CN100399026C - 幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 - Google Patents
幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 Download PDFInfo
- Publication number
- CN100399026C CN100399026C CNB2003101224143A CN200310122414A CN100399026C CN 100399026 C CN100399026 C CN 100399026C CN B2003101224143 A CNB2003101224143 A CN B2003101224143A CN 200310122414 A CN200310122414 A CN 200310122414A CN 100399026 C CN100399026 C CN 100399026C
- Authority
- CN
- China
- Prior art keywords
- urease
- antigen
- bacterium
- film
- minutes
- 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
- 108010046334 Urease Proteins 0.000 title claims abstract description 48
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 20
- 210000002966 serum Anatomy 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 17
- 241000193830 Bacillus <bacterium> Species 0.000 title 1
- 239000000427 antigen Substances 0.000 claims abstract description 37
- 102000036639 antigens Human genes 0.000 claims abstract description 33
- 108091007433 antigens Proteins 0.000 claims abstract description 33
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000003745 diagnosis Methods 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 23
- 238000001514 detection method Methods 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 229940037467 helicobacter pylori Drugs 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 239000000020 Nitrocellulose Substances 0.000 claims description 9
- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- 229920001220 nitrocellulos Polymers 0.000 claims description 9
- 239000007921 spray Substances 0.000 claims description 9
- 239000000084 colloidal system Substances 0.000 claims description 8
- 239000010931 gold Substances 0.000 claims description 8
- 229910052737 gold Inorganic materials 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 241000590002 Helicobacter pylori Species 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000013049 sediment Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 239000003365 glass fiber Substances 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000003317 immunochromatography Methods 0.000 claims description 2
- 238000001262 western blot Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 1
- 239000012528 membrane Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 5
- 238000002523 gelfiltration Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000009007 Diagnostic Kit Methods 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 208000007882 Gastritis Diseases 0.000 description 2
- 208000008469 Peptic Ulcer Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000002274 desiccant Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 208000011906 peptic ulcer disease Diseases 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 201000000498 stomach carcinoma Diseases 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- DGZSVBBLLGZHSF-UHFFFAOYSA-N 4,4-diethylpiperidine Chemical compound CCC1(CC)CCNCC1 DGZSVBBLLGZHSF-UHFFFAOYSA-N 0.000 description 1
- LVSPDZAGCBEQAV-UHFFFAOYSA-N 4-chloronaphthalen-1-ol Chemical class C1=CC=C2C(O)=CC=C(Cl)C2=C1 LVSPDZAGCBEQAV-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- CQVDKGFMVXRRAI-UHFFFAOYSA-J Cl[Au](Cl)(Cl)Cl Chemical compound Cl[Au](Cl)(Cl)Cl CQVDKGFMVXRRAI-UHFFFAOYSA-J 0.000 description 1
- 206010019375 Helicobacter infections Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 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 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 108010088160 Staphylococcal Protein A Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229920006351 engineering plastic Polymers 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 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 description 1
- 239000000758 substrate Substances 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及用于Hp菌患者血清抗体快速诊断的试剂盒产品，该试剂盒以幽门螺杆菌尿素酶为抗原，基于膜免疫层析的原理，采用胶体金双抗原夹心法来制备。
Description
发明领域：
本发明涉及用胶体金双抗原夹心法制备的幽门螺杆菌尿素酶血清抗体快速诊断试剂盒。
现有技术：
幽门螺杆菌(Helicobacter pylori，Hp)是慢性胃炎、消化性溃疡的重要致病因子，在胃癌的癌变模式中也起着先导作用，胃癌的发生与幽门螺杆菌感染的时间长短具有直接关系。1994年，世界卫生组织国际癌症研究机构正式将幽门螺杆菌列为I类(即肯定的)致癌源。我国人群Hp菌感染率高达50％左右，每年有1200万的新增感染人群。以往诊断Hp感染常用胃粘膜组织病理学检查、胃粘膜组织Hp菌培养和采用13C、14C呼吸试验以及14N排泄试验。病理组织学检查及细菌培养除要求较高的试验条件外，尚要以接受胃镜检查为前提，不能被广泛应用，复查时更不易被病人接受；呼吸试验和尿素排泄试验虽具有灵敏和特异的特点，但因价格昂贵，不利于临床上推广普及。二十世纪八十年代末，国内建立了Hp血清抗体检测方法，但因采用Hp全菌抗原、Hp全菌超声破碎抗原、酸提取抗原和高分子量相关抗原等，明显存在因交叉反应所造成的假阳性和因菌株间抗原性差并而造成的假阴性，可靠性较差，以致某些临床单位出现对胃炎及消化性溃疡病人不经进一步诊断而盲目开展抗Hp菌治疗的趋势。二十世纪九十年代，中国预防医学科学院流行病学微生物学研究所发明了人血清抗幽门螺杆菌尿素酶抗体体外检测试剂盒(申请号95108003.2，公开号CN1140257A)，该发明采用酶联免疫法，用Hp尿素酶抗原包被酶标反应板，其酶标抗体为HRP-SPA。该方法虽在一定程度上弥补了过去Hp血清抗体检测方法的缺陷，但仍存在一些出于该方法本身的缺陷，而无法解决的问题，如只能检测血清中的IgG抗体、时间长(2-3小时)、步骤多(2-3步)、需特殊仪器(酶联检测仪)、保存时间短(3个月)、须同时操作多人份血清、病人获得检测结果时间长等。
本发明旨在为临床诊断Hp感染、开展抗Hp感染治疗的随访以及Hp感染的流行病学调查提供一种灵敏、快速、特异的诊断试剂盒。
发明内容：
本发明的目的是提供用于Hp菌患者血清抗体快速诊断的试剂盒产品，该试剂盒以幽门螺杆菌尿素酶为抗原，基于膜免疫层析的原理，采用胶体金双抗原夹心法来制备。
该试剂盒包括粘贴于检测板条上的检测膜，试剂垫和吸收垫，其特征在于检测膜上包被有Hp菌尿素酶抗原、试剂垫包被了胶体金标记的Hp菌尿素酶抗原。
该产品可同时检测血清中IgG、IgM型抗体和唾液等分泌物中的IgA型抗体；具有灵敏度高、特异性强、操作简便、无需专用的仪器和设备、结果直观等特点。
本发明的另一个目的是提供上述试剂盒的制备方法，包括：
A、Hp菌尿素酶抗原的制备；
B、喷膜，将抗原喷涂到硝酸纤维素膜上；
C、标记，用胶体金标记抗原；
D、组装，在检测板条上组装检测膜、试剂垫、吸收垫；
E、切条，用切条机切成小条；
F、成品，将小条放入扣盒中，并密封。
本发明的实施方式：
(一)、Hp尿素酶抗原的制备：
细菌培养：将Hp菌株接种于含10％脱纤维羊血的布氏琼脂平皿中置混合气体(5％氧气，10％二氧化碳，85％氮气)环境中，37℃培养三天后收菌。
细菌破碎及预处理：将收集的Hp菌用生理盐水洗涤一遍后，用凝胶过滤洗脱液(0.05M Tris-Cl，pH0.8，含0.005％的硫柳汞)将菌稀释至适当浓度(约14亿菌/ml)，经超声破碎(在冰浴下进行)后，高速离心(12000rpm，30’)后取上清备用。
凝胶过滤过程及洗脱峰尿素酶活性检测：凝胶过滤介质选用Sephacryl S 300(Amerson-Pharmacia公司产品)，柱型2.6×75cm，抗原上柱体积15-30ml，洗脱液为0.05M Tris-Cl，pH8.0，洗脱速度为16滴/分钟，收集、检测及记录自动进行，每管收集50滴，每隔5管测定一次尿素酶活性，尿素酶试剂由本公司配制。
尿素酶提纯效果分析：将凝胶过滤后尿素酶活性峰成分进行聚丙烯酰胺凝胶电泳(SDS-PAGE)观察尿素酶提纯效果。SDS-PAGE按参考文献进行，浓缩胶采用5％浓度，分离胶采用11％浓度，电泳采用Tris-甘氨酸缓冲系统，凝胶经考马斯亮兰R-250染色，收集高纯度部分备用。
纯化尿素酶成分抗原性Western-blot分析：将纯化的尿素酶成分，经SDS-PAGE电泳后，将各蛋白转移到硝酸纤维素膜(简称NC膜)上，NC膜经5％脱脂奶粉封闭过夜后，放入1∶50稀释的兔抗Hp抗血清中，室温震荡2小时，用0.05％Tween20-TBS洗NC膜三次后，浸泡NC膜于1∶200稀释的辣根过氧化物酶标记的葡萄球菌A蛋白(HRP-SPA)中，室温振荡2小时，用Tween20-TBS洗NC膜三次后，用底物显色液(6mg 4-氯-1-萘酚溶于2ml冷甲醇，与含6ul 30％H2O2的10ml TBS混合)浸泡待检NC膜，室温震荡10-30分钟，观察其抗原性，将抗原性良好的尿素酶成分用于试剂盒用抗原。纯化后的抗原保存于-20℃以下。
(二)检测膜的喷涂与处理：
将硝酸纤维素膜(孔径为gum)平放于BioDot X-Y喷涂仪上。将纯化的尿素酶抗原用PBS稀释为1mg/ml，加入试剂瓶中。设程序为尿素酶喷涂程序(生物喷头，线状，角度：0，速度：50cm/秒，频率：150Hz，开放时间：4％，X0＝0，Y0＝3.5mm，长度：30mm，高度：2mm，压力：4psi)，喷涂。将膜置于室温晾干后放入干燥箱待用。
(三)、胶体金的制备和标记：
采用枸橼酸三钠还原法制备胶体金。取750ml去离子水在快速磁力搅拌条件下加热至95℃。加入7ml 1％的四氯化金溶液，继续在快速搅拌中煮沸5分钟。而后，加入8.4m l的1％的枸橼酸钠溶液，并在剧烈搅拌中煮沸5分钟。将经枸橼酸钠还原的四氯化金(胶体金)溶液移至冷水中，快速搅拌中冷却30分钟。用分光光度计分别测定溶液在波长540nm的吸光值。OD540必须在0.9-1.2之间。
用100mM的碳酸钾溶液将上步所获的胶体金溶液的pH调至6.5。取250ul胶体金溶液，加入1mg/ml的尿素酶抗原1ul，混匀后加入27.5u l10％NaCl并观察颜色反应。遇有颜色变紫，则加大尿素酶抗原用量至不再出现颜色反应为止。
按照试验所得尿素酶标记量，计算出所标记液体的尿素酶总需求量。在快速搅拌的条件下加入尿素酶溶液。在室温下搅拌30分钟后加入2％的牛血清白蛋白，并继续在室温下搅拌15分钟。而后，将溶液置于4℃下离心1小时，离心力为10000g。小心弃去上清，尽量避免搅动沉淀物。加入42ml 100mM Na2HPO4，pH7.4，其中含2％牛血清白蛋白。重新悬浮沉淀物后，在4℃离心20分钟，离心力10000g。弃去上清，保留沉淀物。用42ml含2％牛血清白蛋白的10mM的Na2HPO4重新悬浮沉淀物。用0.22μm的硝酸纤维膜过滤除菌后存放于4℃，备用。
(四)、试剂垫的制备：
试剂垫(玻璃纤维膜)是检测板条上存留胶体金标记尿素酶试剂的部位，将制备好的胶体金溶液均匀喷涂在试剂垫上，晾干后切成30×1.4cm的窄条备用。
(五)、检测板条的制作：
取具有粘性表面的8×30cm塑料薄板，将检测膜贴于距薄板顶端3em处。然后在检测膜上端贴一3×30cm的厚滤纸条构成吸收垫，在检测膜下端贴试剂垫。试剂垫下端贴无胶体金标记尿素酶的玻璃纤维，各条之间重叠1mm，制作好的长板用切割机切成8×0.4cm的窄条，即为检测板条。
(六)、诊断试剂盒的组装：
将切割好的板条放入专用的塑料盒中，再将塑料盒放入铝箔袋内。加入1g的袋装干燥剂和一次性滴管后，以热压封口并打印生产日期。完整的试剂盒包括试纸条、工程塑料扣盒、干燥剂、一次性滴管、使用说明书。
(七)、阳性对照血清：
选用抗幽门螺杆菌尿素酶阳性血清，置-20℃以下保存。
(八)阴性对照血清：
选用抗幽门螺杆菌尿素酶ELISA法检测为阴性的5份以上正常人血清混合，置-20℃以下保存。
(九)血清标本检测：
加100ul待检血清于诊断试剂盒的样品孔内。过20分钟后观察结果：
1、当检测尿素酶阳性血清时，检测膜上出现红色条带。
2、当检测尿素酶阴性血清时，检测膜上不出现红色条带。
本发明优点：
其尿素酶抗原来源于国内人群感染的菌株，具有中国人群特异性。
采用双抗原夹心法，灵敏度高，特异性强。可同时检查血清中抗Hp尿素酶IgG、IgM、IgA抗体。Hp感染的早期往往只有IgM的产生，因此，单纯检查IgG，必然会造成感染早期病人的漏检；由于可同时检查IgA，因此，对样本的选择范围较宽，如可用于唾液标本。
采用胶体金技术，结果直观(肉眼观察)、快速(10-20分钟出结果)。
采用胶体金技术，操作简便，无需专用仪器和设备，1人份即可独立检测，也可多人份操作。
采用胶体金技术，操作简便，可用于床旁检验，复检极为方便。血清学检查，为无创伤性检查，患者无痛苦，尤其适合于儿童检查。
Claims (3)
1.一种用于Hp菌患者血清抗体快速诊断的试剂盒产品，其特征在于：该试剂盒以幽门螺杆菌尿素酶为抗原，基于膜免疫层析的原理，采用胶体金双抗原夹心法来制备；所述的试剂盒包括粘贴于检测板条上的检测膜、试剂垫和吸收垫；所述的检测膜为包被有Hp菌尿素酶抗原的硝酸纤维素膜，其中Hp菌尿素酶抗原是通过下述步骤喷涂到硝酸纤维素膜上：用PBS稀释纯化的尿素酶抗原至1mg/ml，将喷涂仪程序设定为生物喷头，线状，角度：0，速度：50cm/秒，频率：150Hz，开放时间：4％，X0＝0，Y0＝3.5mm，长度：30mm，高度：2mm，压力：4psi，喷涂，晾干后即得；所述的试剂垫为包被了胶体金标记的Hp菌尿素酶抗原的玻璃纤维素膜，其中胶体金标记的Hp菌尿素酶抗原是通过下述方法制备的：快速搅拌胶体金的条件下加入标记量的尿素酶溶液，室温搅拌30分钟后加入2％的牛血清白蛋白，并继续搅拌15分钟后，于4℃下离心1小时，离心力为10000g，弃上清，加入42ml 100mM Na2HPO4，pH7.4，其中含2％牛血清白蛋白，4℃离心20分钟，离心力10000g，弃上清，用42ml含2％牛血清白蛋白的10mM的Na2HPO4重新悬浮沉淀物，用0.22μm的硝酸纤维膜过滤除菌后存放于4℃，即得胶体金标记的Hp菌尿素酶抗原。
2.权利要求1的试剂盒产品的制备方法，包括以下步骤：
Hp菌尿素酶抗原的制备；
喷膜，将抗原喷涂到硝酸纤维素膜上；
标记，用胶体金标记抗原；
组装，在检测板条上组装检测膜、试剂垫、吸收垫；
切条，用切割机切成小条；
成品，将小条放入扣盒中，并密封；
其中所述的喷膜步骤的具体过程为：用PBS稀释纯化的尿素酶抗原至1mg/ml，将喷涂仪程序设定为生物喷头，线状，角度：0，速度：50cm/秒，频率：150Hz，开放时间：4％，X0＝0，Y0＝3.5mm，长度：30mm，高度：2mm，压力：4psi，喷涂，晾干后备用；
所述的标记步骤的具体过程为：快速搅拌胶体金的条件下加入标记量的尿素酶溶液，室温搅拌30分钟后加入2％的牛血清白蛋白，并继续搅拌15分钟后，于4℃下离心1小时，离心力为10000g；弃上清，加入42ml 100mM Na2HPO4，pH7.4，其中含2％牛血清白蛋白，4℃离心20分钟，离心力10000g；弃上清，用42ml含2％牛血清白蛋白的10mM的Na2HPO4重新悬浮沉淀物，用0.22μm的硝酸纤维膜过滤除菌后存放于4℃，备用。
3.根据权利要求2的制备方法，其中Hp菌尿素酶抗原的制备包括：
细菌培养；
细菌破碎、离心和提取上清；
提纯；
纯化尿素酶成分抗原性Western-blot分析，选择抗原性良好的尿素酶成分用于试剂盒用抗原。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101224143A CN100399026C (zh) | 2003-12-23 | 2003-12-23 | 幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101224143A CN100399026C (zh) | 2003-12-23 | 2003-12-23 | 幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1554949A CN1554949A (zh) | 2004-12-15 |
| CN100399026C true CN100399026C (zh) | 2008-07-02 |
Family
ID=34338654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101224143A Expired - Fee Related CN100399026C (zh) | 2003-12-23 | 2003-12-23 | 幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100399026C (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288757A (zh) * | 2011-07-12 | 2011-12-21 | 珠海市银科医学工程有限公司 | 无创伤一步法胃幽门螺旋杆菌检测试剂盒及检测方法 |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293305A (zh) * | 2012-03-05 | 2013-09-11 | 美利泰格诊断试剂(嘉兴)有限公司 | 口腔螺旋杆菌感染检测方法及用于检测的唾液试板 |
| CN107356755A (zh) * | 2017-07-25 | 2017-11-17 | 江斌 | 幽门螺杆菌抗体金检测试剂盒 |
| CN111983229A (zh) * | 2020-09-07 | 2020-11-24 | 三门县人民医院 | 胶体金定量检测幽门螺杆菌抗体试剂条及检测方法 |
| CN113341142A (zh) * | 2021-06-07 | 2021-09-03 | 北京捷乐生物科技有限公司 | 一种胃幽门螺杆菌抗体检测方法及试剂盒 |
| CN115266286B (zh) * | 2022-07-29 | 2023-06-02 | 浙江欧赛思生物科技有限公司 | 一种新型髓鞘染色试剂盒及其染色方法 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1140257A (zh) * | 1995-07-06 | 1997-01-15 | 中国预防医学科学院流行病学微生物学研究所 | 一种人血清抗幽门螺杆菌尿素酶抗体体外检测剂盒 |
| US5837240A (en) * | 1995-04-28 | 1998-11-17 | Oravax-Merieux Co. | Multimeric, recombinant urease vaccine |
| CN1390949A (zh) * | 2002-06-25 | 2003-01-15 | 上海晶泰生物技术有限公司 | 一步多检的胶体金检测微生物的试剂盒 |
-
2003
- 2003-12-23 CN CNB2003101224143A patent/CN100399026C/zh not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837240A (en) * | 1995-04-28 | 1998-11-17 | Oravax-Merieux Co. | Multimeric, recombinant urease vaccine |
| CN1140257A (zh) * | 1995-07-06 | 1997-01-15 | 中国预防医学科学院流行病学微生物学研究所 | 一种人血清抗幽门螺杆菌尿素酶抗体体外检测剂盒 |
| CN1390949A (zh) * | 2002-06-25 | 2003-01-15 | 上海晶泰生物技术有限公司 | 一步多检的胶体金检测微生物的试剂盒 |
Non-Patent Citations (4)
| Title |
|---|
| 人幽门螺杆菌尿素酶抗体诊断试剂盒的研制. 鲁东水.免疫学杂志，第18卷第4期. 2002 |
| 人幽门螺杆菌尿素酶抗体诊断试剂盒的研制. 鲁东水.免疫学杂志，第18卷第4期. 2002 * |
| 幽门螺杆菌根除治疗前后快速尿素酶试验诊断的准确性. 孙为豪.中华消化内镜杂志，第20卷第2期. 2003 |
| 幽门螺杆菌根除治疗前后快速尿素酶试验诊断的准确性. 孙为豪.中华消化内镜杂志，第20卷第2期. 2003 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288757A (zh) * | 2011-07-12 | 2011-12-21 | 珠海市银科医学工程有限公司 | 无创伤一步法胃幽门螺旋杆菌检测试剂盒及检测方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1554949A (zh) | 2004-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100255086B1 (ko) | 치근막질병의특이적이고민감한진단방법과진단키트 | |
| US20160047807A1 (en) | Method for Detection of Legionella Bacteria Employing Purified Antigen-Specific Antibodies | |
| US11506674B2 (en) | Monoclonal antibody against D-dimer and diagnosis agent for detecting D-dimer, crosslinked fibrin and its derivatives containing D-dimer by using the antibody | |
| CN109781980A (zh) | 非洲猪瘟病毒快速检测卡及其应用 | |
| US6794153B2 (en) | Helicobacter pylori antigens in blood | |
| EP2833147A1 (en) | Detection kit for influenza a virus | |
| CN101025418A (zh) | 用于诊断ra的多联抗原膜片法检测试剂盒及其制作方法 | |
| CN104459144B (zh) | 一种猪伪狂犬病毒强毒株和疫苗毒株鉴别检测试纸 | |
| JP7489959B2 (ja) | 細菌性膣炎の診断 | |
| AU2010251237B2 (en) | Method for detecting a wound infection | |
| CN100399026C (zh) | 幽门螺杆菌尿素酶血清抗体快速诊断试剂盒及其制备方法 | |
| KR20170106407A (ko) | 마이코플라즈마 뉴모니아의 면역학적 검출법 및 키트 | |
| CN101981452B (zh) | 肺炎球菌检测方法 | |
| JP2779178B2 (ja) | 標的リガンド用アッセイの感度を高める方法 | |
| JP4268358B2 (ja) | 抗体および免疫学的測定方法 | |
| CN105753981A (zh) | 抗人呼吸道合胞病毒n蛋白抗体及应用该抗体的免疫层析试剂盒 | |
| 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 | |
| CN105753982A (zh) | 抗人肺炎链球菌fam1家族PspA蛋白抗体及应用该抗体的免疫层析试剂盒 | |
| CN1140257A (zh) | 一种人血清抗幽门螺杆菌尿素酶抗体体外检测剂盒 | |
| WO2015093439A1 (ja) | ヘリコバクター・ピロリの検出方法 | |
| CN105968197B (zh) | 一种抗3型解脲支原体mb蛋白抗体及应用该抗体的免疫层析试剂盒 | |
| CN105801692B (zh) | 抗3型解脲支原体mb蛋白抗体及应用该抗体的免疫层析试剂盒 | |
| KR102021539B1 (ko) | 모기 매개 바이러스 진단용 펩타이드 및 이의 용도 | |
| KR102021540B1 (ko) | 모기 매개 바이러스 진단용 펩타이드 및 이의 용도 | |
| US5716792A (en) | Streptococcus suis adhesin protein and method for producing it |
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 | ||
| C56 | Change in the name or address of the patentee |
Owner name: SHENZHEN CITY KANGMEI BIOLOGICAL TECHNOLOGY CO.,LT Free format text: FORMER NAME OR ADDRESS: SHENZHEN KANG MEI INDUSTRIAL DEVELOPMENT CO., LTD. |
|
| CP03 | Change of name, title or address |
Address after: Guangdong province Shenzhen City Futian District No. 178 Huarong Building mintian Road, room 1808 Patentee after: Shenzhen Kangmei biological science and Technology Co.,Ltd. Address before: 6013, Shennan Road, Futian District, Guangdong, Shenzhen City, Shenzhen Kang Mei Industrial Development Co., Ltd. Patentee before: KANGMEI INDUSTRY DEV Co.,Ltd. SH |
|
| ASS | Succession or assignment of patent right |
Owner name: SHENZHEN KANG MEI BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHENZHEN CITY KANGMEI BIOLOGICAL TECHNOLOGY CO.,LTD. Effective date: 20080919 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20080919 Address after: Guangdong province Shenzhen City Futian District No. 178 Huarong Building mintian Road, room 1807 Patentee after: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. Address before: Guangdong province Shenzhen City Futian District No. 178 Huarong Building mintian Road, room 1808 Patentee before: Shenzhen Kangmei biological science and Technology Co.,Ltd. |
|
| C56 | Change in the name or address of the patentee | ||
| CP03 | Change of name, title or address |
Address after: Guangdong city of Shenzhen province Futian District Che Kung Temple Tian An Innovation and Technology Square, a A seat 1006B Patentee after: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. Address before: Guangdong province Shenzhen City Futian District No. 178 Huarong Building mintian Road, room 1807 Patentee before: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. |
|
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: BEIJING KANGMEI TIANHONG BIOTECHNOLOGY Co.,Ltd. Assignor: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. Contract record no.: 2010440020211 Denomination of invention: Quick aiagnostic reagent kid for pyloric spiral bacillus urease serum antibody and its preparing method Granted publication date: 20080702 License type: Exclusive License Open date: 20041215 Record date: 20101109 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170303 Address after: 100095, Haidian District, Beijing high road, No. 1, building No. 16 Patentee after: BEIJING KANGMEI TIANHONG BIOTECHNOLOGY Co.,Ltd. Address before: 518040 Guangdong, Shenzhen, Che Kung Temple, Tian An Innovation Technology Plaza, phase A, block 1006-B, Patentee before: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. |
|
| DD01 | Delivery of document by public notice |
Addressee: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. Document name: Notification of Passing Examination on Formalities |
|
| DD01 | Delivery of document by public notice | ||
| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: BEIJING KANGMEI TIANHONG BIOTECHNOLOGY Co.,Ltd. Assignor: SHENZHEN KANGMEI BIOTECHNOLOGY Co.,Ltd. Contract record no.: 2010440020211 Date of cancellation: 20210331 |
|
| EC01 | Cancellation of recordation of patent licensing contract | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Rapid diagnostic kit for Helicobacter pylori urease antibody and its preparation Effective date of registration: 20210430 Granted publication date: 20080702 Pledgee: Shenzhen hi tech investment small loan Co.,Ltd. Pledgor: BEIJING KANGMEI TIANHONG BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021440020034 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230428 Granted publication date: 20080702 Pledgee: Shenzhen hi tech investment small loan Co.,Ltd. Pledgor: BEIJING KANGMEI TIANHONG BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021440020034 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080702 |