CN108152514A - 一种中性粒细胞明胶酶相关载脂蛋白ngal纳米高效检测试纸 - Google Patents
一种中性粒细胞明胶酶相关载脂蛋白ngal纳米高效检测试纸 Download PDFInfo
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- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- 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
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Abstract
本发明公开了一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,包括设在固定板上依次相连的样品垫、金标垫、硝酸纤维素膜和吸收垫;在硝酸纤维素膜上设有检测线抗体和质控线抗体;在金标垫上设有金标抗体,所述的样品垫为玻璃纤维棉制成的垫,所述的金标垫为羧化纤维素膜制成的垫,所述的金标抗体为显色抗体,检测线抗体为捕捉抗体,显色抗体与捕捉抗体为2株可互相配对形成夹心的抗人H‑FABP单克隆抗体,所述的金标抗体用40nm纳米金壳标记,所述金标垫上设有功能层。本发明通过特制的含有特定功能层的金标垫,使得金标释放速度放缓,得到缓冲,从而使样本中被分析物与金标抗体的浓度比值增大,从而提高了试纸条检测的灵敏度。
Description
技术领域
本发明属于生物技术应用领域,具体地说是一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸。
背景技术
NGAL(NeutrophiLGeLatinaseAssociatedLipocaLin)的分子量22KD、也有报告25KD,主要存在于中性类细胞内,其他组织中存量很少。各种原因(创伤、中毒、感染、疾病)导致急性和慢性肾损伤时,肾脏组织NGAL量明显升高释放入血液。检测血和/或尿NGAL是判断和评估急性和慢性肾损伤的生物标志物,是肾实质结构损伤的指标。类似诊断急性心肌梗死损伤的金标准心肌肌钙蛋白I、T。目前临床诊断急性和慢性肾损伤(功能不全)主要是检测血肌酐(creatine),尿素氮、尿微量蛋白,均为肾损伤的间接功能指标。急性肾损伤后,2-3天血肌酐方出现。NGAL在急性肾损伤2-3小时后在血和尿中就明显升高,检测NGAL提前和极大提高了诊断肾实质损伤的能力的时间,对合理诊治,降低死亡率提供了客观依据。
美国医院住院病人中急性肾损伤肾功能损伤患者约占总数5-7%,在医院重症病房(ICM)病人中比例高达25%,死亡率高达50-80%。急性和慢性肾损伤涉及心脏手术、化疗和放疗、重症高血压、糖尿病、败血症、心力衰竭、以及各种类型心肾综合征。该类病在美国麻省23家医院急性肾损伤(AKI)年花费保守估计80亿美元。因此,应用本申请专利检测NGAL将发挥巨大的社会和经济效益。。
在公告号为CN102955036B文件中,在检测样本时,试纸条表面的不干胶因刀片两边的受力不同,导致试金标垫因厚度不同而导致样本在金标垫上的流速不同,导致带有金标的抗体与样本中的被分析物结合不均匀,最终会导致检测结果的显示—检测线或控制线产生断线,即线条显示不完全的现象。此外,样品中样品添加集中添加量集中,液体流速快,样本很快的冲到金标垫上,即有大量的标记物被带到了金标垫,反而使金标垫的检测灵敏度降低,且检测速度偏慢。
发明内容
发明目的:针对现有技术中存在的不足,本发明的目的是提供一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,使其具有操作简便、灵敏度<3ng/mL、结果稳定等优点。
发明内容:本发明提供了一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,包括设在固定板上依次相连的样品垫、纳米材料垫、硝酸纤维素膜和吸收垫;所述硝酸纤维素膜上设有检测线抗体和质控线抗体;所述纳米材料垫为羧化纤维素膜制成的垫,纳米材料垫设有金包银纳米金壳复合物,所述的样品垫为玻璃纤维棉制成的垫,所述纳米材料垫上设有功能层;
进一步地,所述功能层以质量分数计由12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%组成;络合剂为酒石酸钾钠;
进一步地,金包银纳米金壳复合物是通过特定单链DNA将50nm和10nm纳米金包银壳标志物相联的复合物,所述特定单链DNA的核苷酸序列如SEQIDNO:1或SEQIDNO:2或SEQIDNO:3或SEQIDNO:4,所述特定单链DNA长度为30-80bp,特定单链DNA3’端标有生物素,特定单链DNA5’端连有巯基-SH;
上述的中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸的制备方法,包括以下步骤:
(1)纳米金壳溶液的制备;
(2)金壳标记制备和确认金包银壳纳米材料尺寸和纯度确定;
(3)纳米材料垫的制备:将羧化纤维素膜预处理后,烘干,通过真空环境中通过红外喷涂设备分别将配制好的12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%喷涂至羧化纤维素膜好形成纳米材料;
(4)金包银纳米金壳复合物的制备,即将信号抗体与10nm和50nm金包银壳标记物相联的复合物;用0.1M K2CO3调节pH8.5-9.0;将NGAL信号单抗与纳米壳比例调整为4:100(μg/ml);将单链DNA3'端标有生物素的30-80bp SSDNA与纳米壳信号抗体价链联接反应6-8小时,25℃,其终浓度为0.5-2.0μM;将SSDNA与纳米壳信号抗体复合物加进PEG8000终浓度为0.2-1.0%,2MNaCl/20mM phosphate调节SSDNA纳米壳复合体pH至7.4;将上述pH7.4复合体与链亲和素、辣根过氧化酶,终浓度0.4μM,在25℃条件下,孵育4小时,离心,沉淀清洗3次,保存4℃备用;
(5)金壳纳米材料垫制备;
(6)硝酸纤维素膜预处理和备用;
(7)制备和组装金壳纳米材料垫;
(8)检测线制备;
(9)质控线制备;
(10)试纸条装配。
有益效果:本发明的中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,结果易观察,灵敏度高,5分钟内就能判读结果,具有良好的稳定性和重复性。操作简便,无需特殊仪器设备,可应用于各种原因导致的急性肾损伤的早期诊断,慢性肾功能不全透析及治疗效果评判。相对于现有技术其检测效率大幅提升。
本发明通过特制的含有特定功能层的纳米材料垫,使样本中被分析物与金标抗体的浓度比值增大,从而提高了试纸条检测的灵敏度。
此外,通过特定功能层的纳米材料垫,通过特定比例的12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%的作用,避免了纳米材料垫因厚度不同而导致样本在纳米材料垫上的流速不同,导致抗体与样本中的被分析物结合不均匀,最终会导致检测结果的显示—检测线或控制线产生断线,即线条显示不完全的现象。
附图说明
图1是试纸条的结构示意图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
如图1所示,实施例制备的均是一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,包括设在固定板上依次相连的样品垫1、纳米材料垫2、硝酸纤维素膜3和吸收垫4;所述硝酸纤维素膜3上设有检测线抗体5和质控线抗体6;所述纳米材料垫2为羧化纤维素膜制成的垫,纳米材料垫2设有金包银纳米金壳复合物,所述的样品垫1为玻璃纤维棉制成的垫,所述纳米材料垫上设有功能层。所述功能层以质量分数计由12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%组成。金包银纳米金壳复合物是通过特定单链DNA将50nm和10nm纳米金包银壳标志物相联的复合物,所述特定单链DNA的核苷酸序列如SEQIDNO:1或SEQIDNO:2或SEQIDNO:3或SEQIDNO:4,所述特定单链DNA长度为30-80bp,特定单链DNA3’端标有生物素,特定单链DNA5’端连有巯基-SH。
本实验用到的材料:检测线抗体为抗人NGAL单克隆抗体BOT06为捕捉抗体,质控线抗体为羊抗鼠IgG,金壳标记抗体包含有显色抗体为鼠抗人NGAL抗体BOT05,购买于Genscript The Biology CRO南京公司。
金包银纳米金壳复合物所用到的特定单链DNA是由本公司设计合成制得。其他材料均自市场上购买。本专利是针对本公司专利公告号为CN102955036B的改进,某些现有技术参照其内容作省略。
实施例1
(1)纳米金壳溶液的制备:
以柠檬酸钠和鞣酸为双稳定剂和还原剂,制备单分散的,尺寸可调的准球形银纳米颗粒溶液。在50-70℃的水浴下,将1.5-2.0ml的1%AgNO3溶液加入到50-70ml去离子水中,并加热至恒温。该溶液体系记为A;在50-70℃的水浴下,将0.05-5.0ml的0.5%的鞣酸溶液和1-3ml的1%的柠檬酸钠溶液加入到20-40ml的去离子水中,并加热到恒温,该溶液记为B。在剧烈搅拌下,将B导入A中,并持续搅拌到溶液呈现亮黄色,此后将反应溶液加热到沸腾,并保持回流,持续10-30分钟,室温冷却后定容到100ml。
以单分散的银纳米颗粒为牺牲模版,通过置换反应制备但分散的金纳米壳溶液:取20-40ml银纳米颗粒溶液,在12000-15000g,4℃下离心15-30分钟,吸出上清液,再以去离子水重新分散,并定容到100ml。将此100ml的银纳米颗粒溶液迅速加热到沸腾,在保持回流和剧烈搅拌下加入2mM HAuCl4(或NaAuCl4/KAuCl4)1-4ml,反应液颜色逐渐由黄色转变为墨绿色,之后在继续搅拌的情况下室温冷却。
(2)显色抗体的金标记:
用0.05-0.20MK2CO3调节纳米金壳溶液至pH为7.4-9.0。于80-120ml纳米金壳溶液中逐滴加入显色抗体(鼠抗人NGAL抗体BOT05)2.0-4.0mg,搅拌3-10分钟),静置20-60分钟。再加入15-35ml,2-10%BSA和20mM Tris-HCl pH7.5-9.0,2-8℃封闭过夜,得反应液;反应液于2-8℃,3000-5000g,离心20-40分钟,沉淀用100-150ml,0.5-2%BSA和20mM Tris-HClpH8.0-9.0重悬,重复离心两次,离心速度依次为4000-2000g和3000-1000g得沉淀。用1000-1500μl重悬液(成份为20-40%海藻糖,0.5-2.0%BSA,0.005-0.02%Tween-20,0.01-0.03%NaN3,10-30mM Tris-HCl,pH8.0-9.0)悬浮沉淀,得金标抗体;
(3)纳米材料垫制备及喷垫:
纳米材料垫的制备:将羧化纤维素膜预处理后,烘干,通过真空环境中通过红外喷涂设备分别将配制好的12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂7%、硫酸铜0.4%、络合剂0.6%喷涂至羧化纤维素膜好形成纳米材料;烘干后,将金标抗体喷印在纳米材料垫上;
(4)检测线制备:
取1株抗人NGAL单克隆抗体BOT06(可与鼠抗人NGAL抗体BOT05配对形成夹心)做检测线抗体,以缓冲液5-20%海藻糖,3-8%甲醇稀释为
0.5-2.0mg/mL;用划膜仪将抗体划在硝酸纤维素膜上,浓度为1μl/cm形成检测线,真空干燥;
(5)质控线制备:
羊抗鼠IgG用缓冲液5-20%海藻糖,3-8%甲醇稀释为0.2-1.0mg/ml;用划膜仪将抗体划在硝酸纤维素膜上,浓度为0.5μl/cm形成质控线,真空干燥;
(6)试纸条装配:
将样品垫1、纳米材料垫2、硝酸纤维素膜3和吸水纸4按顺序粘贴在单面压敏胶的塑料板上,在硝酸纤维素膜3上设有检测线抗体5和质控线抗体6;切割粘贴后的塑料板,装入检测卡;用带有干燥剂的热封锡箔袋包装,室温保存;
(7)检测:
将样品滴加入加样孔,10-20分钟时观察,判读试纸条检测结果的标准为:阳性,检测线处和质控线处均有显色条带;阴性,检测线处无显色条带,质控线处有显色条带;试纸条失效,质控线处无显色条带。
实施例2:
其步骤相同,步骤(3)如下:
(3)纳米材料垫制备及喷垫:
纳米材料垫的制备:将羧化纤维素膜预处理后,烘干,通过真空环境中通过红外喷涂设备分别将配制好的12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6.5%、硫酸铜0.6%、络合剂0.9%喷涂至羧化纤维素膜好形成纳米材料;烘干后,将金标抗体喷印在纳米材料垫上;
将100μL配制的10ng/ml、50ng/ml、100ng/ml和200ng/mlNGAL标准品样品滴加入实施例1和2的样品孔,将样品滴加入加样孔,4分钟时出现结果,判读试纸条检测结果的标准为:阳性,检测线处和质控线处均有显色条带;阴性,检测线处无显色条带,质控线处有显色条带;试纸条失效,质控线处无显色条带。且显色灵敏度<3ng/mL,无断线现象。
以上所述仅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,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,其特征在于:包括设在固定板上依次相连的样品垫(1)、纳米材料垫(2)、硝酸纤维素膜(3)和吸收垫(4);所述硝酸纤维素膜(3)上设有检测线抗体(5)和质控线抗体(6);所述纳米材料垫(2)为羧化纤维素膜制成的垫,纳米材料垫(2)设有金包银纳米金壳复合物,所述的样品垫(1)为玻璃纤维棉制成的垫,所述纳米材料垫上设有功能层。
2.根据权利要求1所述的一种中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸,其特征在于:所述功能层以质量分数计由12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%组成。
3.一种制备权利要求1所述的中性粒细胞明胶酶相关载脂蛋白NGAL纳米高效检测试纸的制备方法,其特征在于,包括以下步骤:
(1)纳米金壳溶液的制备;
(2)金壳标记制备和确认金包银壳纳米材料尺寸和纯度确定;
(3)纳米材料垫的制备:将羧化纤维素膜预处理后,烘干,通过真空环境中通过红外喷涂设备分别将配制好的12%纯硝酸银、70%明胶、6%的司本80、4%的环糊精,碱剂6-8%、硫酸铜0.1-0.4%、络合剂0.5-1%喷涂至羧化纤维素膜好形成纳米材料;
(4)金包银纳米金壳复合物的制备,即将信号抗体与10nm和50nm金包银壳标记物相联的复合物;用0.1M K2CO3调节;pH8.5-9.0;将NGAL信号单抗与纳米壳比例调整为4:100(μg/ml);将单链DNA3'端标有生物素的30-80bpSSDNA与纳米壳信号抗体价链联接反应6-8小时,25℃,其终浓度为0.5-2.0μM;将SSDNA与纳米壳信号抗体复合物加进PEG8000终浓度为0.2-1.0%,2MNaCl/20mM phosphate调节SSDNA纳米壳复合体pH至7.4;将上述pH7.4复合体与链亲和素、辣根过氧化酶,终浓度0.4μM,在25℃条件下,孵育4小时,离心,沉淀清洗3次,保存4℃备用;
(5)金壳纳米材料垫制备;
(6)硝酸纤维素膜预处理和备用;
(7)制备和组装金壳纳米材料垫;
(8)检测线制备;
(9)质控线制备;
(10)试纸条装配。
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CN102955036A (zh) * | 2012-11-27 | 2013-03-06 | 南京医科大学第二附属医院 | 一种高敏感性中性粒细胞明胶酶相关载脂蛋白ngal的纳米材料检测试纸条及其制备方法 |
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WO1996024060A1 (en) * | 1995-02-03 | 1996-08-08 | British Biocell International Limited | Assay device and method |
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CN102955036B (zh) * | 2012-11-27 | 2015-11-18 | 南京医科大学第二附属医院 | 一种高敏感性中性粒细胞明胶酶相关载脂蛋白ngal的纳米材料检测试纸条及其制备方法 |
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