CN101320039A - 一种地西泮胶体金法检测试纸及制备方法 - Google Patents
一种地西泮胶体金法检测试纸及制备方法 Download PDFInfo
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Abstract
一种地西泮胶体金法检测试纸及制备方法,属于生物学免疫方法的测定技术领域。本发明检测试纸由样品垫、结合释放垫、硝酸纤维素膜、吸水垫和塑料底板组成,塑料底板上依次粘贴样品垫、结合释放垫、硝酸纤维素膜和吸水垫,结合释放垫上包被了抗地西泮多抗-胶体金标记物,硝酸纤维素膜上依次包被有地西泮偶联抗原和羊抗兔IgG抗体,以地西泮偶联抗原作为测试线,以羊抗兔IgG抗体作为质控线。本发明具有使用方便、经济快捷、制作容易、成本低廉的特点。
Description
技术领域
一种地西泮胶体金法检测试纸及制备方法,属于生物学免疫方法的测定技术领域。
背景技术
地西泮(Diazepam)为苯二氮卓类镇静安眠药,化学名称为:1-甲基-5-苯基-7-氯-1,3-二氢-2H-1,4-苯并二氮卓-2-酮。曾用作动物饲料添加剂,作为生长促进剂,具有使动物嗜睡少动,生长快且有改变肉质的作用。也用在屠宰和动物检疫之前,或将动物转移屠宰之前,起镇静作用,以减轻动物紧张状态,降低动物伤害和死亡率。此类药物常见嗜睡,可见无力、头痛、晕眩、恶心、便秘,偶见皮疹、肝损害、骨髓抑制等不良反应。若随意在饲料中添加此类药物,蓄积的药物通过食物链进入人体,将造成巨大的危害。为此许多国家将此类药物列入禁用药物名单。目前,国内外有关地西泮的检测主要有高效液相色谱法(HPLC)、气相色谱法(GC)、薄层色谱法(TLC)、气-质联用法(GC-MS)、液-质联用法(LC-MS)等,但这些方法不仅需要昂贵的仪器设备,对检材的要求也比较高,需要进一步的提纯处理才能进行,且不能适应高通量、现场的快速检测。因此实现具有快速,便携优点的免疫检测方法的多残留检测具有现实意义。
发明内容
本发明的目的是提供一种地西泮胶体金法检测试纸及制备方法,为了克服现有检测技术的缺陷,提供一种便携,快速适合进行现场检测地西泮的免疫层析色谱检测法(gold immunochromatography assay;GICA)。
本发明的技术方案:一种地西泮胶体金法检测试纸,由样品垫、结合释放垫、硝酸纤维素膜、吸水垫和塑料底板组成,塑料底板上依次粘贴样品垫、结合释放垫、硝酸纤维素膜和吸水垫,结合释放垫上包被了抗地西泮多抗-胶体金标记物,硝酸纤维素膜上依次包被有地西泮偶联抗原和羊抗兔IgG抗体,以地西泮偶联抗原作为测试线,以羊抗兔IgG抗体作为质控线;
制备步骤为:
(1)将地西泮与牛血清蛋白分子进行偶联,作为地西泮偶联抗原;
(2)用地西泮偶联抗原按常规方法免疫制得抗地西泮多抗;
(3)用柠檬酸三钠还原剂将氯金酸还原制成20nm-40nm胶体金颗粒;
(4)将3mL胶体金调至pH 9,搅拌下逐滴加入蛋白浓度为0.2mg/mL的抗地西泮多抗0.05mL,放置30min后加入5%牛血清蛋白BSA溶液使终浓度为1%,放置至少30min后,10000转离心50min,移去上清液后用含5%蔗糖的0.002mol/LpH 9.0的硼酸盐缓冲液3mL重悬,重复2次,最后用0.3mL缓冲液溶解,得到稳定的抗地西泮多抗-胶体金标记物;
(5)将抗地西泮多抗-胶体金标记物包被在胶体金结合释放垫上,将地西泮偶联抗原和羊抗兔IgG抗体分别包被在硝酸纤维素膜上,地西泮偶联抗原作为测试线和羊抗兔IgG抗体作为质控线,在37℃烘箱干燥;
(6)试纸条的组装:将样品垫、结合释放垫、硝酸纤维素膜、吸水垫由一端依次黏附在塑料底板上,即得到用于免疫检测的地西泮胶体金法检测试纸。
本发明检测原理是利用样品垫形成的毛细管虹吸效应,使被检测物质首先与胶体金标记的抗地西泮多抗发生竞争形式的结合,其后果是,当胶体金标记的抗地西泮多抗过量时,多余的多抗漂移到测试线,与地西泮偶联抗原结合并显色;而与检测物结合的胶体金标记的抗地西泮多抗,其V区结合位点被检测物质占据,只能跨越测试线漂移到质控线以C区位点与羊抗兔IgG抗体非特异性结合,同测试线进行比色而得到检测结果。
本发明的有益效果:本发明应用层析式一步竞争法原理,试纸中上下线比色来半定量检测血清或尿液样品中的地西泮残留量,在5-10min内,快速准确地检测出样品是否含有地西泮,以确定地西泮是否超标,能够满足食品安全对地西泮残留量检测需求,适用于屠宰企业及政府检测机构。
本发明与现有技术相比,其效果不言而喻,即具有使用方便、经济快捷、制作容易、成本低廉的特点。
附图说明
图1本发明地西泮胶体金法检测试纸的侧视图。
具体实施方式
实施例1:地西泮检测试纸的制备
(1)将地西泮与牛血清蛋白分子偶联,作为地西泮偶联抗原,
(2)用地西泮偶联抗原免疫获得抗地西泮多抗:
(3)用柠檬酸三钠还原剂将氯金酸还原制成20nm-40nm胶体金颗粒;
(4)将3mL胶体金调至pH 9,搅拌下逐滴加入蛋白浓度为0.2mg/mL的抗地西泮多抗0.05mL,放置30min后加入5%牛血清白蛋白(BSA)溶液使终浓度为1%,放置至少30min后,10000转离心50min,移去上清液后用0.002mol/L pH9.0的硼酸盐缓冲液(含5%蔗糖)3mL重悬,重复2次,最后用0.3mL缓冲液溶解,得到稳定的抗地西泮多抗-胶体金标记物;
(5)将抗地西泮多抗-胶体金标记物包被在胶体金结合释放垫上,将地西泮偶联抗原和羊抗兔IgG抗体分别包被在硝酸纤维素膜上,作为测试线和质控线,在37℃烘箱干燥。
(6)试纸条的组装:将样品垫、结合释放垫、硝酸纤维素膜、吸水垫由一端依次黏附在塑料底板上,即得到用于检测的地西泮胶体金法检测试纸。
实施例2:试纸要求
(1)阴性参考品符合率
用磷酸盐缓冲液(PBS:0.01mol/L,pH 7.4)配制浓度为10ng/mL的盐酸异丙嗪标准品溶液进行10次平行检测,以正常或矫正目力观察结果,反应时间在5min时观察结果,应为阴性。
用磷酸盐缓冲液(PBS:0.01mol/L,pH 7.4)配制浓度为10ng/mL的巴比妥标准品溶液进行10次平行检测,以正常或矫正目力观察结果,反应时间在5min时观察结果,应为阴性。
(2)阳性参考品符合率
用磷酸盐缓冲液(PBS:0.01mol/L,pH 7.4)配制浓度为50、100、150ng/mL的地西泮标准品进行平行检测,各浓度进行10次平行实验,反应时间在5min时观察结果,不得出现阴性。
(3)最低检出量
用磷酸盐缓冲液(PBS:0.01mol/L,pH 7.4)分别配制浓度为0、2、5、10、20、50、100ng/mL的地西泮标准品溶液,各浓度进行10次平行实验,反应时间在5min时,以正常或矫正目力观察结果,最低检出量不高于50ng/mL。
(4)再现性
用磷酸盐缓冲液(PBS:0.01mol/L,pH 7.4)分别配制浓度为50ng/mL的地西泮标准品溶液,进行10次平行实验,反应时间在5min时,以正常或矫正目力观察结果,结果一致,显色度均一。
(5)稳定性测试
37℃放置10天后,各项指标应符合以上要求。
实施例3:试纸使用方法
(1)样品制备
血清:抽取待检动物血液,离心或静置后取透明上清液使用;若血清有过度溶血现象,将血清用蒸馏水稀释一倍后再进行实验,否则试剂片红色太深,影响测试结果。
尿液:用尿液直接进行测试,如浑浊,先离心取上清液或以蒸馏水1∶1稀释。
(2)操作
将试纸箭头端浸泡在待测样本中,不要超过样品垫1cm。浸入30sec取出放平,10min读取结果。
(3)检测结果
阴性:
两条带都显色,且测试线颜色深于质控线颜色,说明样品中不含地西泮。
两条带都显色,且测试线颜色与质控线颜色相同或接近,说明样品的地西泮含量低于50ppb(50ng/mL)。
阳性:
两条带都显色,但测试线颜色浅于质控线颜色,说明检测样品中地西泮含量超过50ppb(50ng/mL)。
测试线不显色,质控线显色,说明样品中的地西泮含量高于100ppb(100ng/mL)。
Claims (2)
1、一种地西泮胶体金法检测试纸,其特征在于由样品垫、结合释放垫、硝酸纤维素膜、吸水垫和塑料底板组成,塑料底板上依次粘贴样品垫、结合释放垫、硝酸纤维素膜和吸水垫,结合释放垫上包被了抗地西泮多抗-胶体金标记物,硝酸纤维素膜上依次包被有地西泮偶联抗原和羊抗兔IgG抗体,以地西泮偶联抗原作为测试线,以羊抗兔IgG抗体作为质控线。
2、权利要求1所述地西泮胶体金法检测试纸的制备方法,其特征在于制备步骤为:
(1)将地西泮与牛血清蛋白分子进行偶联,作为地西泮偶联抗原;
(2)用地西泮偶联抗原按常规方法免疫制得抗地西泮多抗;
(3)用柠檬酸三钠还原剂将氯金酸还原制成20nm-40nm胶体金颗粒;
(4)将3mL胶体金调至pH 9,搅拌下逐滴加入蛋白浓度为0.2mg/mL的抗地西泮多抗0.05mL,放置30min后加入5%牛血清蛋白BSA溶液使终浓度为1%,放置至少30min后,10000转离心50min,移去上清液后用含5%蔗糖的0.002mol/LpH 9.0的硼酸盐缓冲液3mL重悬,重复2次,最后用0.3mL缓冲液溶解,得到稳定的抗地西泮多抗-胶体金标记物;
(5)将抗地西泮多抗-胶体金标记物包被在胶体金结合释放垫上,将地西泮偶联抗原和羊抗兔IgG抗体分别包被在硝酸纤维素膜上,地西泮偶联抗原作为测试线和羊抗兔IgG抗体作为质控线,在37℃烘箱干燥;
(6)试纸条的组装:将样品垫、结合释放垫、硝酸纤维素膜、吸水垫由一端依次黏附在塑料底板上,即得到用于免疫检测的地西泮胶体金法检测试纸。
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CN112180081A (zh) * | 2020-09-07 | 2021-01-05 | 天津浩泰科技有限公司 | 一种小分子免疫层析检测方法以及试纸条和试剂盒 |
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CN100513423C (zh) * | 2007-11-02 | 2009-07-15 | 江南大学 | 一种地西泮人工抗原的制备方法 |
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CN112180081A (zh) * | 2020-09-07 | 2021-01-05 | 天津浩泰科技有限公司 | 一种小分子免疫层析检测方法以及试纸条和试剂盒 |
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