CN116465876A - 一种大田软海绵酸的快速检测方法及其试剂盒 - Google Patents
一种大田软海绵酸的快速检测方法及其试剂盒 Download PDFInfo
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Abstract
本发明属于水产品安全检测领域,具体涉及一种大田软海绵酸的快速检测方法及其试剂盒,所述试剂盒包括96孔酶标板架,12条酶标板条、6瓶不同浓度的OA标准品、1瓶偶联供体微球的OA‑BSA、1瓶偶联受体微球的OA抗体、缓冲液。本发明采用直接竞争,以表面包被有亲和涂层的聚苯乙烯微球为载体,当分子间存在相互作用使微球相互接近时,通过激发光激发,产生荧光,从而可以实现大田软海绵酸的快速检测。
Description
技术领域
本发明属于水产品安全检测领域,具体涉及一种大田软海绵酸的快速检测方法及其试剂盒。
背景技术
大田软海绵酸是腹泻性贝类毒素的一种,具有分布广泛,危害大等特点,它可以引起人体腹泻、呕吐等症状。除此之外,越来越多的研究表明OA具有细胞毒性、神经毒性、胚胎毒性、肝毒性等多种毒性。其致毒机制与其可以特异性抑制几种丝氨酸/苏氨酸蛋白磷酸酶有关。基于OA的这些危害,对OA进行快速、灵敏的检测显得尤为重要。目前应用最多的检测方法有液相色谱-串联质谱法、高效液相色谱法、生物传感器法、酶联免疫吸附法、蛋白磷酸酶抑制法等。但这些方法都有自己的缺点,如液相色谱-串联质谱法对环境的温度、湿度等要求高,测试速度慢,而且仪器昂贵,操作复杂,影响分析工作的效率,而生物传感器法的检测结果易受环境影响,可能存在灵敏度和稳定性等问题,蛋白磷酸酶抑制法易受样品基质的干扰,容易出现假阳性结果,进而降低检测结果的准确率。
均相光激化学发光免疫分析技术是一种以表面包被有亲和涂层的受体微球和供体微球为载体的均相免疫检测技术。该技术灵敏度高、特异性强、背景影响低、样品要求低、消耗少、分析操作简单并且检测范围广泛,既适用于传统ELISA和其他化学发光技术可以检测的大分子间相互作用,更可以胜任小分子相互作用的高通量检测。但是,目前并未查阅到该方法运用于大田软海绵酸检测的报道。
发明内容
本发明的目的在于提供一种大田软海绵酸的快速检测方法及其试剂盒,具有检测快速、操作简单、检测成本低的特点。
为实现上述目的,本发明采取的技术方案为:
一种大田软海绵酸的快速检测试剂盒,所述试剂盒包括96孔酶标板架,12条酶标板条、6瓶不同浓度的OA标准品、1瓶偶联供体微球的OA-BSA、1瓶偶联受体微球的OA抗体、缓冲液。其检测原理为:供体微球与受体微球在抗原抗体结合的作用下,使二者之间的距离小于200nm,在680nm的激发光的照射下,能量随单体氧分子扩散,传递至受体微球上的稀土原子铕,发射出615nm、半衰期为0.3s的激发光,供仪器检测。由此绘制标准曲线,由标准曲线及检测样品时的荧光值,即可得到贝类样品中OA的浓度。
其中,所述偶联供体微球的OA-BSA及偶联受体微球的OA抗体制备如下:取OA-BSA及OA抗体各0.1mg,采用超滤管转换缓冲条件至MES缓冲液;取供体微球及受体微球1mg,用MES洗涤微球;将OA-BSA与供体微球置于同一离心管,OA抗体与受体微球置于同一离心管,在37℃的条件下避光震荡18-20h,使之偶联;分别向两离心管内分别加入10μL0.2mol/L的NaBH4用于还原醛基,继续在37℃的条件下避光震荡2h;向偶联好的微球中分别加入500μL封闭缓冲液后13000r/min离心10min,弃去上清液;再分别加入350μL封闭缓冲液,超声震荡混匀后置于37℃震荡2h,离心取上清;最后加入1mL封闭缓冲液将微球偶联物配置为1mg/mL溶液。
进一步地,所述试剂盒内包括1个96孔酶标板架和适配的酶标板条。
进一步地,所述试剂盒内包括6瓶浓度不同的OA标准品,浓度分别为0,0.02,0.2,2,20,200ng/mL和1瓶偶联供体微球的OA-BSA1mg/mL和1瓶偶联受体微球的OA抗体1mg/mL。
进一步地,所述缓冲液为pH=7.8的包含NaCl、BSA、二乙烯三胺五乙酸(DTPA)、Tween-80、以及含NaN3的Tris-HCl的混合溶液。
进一步地,所述封闭液为6gTris-base,9gNaCl,0.5mLProcline300,2gBSA的混合溶液。
进一步地,所述缓冲液的制备如下:将8mmol/L的NaCl、0.1%的BSA、50μmol/L的二乙烯三胺五乙酸(DTPA)、0.1mol/L的Tween-80和含0.1%NaN3的Tris-HCl混合并调节pH至7.8。
本发明还提供了一种大田软海绵酸的快速检测方法,其采用上述的试剂盒实现大田软海绵酸的检测,具体的,所述检测方法使用竞争法,包括以下步骤:取酶标板条置于板架上,依次加入用缓冲液稀释好的偶联供体微球的OA-BSA、OA标准品以及偶联受体微球的OA抗体,OA标准品与偶联供体微球的OA-BSA竞争结合偶联受体微球的OA抗体,在37℃的条件下孵育10min,若偶联供体微球的OA-BSA与偶联受体微球的OA抗体结合,会使两微球间的距离小于200nm,在激发光的照射下产生荧光,反之则不产生荧光,用均相光激化学发光免疫分析仪检测其荧光强度;根据OA标准品浓度及其对应的荧光强度绘制标准曲线,然后将待测样品的荧光强度代入对应的标准曲线中,便可得到所述待测样品中OA的含量。
本发明采用直接竞争,以表面包被有亲和涂层的聚苯乙烯微球为载体,当分子间存在相互作用使微球相互接近时,通过激发光激发,产生荧光,从而可以实现大田软海绵酸的快速检测,操作简单的同时,降低了检测成本。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1为本发明实施例1中的标准曲线图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本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。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进。这些都属于本发明的保护范围。
实施例1OA均相光激化学发光免疫分析试剂盒的制备
偶联供体微球的OA-BSA及偶联受体微球的OA抗体的制备:分别取OA-BSA和OA抗体各0.1mg,采用超滤管转换缓冲条件至MES溶液;分别取供体微球和受体微球各1mg,均MES溶液洗涤;将OA-BSA与供体微球,OA抗体与受体微球置于同一离心管中,37℃避光震荡18-20h;不洗涤包被后的微球,分别加入10μL0.2mol/L的NaBH4溶液,37℃避光震荡2h;向两离心管中分别加入500μL封闭液,超声洗涤、离心去上清;再加入350μL封闭液,超声混匀,37℃避光震荡2h,离心去上清;最后加入1mL封闭缓冲液,将其配置为1mg/mL的偶联溶液。
缓冲液的制备:将8mmol/L的Nacl、0.1%的BSA、50μmol/L的二乙烯三胺五乙酸(DTPA)、0.1mol/L的Tween-80和含0.1%NaN3的Tris-HCl混合并调节pH至7.8。
封闭液的配置:6gTris-base,9gNaCl,0.5mLProcline300,2gBSA的混合溶液。
试剂盒的配置:
(1)96孔板×1块;
(2)适配96孔板的板条×12条;
(3)大田软海绵酸标准品×6瓶,浓度分别为0,0.02,0.2,2,20,200ng/mL;
(4)偶联供体微球的OA-BSA×1瓶,1mL;
(5)偶联受体微球的OA抗体×1瓶,1mL;
(6)缓冲液×1瓶,10mL。
其检测原理为:供体微球与受体微球在抗原抗体结合的作用下,使二者之间的距离小于200nm,在680nm的激发光的照射下,能量随单体氧分子扩散,传递至受体微球上的稀土原子铕,发射出615nm、半衰期为0.3s的激发光,供仪器检测。由此绘制标准曲线(如图1),其回归方程为:y=-0.85052-1.43944x,R2=0.99082。由标准曲线及检测样品时的荧光值,即可得到贝类样品中OA的浓度。
实施例2OA均相光激化学发光免疫分析试剂盒的应用
所述的OA均相光激化学发光免疫分析试剂盒的检测方法为:
样品处理方法:将剪碎的试样均质,准确称取10g(精确至0.1g),加入50mL90%的甲醇溶液,均质1~2分钟,6000r/min离心10分钟,转移出上清液,根据上清液体积加入2倍体积的PBS溶液,混合均匀,吸取25μL试样稀释液进行测定。
用缓冲液稀释偶联OA-BSA的供体微球以及偶联OA抗体的受体微球,依次向96孔板中加入25μL稀释好的偶联OA-BSA的供体微球、OA标准品以及偶联OA抗体的受体微球,OA标准品与偶联供体微球的OA-BSA竞争结合偶联受体微球的OA抗体,,37℃避光震荡反应10min,若偶联供体微球的OA-BSA与偶联受体微球的OA抗体结合,会使两微球间的距离小于200nm,在激发光的照射下产生荧光,反之则不产生荧光,用均相光激化学发光免疫分析仪检测其荧光值并绘制标准曲线,根据标准曲线得到样品中OA的浓度。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质内容。
Claims (8)
1.一种大田软海绵酸的快速检测试剂盒,其特征在于:所述试剂盒包括96孔酶标板架,酶标板条、OA标准品、偶联供体微球的OA-BSA、偶联受体微球的OA抗体、缓冲液,取OA-BSA及OA抗体各0.1mg,采用超滤管转换缓冲条件至MES缓冲液;取供体微球及受体微球1mg,用MES洗涤微球;将OA-BSA与供体微球置于同一离心管,OA抗体与受体微球置于同一离心管,在37℃的条件下避光震荡18-20h,使之偶联;分别向两离心管内分别加入10μL0.2mol/L的NaBH4用于还原醛基,继续在37℃的条件下避光震荡2h;向偶联好的微球中分别加入500μL封闭缓冲液后13000r/min离心10min,弃去上清液;再分别加入350μL封闭缓冲液,超声震荡混匀后置于37℃震荡2h,离心取上清;最后加入1mL封闭缓冲液将微球偶联物配置为1mg/mL溶液。
2.如权利要求1所述的一种大田软海绵酸的快速检测试剂盒,其特征在于:所述试剂盒内包括1个96孔酶标板架和适配的酶标板条。
3.如权利要求1所述的一种大田软海绵酸的快速检测试剂盒,其特征在于:所述试剂盒内包括6瓶浓度不同的OA标准品,浓度分别为0,0.02,0.2,2,20,200ng/mL和1瓶偶联供体微球的OA-BSA1mg/mL和1瓶偶联受体微球的OA抗体1mg/mL。
4.如权利要求1所述的一种大田软海绵酸的快速检测试剂盒,其特征在于:所述缓冲液为pH=7.8的包含NaCl、BSA、二乙烯三胺五乙酸(DTPA)、Tween-80、以及含NaN3的Tris-HCl的混合溶液。
5.如权利要求1所述的一种大田软海绵酸的快速检测试剂盒,其特征在于:所述封闭液为6gTris-base,9gNaCl,0.5mLProcline300,2gBSA的混合溶液。
6.如权利要求1所述的一种大田软海绵酸的快速检测试剂盒,其特征在于:所述缓冲液的制备如下:将8mmol/L的NaCl、0.1%的BSA、50μmol/L的二乙烯三胺五乙酸(DTPA)、0.1mol/L的Tween-80和含0.1%NaN3的Tris-HCl混合并调节pH至7.8。
7.一种大田软海绵酸的快速检测方法,其特征在于,采用如权利要求1-6任一项所述的试剂盒实现大田软海绵酸的检测。
8.如权利要求7所述的大田软海绵酸的快速检测方法,其特征在于,所述检测方法使用竞争法,包括以下步骤:取酶标板条置于板架上,依次加入用缓冲液稀释好的偶联供体微球的OA-BSA、OA标准品以及偶联受体微球的OA抗体,OA标准品与偶联供体微球的OA-BSA竞争结合偶联受体微球的OA抗体,在37℃的条件下孵育10min,若偶联供体微球的OA-BSA与偶联受体微球的OA抗体结合,会使两微球间的距离小于200nm,在激发光的照射下产生荧光,反之则不产生荧光,用均相光激化学发光免疫分析仪检测其荧光强度;根据OA标准品浓度及其对应的荧光强度绘制标准曲线,然后将待测样品的荧光强度代入对应的标准曲线中,便可得到所述待测样品中OA的含量。
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