断,尤其是确诊该病,还主要依赖于病原学及影像学诊断。但影像学诊断需要影像学背景知识,操作复杂,却又不易识别较小和非典型的病灶,尤其又不易与脓肿或肿瘤进行区别当前,基于纯化的天然抗原Em2的酶联免疫吸附试验(Em2-ELISA)的诊断方法已被世界卫生组织推荐用于AE的血清诊断。
发明内容
本发明为克服现有技术的上述缺陷,提供一种检测棘球蚴病的胶体金试纸条及其制备方法。
本发明的目的一是提供一种检测棘球蚴病的胶体金试纸条的制备方法,具体包括如下步骤:
(1)胶体金的制备:利用柠檬酸钠还原法制备胶体金溶液。在100 mL去离子水中加入1mL 1%的氯金酸(HAuCl4)溶液,置于恒温磁力搅拌器上匀速搅拌混匀,开启加热至溶液沸腾1 min,迅速加入新配制的1%柠檬酸钠溶液2.1 mL,继续搅拌加热5 min,可见溶液逐渐变为蓝黑色,然后紫黑色,再加热出现红色,继续沸煮出现透明的橙红色,继续沸煮7~10 min,颜色稳定为酒红色。用铝箔纸盖住瓶口,自然冷却至室温,加去离子水定容至 100 mL。倒入棕色瓶,4℃避光保存;
(2)胶体金标记二抗(山羊抗人IgG Fc)及纯化:
取1.5 mL胶体金溶液,用0.1 mol/L K2CO3溶液调节pH值至6.5,加入15 μg的山羊抗人IgG Fc抗体,混合均匀,搅拌混匀室温反应40 min。搅拌下加入30 μL 10%的BSA终止,静置30 min;
纯化胶体金标记抗体,首先4℃温度下5000 rpm离心30 min,弃去由凝聚的胶体金颗粒形成的沉淀。然后 4℃温度下9000 rpm离心30 min,仔细吸去上清,沉淀物用金标结合物复溶液稀释至0.15ml,4℃避光保存备用,有效期7天;
(3)金标垫、样品垫处理:
将玻璃纤维膜浸泡于封闭液中30 min,37~40℃烘干,密封备用。
将样品垫浸泡于0.01 M pH7.4磷酸缓冲液中30 min,37±2℃烘干,12~18 h,装袋备用,有效期24个月;
(4)喷金与划膜
将纯化好的山羊抗人IgG Fc-胶体金复合物用喷膜机以2.0 µL/cm均匀的喷在处理过的金标垫上,37℃烘干24 h,装袋,密封备用。有效期24个月;
将浓度为0.5 mg/mL的EmAgB3-GGGS-Em18重组蛋白为抗原以1.0 µL/cm划T线,浓度为1mg/mL的人IgG1以1.0 µL/cm划C线,37℃烘干24 h,待用;
(5)组装
将各个部分组装成胶体金试纸,切成宽度0.4 cm的试纸条。
本发明的目的二在于提供一种采用上述方法制备的胶体金试纸条,该试纸条可用于检测棘球蚴病。
免疫胶体金技术是以胶体金作为示踪标记物应用于抗原抗体反应检测的一种免疫标记技术。该技术使用方便、快捷,不需要特殊的仪器和操作要求,成本低廉、便于基层和现场使用,整个反应一般在15 min内即可完成定性判定。另外,使用范围广泛,可适应多种检测条件。标记物稳定,建立的试纸条在4℃储存可长达两年以上,而无信号衰减。胶体金本身为红色,不需要加入额外的试剂,省却了诸如酶标试剂等致癌性底物和终止液,对人体不会造成危害。鉴于胶体金免疫试纸条的优点、技术成熟度和本病的危害,本发明利用克隆表达和纯化后的EmAgB3-GGGS-Em18蛋白,提供了胶体金免疫试纸条及其制备方法,为AE的早期诊断和防治提供了简便易用的诊断工具。
利用克隆表达和纯化的EmAgB3-GGGS-Em18蛋白建立了胶体金免疫试纸条的诊断方法,且建立的诊断方法均具有良好的敏感性和特异性,对血清的检测下限可达到10-6,检测准确率高。且本发明提供的试纸条,用于检测时操作简便、迅速、检测样本不需要特殊前处理、不依赖于任何仪器(如酶标仪等),结果判断更为直观,便于基层现场使用,为该病的的大规模筛查提供了血清学诊断工具。
保藏说明:
培养物名称:Escherichia coli pET28a-BL21(DE3)/01/EmAgB3-GGGS-Em18
保藏机构:中国典型培养物保藏中心
保藏机构简称:CCTCC
地址:中国湖北省武汉市武汉大学
保藏日期:2019年8月 12 日
保藏中心登记入册编号:CCTCC NO: M 2019622。
附图说明
图1为实施例1中胶体金免疫层析试纸条组装示意图。图2为实施例3中基特异性检测结果。图3为实施例4中敏感性检测结果。
图1中,1为PVC板,2为NC膜,3为吸水纸,4为金标垫,5为样品垫,6为 T线,7为 C线。
图2中,B为空白对照;P为阳性对照;N为阴性对照;1-7:分别为脑囊虫病、裂头蚴病、华支睾吸虫病、细粒棘球蚴病、肝片吸虫病、日本血吸虫病及肺吸虫病阳性血清。
图3中,1-5为阳性血清,稀释度分别为为10-3、10-4、10-5、10-6、10-7。
具体实施方式
下面结合具体实施实例对本发明进行详细说明。以下实施实例有助于此领域的技术人员进一步理解本发明,但不以任何形式限制本发明。
下述实施例中所使用的EmAgB3-GGGS-Em18蛋白现保藏于中国典型培养物保藏中心,保藏编号CCTCC NO: M 2019622,保藏日期2019年8月 12 日。
下述实施例中所使用人阴、阳性血清及待检血清和细粒棘球蚴病阳性血清来自青海省人民医院临床收集的病人血清,且均经过手术、影像学和血清学鉴定确诊的血清。其它对照血清脑囊虫病、裂头蚴病、华支睾吸虫病、肝片吸虫病、日本血吸虫病及肺吸虫病来自韩国成均馆大学。
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所使用的溶液配制如下:
1%的氯金酸(HAuCl4)溶液:准确称取2.0 g四氯金酸(HAuC14),溶于去离子水并定容至200 mL,置棕色试剂瓶中4℃避光保存备用。
1%柠檬酸三钠水溶液:准确称取0.5 g Na3C6H5O2·2H2O溶于去离子水中,待充分溶解后定容至50 mL,用0.22 μm滤膜过滤(现配现用)。
0.l mol/L K2CO3溶液:称取13.8 g K2CO3,充分溶解于1000 mL的去离子水中,用0.22 μm滤膜过滤,置4℃保存备用。
抗原抗体稀释液(含6%甲醇的0.01 M pH7.2 PBS):准确称取8.0 g 的NaCl、0.2 g的 KCl;2.9 g 的Na2HPO4·12H2O;0.2 g 的KH2PO4,量取甲醇60 mL,去离子水定容至1 L,置4℃备用。
金标结合物复溶液的配制:准确称取0.5 g叠氮钠(NaN3)、20 g牛血清白蛋白(BSA)溶于0.01 mol/L pH值7.2的PBS中,搅拌并定容至1000 mL。充分溶解后用0.22 μm滤膜过滤,置2~8℃条件下备用,有效期4个月。
封闭液:准确称取20 g BSA、0.5 g NaN3,吸取1 mL Triton X-100,用0.01 mol/LpH值为7.2的PBS溶液充分溶解并定容至1000 mL,用0.22 μm滤膜过滤后,置2~8℃条件下备用,有效期4个月。
PB 缓冲液(pH7.4):准确称量3.58 g Na2HPO4·12H2O、0.24 g KH2PO4,用去离子水充分溶解后,调节pH值到7.4,最后定容至1000 mL。
实施例1 试纸制备
(1)材料准备
将500 mL烧杯、20 mL小烧杯、转子、棕色瓶、玻璃棒等洗净后放入酸缸中(重铬酸钾/浓硫酸/超纯水=120 g:200 mL:1000 mL)浸泡24 h。取出先用自来水冲洗3-4次,再用超纯水冲洗3~4次,置于37℃烘箱中烘干备用。
(2)胶体金的制备
利用柠檬酸钠还原法制备胶体金溶液:在100 mL去离子水中加入1 mL 1%的氯金酸(HAuCl4)溶液,置于恒温磁力搅拌器上匀速搅拌混匀,开启加热至溶液沸腾1 min,迅速加入新配制的1%柠檬酸钠溶液2.1 mL,继续搅拌加热5 min,可见溶液逐渐变为蓝黑色,然后紫黑色,再加热出现红色,继续沸煮出现透明的橙红色,继续沸煮7~10 min,颜色稳定为酒红色。用铝箔纸盖住瓶口,自然冷却至室温,加去离子水定容至 100 mL。倒入棕色瓶,4℃避光保存。
(3)胶体金标记二抗(山羊抗人IgG Fc)及纯化
取1.5 mL烧制好的胶体金溶液,用0.1 mol/L K2CO3溶液调节pH值至6.5,加入15 μg的山羊抗人IgG Fc抗体,混合均匀,搅拌混匀室温反应40 min。搅拌下加入30 μL 10%的BSA终止,静置30 min。
采用两次离心法纯化胶体金标记抗体,首先4℃温度下5000 rpm离心30 min,弃去由凝聚的胶体金颗粒形成的沉淀。然后 4℃温度下9000 rpm离心30 min。仔细吸去上清,沉淀物用金标结合物复溶液稀释至原来体积的十分之一,即0.15ml,4℃避光保存备用,有效期7天。
(4)金标垫、样品垫处理
将玻璃纤维膜浸泡于封闭液中30 min,37~40℃烘干,密封备用。
将样品垫浸泡于0.01 M pH7.4磷酸缓冲液中30 min,37±2℃烘干,12~18 h。装袋备用。有效期24个月。
(5)喷金与划膜
将纯化好的山羊抗人IgG Fc-胶体金复合物用喷膜机以2.0 µL/cm均匀的喷在处理过的金标垫上,37℃烘干24 h,装袋,密封备用。有效期24个月。
选取PALL170的NC膜用划膜喷金机将浓度为0.5 mg/mL的重组蛋白EmAgB3-GGGS-Em18以1.0 µL/cm划T线,浓度为1 mg/mL的人IgG1以1.0 µL/cm划C线,37℃烘干24 h,待用。
(6)组装
如图1所示将胶体金试纸条各个部分组装,并检测配对效果。应用切条机切成宽度0.4cm的试纸条。组装好的胶体金试纸条T线包被了EmAgB3-GGGS-Em18蛋白。
实施例2测试与结果判定
检测实施例1建立的胶体金试纸条。将待检血清用稀释液10倍稀释后,吸取100 μL稀释后的血清样品加至样品垫上,样品将由样品端向吸水纸端移动,当阳性血清与胶体金垫上的山羊抗人IgG Fc-胶体金复合物相遇,结合成阳性血清-山羊抗人IgGFc-胶体金复合物,胶体金累积到一定的量的时候T线显示红色。阴性血清或多余阳性血清继续往吸水纸端移动,遇到标记C线的人IgG1血清,结合成人IgG1-山羊抗人IgGFc-胶体金复合物,C线显色。整个反应在15 min内完成并判定结果。
反应完成后,若T线和C线同时出现红色条带,则表示待检血清为阳性。若C线出现红色条带,而T线不出现条带,则判定为血清阴性。若C线不出现条带,无论T线是否出现红色条带,均判定试纸条无效,需进行重复性试验。
实施例3特异性试验
选择保存的脑囊虫病、裂头蚴病、华支睾吸虫病、细粒棘球蚴病、肝片吸虫病、日本血吸虫病及肺吸虫病等的阳性血清,应用实施例1建立的胶体金试纸条检测方法进行检测,每份血清重复检测3次,确定上述7种寄生虫病的血清是否与胶体金试纸条方法用重组抗原起交叉反应。
结果如图2所示,阳性对照、阴性对照和空白对照均成立。其它血清均为阴性,表明建立的胶体金试纸条方法具有良好的特异性。
实施例4敏感性试验
将临床收集AE手术病人的阳性血清稀释成不同稀释度10-1、10-2、10-3、10-4、10-5、10-6、10-7,同时设立健康人的血清作为阴性对照,以及样品稀释液作为空白对照,利用实施例1建立的胶体金试纸条进行检测。
结果如图3所示,基于EmAgB3-GGGS-Em18抗原建立的胶体金试纸条的检测下线为10-6。表明建立的胶体金试纸条方法具有良好的敏感性。
实施例5符合性试验
利用实施例1建立的胶体金试纸条检测方法对临床200份医院病人血清进行检测,同时用商品化胶体金试纸条做平行检测,对两种方法的结果进行比较。收集的200份血清已经临床确诊,110份来自AE病人、42份来自CE病人、2份来自AE和CE混合感染的病人,46份来自健康人的血清。
用实施例1建立的胶体金试纸,检出阳性血清数117份,包括AE血清110份、CE血清3份(假阳性)、AE/CE混合感染2份、2份阴性血清(假阳性)。商品试剂盒检测结果为:检出阳性血清数155份,包括AE血清110份、CE血清42份、AE/CE混合感染2份、1份阴性血清(假阳性)。可见用EmAgB3-GGGS-Em18建立的胶体金试纸条方法与商品ELISA试剂盒的符合率为81%。
Claims (2)
1.一种检测棘球蚴病的胶体金试纸条的制备方法,其特征在于所述方法包括如下步骤:
(1)胶体金溶液的制备:在100 mL去离子水中加入1 mL 1%的氯金酸溶液,沸腾1 min,加入1%柠檬酸钠溶液2.1 mL,加热至颜色为酒红色;
(2)胶体金标记山羊抗人IgG Fc及纯化:取步骤(1)所得的胶体金溶液1.5 mL,调节pH值至6.5,加入15μg的山羊抗人IgG Fc抗体,室温下反应40 min,加入30 μL 10%的BSA,静置30 min;4℃ 5000rpm离心30 min,弃沉淀,4℃ 9000rpm离心30 min,吸去上清,沉淀物用金标结合物复溶液稀释至0.15ml,得到纯化的山羊抗人IgG Fc-胶体金复合物;
(3)金标垫、样品垫处理:将玻璃纤维膜浸泡于封闭液中30 min,37~40℃烘干,备用;将样品垫浸泡于0.01 M pH7.4磷酸缓冲液中30 min,37±2℃烘干12~18 h,备用;
(4)喷金与划膜:将步骤(2)获得的山羊抗人IgG Fc-胶体金复合物以2.0 µL/cm喷在步骤(3)处理过的金标垫上,37℃烘干24 h;将浓度为0.5 mg/mL的EmAgB3-GGGS-Em18重组蛋白为抗原以1.0 µL/cm划T线,浓度为1 mg/mL的人IgG1以1.0 µL/cm划C线,37℃烘干24 h;
(5)组装:将各个部分组装成胶体金试纸,切成试纸条。
2.一种采用权利要求1所述的方法制成的试纸条,其特征在于所述试纸条可以用来检测棘球蚴病。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010690018.4A CN111796103A (zh) | 2020-07-17 | 2020-07-17 | 一种检测棘球蚴病的胶体金试纸条及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010690018.4A CN111796103A (zh) | 2020-07-17 | 2020-07-17 | 一种检测棘球蚴病的胶体金试纸条及其制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111796103A true CN111796103A (zh) | 2020-10-20 |
Family
ID=72807570
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010690018.4A Pending CN111796103A (zh) | 2020-07-17 | 2020-07-17 | 一种检测棘球蚴病的胶体金试纸条及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111796103A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113999818A (zh) * | 2021-11-03 | 2022-02-01 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | 一种基于检测循环抗原的诊断包虫病的免疫层析试条 |
CN114573677A (zh) * | 2022-03-21 | 2022-06-03 | 洛阳现代生物技术研究院有限公司 | 细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101948521A (zh) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | 诊断细粒棘球蚴病的重组抗原蛋白、其制备方法和用途 |
CN102608330A (zh) * | 2012-03-12 | 2012-07-25 | 中国疾病预防控制中心寄生虫病预防控制所 | 检测泡型包虫病的免疫层析试条及制备方法 |
-
2020
- 2020-07-17 CN CN202010690018.4A patent/CN111796103A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101948521A (zh) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | 诊断细粒棘球蚴病的重组抗原蛋白、其制备方法和用途 |
CN102608330A (zh) * | 2012-03-12 | 2012-07-25 | 中国疾病预防控制中心寄生虫病预防控制所 | 检测泡型包虫病的免疫层析试条及制备方法 |
Non-Patent Citations (2)
Title |
---|
AHN CS 等: "An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis", 《J CLIN MICROBIOL》 * |
蔡其刚: "青南儿童棘球蚴病调查分析及AE免疫学诊断、代谢组学研究", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113999818A (zh) * | 2021-11-03 | 2022-02-01 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | 一种基于检测循环抗原的诊断包虫病的免疫层析试条 |
CN113999818B (zh) * | 2021-11-03 | 2024-04-12 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | 一种基于检测循环抗原的诊断包虫病的免疫层析试条 |
CN114573677A (zh) * | 2022-03-21 | 2022-06-03 | 洛阳现代生物技术研究院有限公司 | 细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1381864B1 (en) | Detection of candida | |
CN111024954A (zh) | 联合检测covid-19抗原和抗体的胶体金免疫层析装置及其使用法 | |
CN111337689A (zh) | 一种新型冠状病毒检测试剂盒 | |
CN101738473B (zh) | 梅毒螺旋体抗体诊断试剂盒及其制备方法 | |
AU2002248996A1 (en) | Detection of candida | |
KR100766098B1 (ko) | 항체측정방법 및 항체측정장치 | |
CN101858914B (zh) | 梅毒特异性总抗体胶体金免疫层析检测试剂条及其制备方法 | |
CN108387748A (zh) | 用于检测猫血清淀粉样蛋白a的免疫荧光层析检测卡与制备方法 | |
CN111781350A (zh) | 检测新型冠状病毒的胶体金免疫层析试纸条及其制备方法 | |
CN111796103A (zh) | 一种检测棘球蚴病的胶体金试纸条及其制备方法 | |
CN105859843A (zh) | 呼吸道合胞病毒抗原制备方法及利用该抗原制备的呼吸道合胞病毒抗体的快速检测试剂盒 | |
CN111007257A (zh) | 一种猪伪狂犬病毒gE蛋白抗体检测免疫阻断层析试剂盒及其应用 | |
CN101825634A (zh) | 梅毒特异性IgM与IgG抗体联合检测试剂条及其制备方法 | |
CN101825636B (zh) | 梅毒特异性IgM抗体与特异性总抗体联合检测试剂条及其制备方法 | |
CN112326966A (zh) | 一种新型冠状病毒抗原的快速检测试剂盒及其制备方法和应用 | |
CN101825635B (zh) | 梅毒特异性IgG抗体与特异性总抗体联合检测试剂条及其制备方法 | |
CN101858915A (zh) | 梅毒特异性IgG抗体胶体金免疫层析检测试剂条及其制备方法 | |
CN106188248A (zh) | 一种eb病毒抗原制备方法及利用该抗原制备的检测eb病毒抗体的快速检测试剂盒 | |
EP3802591A1 (en) | Antibody pairs for use in a rapid influenza b diagnostic test | |
CN208443851U (zh) | 用于检测猫血清淀粉样蛋白a的免疫荧光层析检测卡 | |
CN103033616A (zh) | 一种肾综合征出血热病毒IgM抗体检测方法及试剂盒 | |
CN105585635B (zh) | 抗人肺炎支原体p1蛋白抗体及应用该抗体的免疫层析试剂盒 | |
CN201697920U (zh) | 梅毒特异性IgM抗体与特异性总抗体联合检测试剂条 | |
CN101762693A (zh) | 一种检测tp抗体的磁性免疫层析试纸条及其制备方法 | |
CN104330569A (zh) | 一种快速检测抗体的试纸条 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201020 |
|
RJ01 | Rejection of invention patent application after publication |