CN114133326A - 一种斑蝥黄胶体金侧向流免疫层析卡的制备及其应用 - Google Patents
一种斑蝥黄胶体金侧向流免疫层析卡的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种斑蝥黄半抗原的结构式,以及其对应的人工抗原、抗体与免疫层析检测卡的制备方法及其在斑蝥黄胶体金侧向流免疫层析法(Gold lateral flow assay,GLFA)检测中的应用。本发明制备的斑蝥黄人工抗体具备高免疫原性,经动物免疫制备得到的抗体对斑蝥黄具备高亲和度结合力,且制备的斑蝥黄免疫层析检测卡可用于禽蛋样本中斑蝥黄的现场快速检测。
Description
技术领域
本发明属于免疫分析化学技术领域,具体涉及一种斑蝥黄人工抗原、抗体及其免疫层析检测卡的制备与斑蝥黄胶体金侧向流免疫层析检测方法的建立。
背景技术
斑蝥黄,又名角黄素,结构式如附图1,是类胡萝卜属的一种。2019年央视315晚会曝光家禽吃了添加斑蝥黄的饲料,蛋黄颜色会变得更深,不法商家借此将普通饲养禽蛋冒充为“土鸡/鸭蛋”。因此,如何对禽蛋产品中的斑蝥黄进行检测成为一个重要的食品安全问题。
目前,国内外对斑蝥黄残留的检测主要为高效液相色谱法和液相色谱-质谱法。这类方法虽具备检测灵敏度高、特异性强等优势,但是检测样本样品前处理复杂、耗时,同时仪器检测方法需要昂贵的大型仪器和设备,配备专业的检测技术人员进行操作和管理,无法进行现场大规模检测,时效性差,难以推广。
胶体金侧向流免疫层析法(Gold lateral flow assay,GLFA)是一种利用抗体抗原特异性结合原理和免疫浓缩手段进行检测分析的免疫标记分析方法,具备高特异性、高灵敏度、检测时限短、显色明显稳定、受基质影响小等优点。然而得到高亲和力的特异性抗体是实现GLFA检测的前提,其中人工抗原的合成是其中重要的一步。
由于斑蝥黄是小分子物质(分子量MW<1kDa),本身不具有诱导机体产生抗体的能力,需要和蛋白载体偶联后,才能具有免疫原性。但斑蝥黄结构中没有可以直接利用的活性基团如-COOH、-NH2、-OH或-SH2等,因此,要实现斑蝥黄和载体蛋白的偶联,首先就要设计合理的斑蝥黄半抗原。
发明内容
本发明的第一目的在于提供一种斑蝥黄半抗原,其结构式如图2所示。该半抗原分子一侧与斑蝥黄完全一致,另一侧具有易于与载体相偶联的-COOH,而且还可以商业化获得,因此是一个非常优秀的半抗原。
本发明的第二目的在于提供一种斑蝥黄抗体的制备方法。
本发明的第三目的在于提供一种斑蝥黄免疫层析检测卡的制备方法及其应用。
为了实现本发明的上述目的,特采用以下技术方案:
(一)本实施方式提供了一种斑蝥黄人工抗原的制备方法,其包括:
(1)用DMF溶解斑蝥黄半抗原(结构式见附图2)、EDC和NHS(质量比为7:10:10),室温下搅拌反应2-4h后得A液;0.75mg/mL的载体蛋白水溶液记为B液;
(2)在4℃搅拌下将A液缓慢加入至B液中,加入体积比为A液:B液=27:8,反应1-12h;
(3)纯化:反应液离心后弃沉淀,上清液在4℃下透析48h后冻干成粉末,-20℃保存。
(二)本实施方式提供了斑蝥黄抗体的制备方法,其包括:
(1)斑蝥黄人工抗原用生理盐水配置成0.5mg/mL浓度,并与等体积的弗氏完全佐剂相互混合;
(2)将二者分别装入不同的注射器针管中,针头用空心软管连通,通过来回推动注射器活塞使人工抗原与佐剂进行乳化;
(3)初次免疫使用弗氏完全佐剂,加强免疫使用弗氏不完全佐剂,按多点式皮下注射的方式进行药。免疫动物包括新西兰大白兔、SD大鼠和昆明小鼠;当免疫动物为新西兰大白兔时免疫剂量为2mL/只;当免疫动物为SD大鼠时免疫剂量为1mL/只;当免疫动物为昆明小鼠时免疫剂量为0.1-0.5mL/只。免疫频率皆为10d一次,免疫周期在60-90d。
(4)多克隆抗体制备:收集免疫动物的抗血清,Protein G亲和层析法纯化后,将洗脱的多克隆抗体液置于4℃环境透析10-14h,冷冻干燥成粉末,-20℃保存。
(三)本实施方式提供了一种斑蝥黄免疫层析检测卡的制备方法,其包括:
(1)胶体金的制备:取约3mL的1%(W/V)四水合氯金酸溶液,与100mL去离子水共同置于圆底烧瓶中,油浴环境下加热至沸腾后,快速加入10-20mL 1%(W/V)的柠檬酸钠溶液,保持微沸状态加热5-30min,待体系颜色稳定为酒红色时停止加热。将胶体金冷却后4℃保存。
(2)胶体金标记斑蝥黄抗体的制备方法:取100mL胶体金,用0.1mol/L碳酸钾溶液调节pH到8。搅拌状态下加入斑蝥黄抗体,搅拌20min后再逐滴加入聚乙二醇20000(PEG20000),搅拌10-15min。反应完成后离心弃上清液,加入10mL PBS缓冲液(含0.4mg/mLPEG)清洗2次。将沉淀用含2%BSA的PBS缓冲液(pH=7.4)溶解,4℃保存备用。
(3)胶体金结合垫的制备:取胶体金标记的抗体离心,取上清液涂于玻璃纤维膜上,室温下晾干,制成胶体金结合垫;
(4)免疫层析膜的包被:按固定浓度在检测线(T)上包被一定体积的斑蝥黄抗体,质控线(C)上包被一定体积的羊抗大鼠IgG,T线和C线的宽度皆在1.5-2.5mm范围内;
(5)免疫层析检测卡的组装:聚氯乙烯衬板作为支撑载体固定于免疫层析检测卡槽下壳体中,然后样品垫、胶体金结合垫、免疫层析膜和吸水滤纸依次排列连接于聚氯乙烯衬板上表面(拼接顺序如附图7所示),再将免疫层析检测卡槽上壳体与下壳体通过卡扣连接,就得到斑蝥黄免疫层析检测卡。常温密封储存。
(四)本实施方式提供了一种斑蝥黄GLFA分析方法,其包括:
(1)检测方法
取斑蝥黄免疫层析检测卡,加样池端微微向下倾斜;用定量管移取禽蛋样品液并快速滴加在检测卡的样品池中,加样完成后静置等待5-30min,观察检测线和质控线的颜色变化。
(2)结果判读
如附图8所示,检测卡结果判读方法为:
A.如果C线显色明显,且T线无肉眼可见颜色,证明该样本为阳性样本,禽蛋中斑蝥黄含量在灵敏度下限以上;
B.如果C线显色明显,且T线有肉眼可见颜色,证明该样本为阴性样本,禽蛋中斑蝥黄含量在灵敏度下限以下;
C.如果C线不显色,表明该检测卡失效。
与现有技术相比,本发明的有益效果为:
第一,本发明首次提供了一种有效的斑蝥黄的半抗原结构式及其人工抗原、抗体的制备方法。
第二,本发明提供了一种斑蝥黄快速免疫层析检测卡制备方法。该免疫层析检测卡制备工艺简单、材料易得,实用性强,尤其适合于工业大生产。
第三,本发明提供了一种斑蝥黄GLFA快速分析方法,该GLFA法具有特异性强、操作简捷、结果判读简单等有钱,尤其是不需要贵重仪器,对于斑蝥黄的残留检测具有重大价值。
为了更清楚地说明本发明实施例或现有技术中的技术方案,以下将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为斑蝥黄结构图;
图2为斑蝥黄半抗原结构图;
图3为斑蝥黄人工抗原1的紫外鉴定图;
图4为斑蝥黄人工抗原2的紫外鉴定图;
图5为斑蝥黄人工抗原1和人工抗原2的SDS-PAGE蛋白电泳图;
图6为斑蝥黄多克隆抗体的SDS-PAGE蛋白电泳法图;
图7为免疫层析检测卡结构图;
图8为GLFA检测结构判定图;图a)阳性样本;图b)阴性样本;图c)、图d)检测卡无效;
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。
下述实施例中的实验方法,如无特殊说明,均为本领域技术人员所熟知的常规方法。下述实施例中所用的试验材料,如无特殊说明,均为从常规生化试剂厂商购买得到。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。以下实施例中所用的磷酸盐缓冲液(简称PBS)均为0.01mol/L、pH=7.4的磷酸盐缓冲液,三乙醇胺缓冲液(简称PBS)均为0.01mol/L、pH=7.4的三乙醇胺缓冲液。牛血清白蛋白简称BSA,卵清白蛋白简称OVA,辣根过氧化物酶简称HRP,碳二亚胺简称EDC,N-羟基丁二酰亚胺简称NHS,N,N-二甲基甲酰胺简称DMF。
实施例1:斑蝥黄人工抗原的合成与鉴定
(1)斑蝥黄人工抗原1的合成:
①称取斑蝥黄7mg、EDC 10mg、NHS 10mg于S DMF中,搅拌反应4h,记为A液;
②称取OVA 6mg溶于8mL水中,记为B液;
③在4℃搅拌下将A液缓慢加入至B液中,反应6h;
④纯化:将反应液于4000r/min下离心10min,取上清液置于透析袋在4℃下透析48h,最终得到斑蝥黄人工抗原,冻干成粉末-20℃进行保存。
(2)斑蝥黄人工抗原2的合成:
①称取斑蝥黄7mg、EDC 10mg、NHS 10mg于2mL DMF中,搅拌反应4h,记为A液;
②称取BSA 6mg溶于8mL水中,记为B液;
③在4℃搅拌下将A液缓慢加入至B液中,反应6h;
④纯化:将反应液于4000r/min下离心10min,取上清液置于透析袋在4℃下透析48h,最终得到斑蝥黄人工抗原,冻干成粉末-20℃进行保存。
(3)紫外-可见分光光度计鉴定:
①操作:将斑蝥黄半抗原、BSA、OVA、人工抗原1和人工抗原2分别稀释一定的浓度,利用紫外可见分光光度计对3者进行紫外吸收扫描。
②结果:附图3显示人工抗原1同时具有斑蝥黄和OVA的特征峰,且具有一定位移,证明OVA与斑蝥黄偶联成功;附图4显示人工抗原2同时具有斑蝥黄和BSA的特征峰,且具有一定位移,证明BSA与斑蝥黄偶联成功。综上所述,斑蝥黄人工抗原1与斑蝥黄人工抗原2制备成功。
(4)SDS-PAGE蛋白电泳法鉴定:
①样品的处理:取2μg/mL的BSA、OVA和人工抗原1和人工抗原2各15μL,与等体积的Loading buffer混匀,煮沸10min。
②上样:使用移液器将待测样品加入样品槽中(吸收待测样品时,避免吸入气泡),每孔上样量为15μL。
③电泳:打开电源,设置电压为180V,直至溴酚蓝指示剂到达凝胶的底部,立即关闭电源。
④染色:取出凝胶,倒入考马斯亮蓝R-250染色液至浸没凝胶,室温下摇床染色2-3h,待整个凝胶被染透后,染色完成。
⑤脱色处理:待染色完成后,弃去染色液,用水清洗表面残留染色液,加入脱色液,置于脱色摇床上脱色过夜,直至凝胶呈现乳白透明状,蓝色条带清晰可见。
⑥结果:如附图5所示,由于偶联半抗原后,人工抗原1的分子量大于其对应的载体蛋白,即OVA,致使OVA的电泳速度略大于人工抗原1,所以OVA的电泳条带与人工抗原1电泳条带位置将近或出现于人工抗原1电泳条带的下方;同理,BSA的电泳条带出现于人工抗原2电泳条带的下方。综上所述,证明斑蝥黄人工抗原1与斑蝥黄人工抗原2制备成功。
实施例2:斑蝥黄多抗血清的制备与评估
(1)抗血清的制备:
①用生理盐水将实例1中值备斑蝥黄人工抗原2配置成0.5mg/mL后与等体积弗氏完全佐剂相互混合。
②将二者装分别装入不同5mL无菌注射器针筒内,并将此二注射器头部用0.5mmPVC透明空心塑料管连接,通过来回推动注射器活塞使抗原与佐剂发生乳化。耗时约10min,使其呈稳定均匀不分层的乳液状态,得到0.25mg/mL的斑蝥黄人工抗原乳液。
③免疫动物为4周龄SD雌性大鼠。初次免疫使用弗氏完全佐剂,加强免疫使用弗氏不完全佐剂。免疫剂量为1mL/只,免疫频率为10d一次;免疫周期共90d。给药时按皮下注射方式,在大鼠脊椎、腿、腋下等部位多点少量进行注射。
④末次免疫后再突击免疫一次,7天后用5%水合氯醛麻醉按300mg/Kg剂量麻醉,心脏采血法取全血,在37℃静置0.5h后3000r/min离心10min,收集上层抗血清。
(2)抗血清效价评估:
采用夹心ELISA法对抗血清效价进行评估。
①取实验例1中制备的人工抗原1用生理盐水配置成5μg/mL浓度,并按100μL/孔加入酶标板,置于4℃包被过夜。
②取出ELISA板,弃去液体,每孔加入100μL PBS(含5%Tween20)进行洗涤,共洗涤3次,3min/次。
③洗涤后每孔加入100μL 5%的脱脂奶粉,37℃,2h。
④待封闭结束后,弃液,PBS(含5%Tween 20)洗涤三次,再加入100μL/孔逐级稀释好的抗血清,37℃孵育1.5h
⑤孵育后弃液,PBS(含5%Tween 20)洗涤三次。
⑥加入HRP标记的羊抗大鼠的IgG(1:1000)稀释,100μL/孔,37℃,2h。
⑦弃HRP标记的羊抗大鼠的IgG,PBS(含5%Tween 20)洗涤3次,加入底物显色液,100μL/孔,37℃避光显色20min。
⑧加入终止液,50μL/孔,酶标分析仪上读取每孔OD450值并记录。
⑨当样本OD450值≥2.1*阴性对照OD450值时,认为样本为阳性样本;取阳性值对应的最大稀释比为抗血清效价。
⑩结果:斑蝥黄抗血清效价≥1:64000,可用于提取多克隆抗体。
实施例3:斑蝥黄多克隆抗体的纯化与评估
(1)斑蝥黄多克隆抗体的纯化:
①平衡:取10mL Protein G Agarose填料装填于12mL亲和纯化柱中,用20倍柱体积的TBS洗涤并平衡纯化柱;
②上样:纯化柱平衡后,把含有待纯化的抗血清上样到纯化柱,4℃等待30min使多克隆抗体与Protein G充分结合后进行过柱;
③洗涤:过柱后用20倍柱体积的TBS洗涤纯化柱,以去除未结合和非特异性结合的蛋白,用紫外分光光度计检测290nm处吸光度来判定是否洗涤干净;
④洗脱:洗涤完后,用10mL洗脱液缓冲液(1mol/L甘氨酸,调节pH至2.7)洗脱结合的多克隆抗体,收集洗脱的多克隆抗体,4℃透析12h以除盐,最后将多克隆抗体冻干成粉末-20℃进行保存。
(2)斑蝥黄多克隆抗体SDS-PAGE蛋白电泳纯度鉴定:
①样品的处理:取样品的处理:取180μg/mL名都的斑蝥黄多克隆抗体、抗血清液、多克隆抗体洗脱液,分别与1×Loading buffer(1:1)混匀,煮沸10min。
②加样:取15μL样品加入加样孔中;
③电泳:浓缩胶运行电压180V,运行50min;
④染色:取出凝胶,倒入考马斯亮蓝R-250染色液至侵没凝胶,室温下摇床染色2-3h,待整个凝胶被染透后,染色完成。
⑤脱色处理:待染色完成后,弃去染色液,用水清洗表面残留染色液,加入脱色液,置于脱色摇床上脱色过夜,直至凝胶呈现乳白透明状,蓝色条带清晰可见。
⑥结果:斑蝥黄多克隆抗体的的SDS-PAGE蛋白电泳法鉴定结果如图6所示,相比抗血清,斑蝥黄多克隆抗体仅在55KDa处有明显条带,说明本方法纯化的斑蝥黄多克隆抗体具备高纯度;且抗血清洗脱液在55KDa处无条带,说明本方法将抗血清中的斑蝥黄多克隆抗体完全分离纯化。
实施例4:一种斑蝥黄免疫层析检测卡制备方法
(1)胶体金的制备:
取3.1mL 1%(W/V)的四水合氯金酸溶液,与100mL去离子水混合后置于圆底烧瓶中,用油浴锅在200r/min转数下一边冷凝回流一边加热,加热至沸腾后,快速加入10mL 1%(W/V)的柠檬酸钠溶液,并保持搅拌和微沸状态加热15min,待体系颜色稳定为酒红色时停止加热。待胶体金冷却后4℃保存。
(2)胶体金标记斑蝥黄多克隆抗体的制备方法:
取胶体金100mL,用0.1mol/L碳酸钾溶液调pH到8.0。边搅拌边加入1.5mg斑蝥黄多克隆抗体,搅拌20min后再逐滴加入2mL 25mg/mL的聚乙二醇20000(PEG20000),搅拌15min。20000r/min离心15min后弃上清液,加入10mL PBS(含0.4mg/mL PEG)清洗2次。将沉淀用5mLPBS(含2%BSA)溶解,4℃保存。
(3)胶体金结合垫的制备:
取10mL胶体金标记抗体,离心后弃沉淀,取上清液用均匀地涂在玻璃纤维膜上,室温下晾干,制成胶体金结合垫;
(4)免疫层析膜的包被:
检测线(T)上按0.075μg/cm剂量包被斑蝥黄多克隆抗体,每条线宽2mm;质控线(C)上按0.75μg/cm剂量包被羊抗大鼠IgG,每条线宽2mm;
(5)免疫层析检测卡的组装:
聚氯乙烯衬板作为支撑载体固定于免疫层析检测卡槽下壳体中,然后样品垫、胶体金结合垫、免疫层析膜和吸水滤纸依次排列连接于聚氯乙烯衬板上表面(拼接顺序如附图7),再将免疫层析检测卡槽上壳体与下壳体通过卡扣连接,就得到斑蝥黄检测卡。常温密封储存。
实施例5:GLFA检测方法建立及应用
(1)检测方法
将斑蝥黄免疫层析检测卡加样池端微微向下倾斜;移取经前处理的禽蛋样品液并快速滴加在检测卡的样品池中,加样完成后静置等待20min,观察检测线和质控线的颜色变化。
(2)结果判读
如附图8所示,检测卡结果判读方法为:
A.如果C线显色明显,且T线无肉眼可见颜色,证明该样本为阳性样本,禽蛋中斑蝥黄含量在灵敏度下限以上;
B.如果C线显色明显,且T线有肉眼可见颜色,证明该样本为阴性样本,禽蛋中斑蝥黄含量在灵敏度下限以下;
C.如果C线不显色,表明该检测卡失效。
Claims (10)
2.如权利要求1所述的斑蝥黄人工抗原,其特征在于:所述载体为蛋白质类载体、多肽类载体或大分子有机化合物类载体。
3.如权利要求2所述的蛋白质类载体,其特征在于:所述载体为牛血清白蛋白、血蓝蛋白或卵清白蛋白。
4.如权利要求2所述的多肽类载体,其特征在于:所述载体为多聚赖氨酸、多聚谷氨酸或多聚混合氨基酸。
5.如权利要求2所述的大分子有机化合物类载体,其特征在于:所述载体为聚乙烯比咯烷酮、淀粉、硫酸葡萄糖、羧甲基纤维素、聚甲基丙酸酯微粒、乳胶或炭末。
6.根据权利要求2所述的斑蝥黄人工抗原的制备方法,其特征在于:包括以下步骤:
(1)按7:10:10的质量比称取斑蝥黄半抗原、碳二亚胺(EDC)和N-羟基丁二酰亚胺(NHS),并将其溶解于N,N-二甲基甲酰胺(DMF)中,室温下搅拌2-4h,最后得到A液;
(2)配置0.75mg/mL浓度的载体蛋白试液,记为B液;
(3)在4℃搅拌下按A液:B液=27:8的体积比缓慢将A液加入至B液中,反应1-12h;
(4)纯化:反应液离心后取其上清液,置于透析袋在0-4℃下透析24-72h,冻干成粉末低温进行保存。
7.如权利要求1所述的斑蝥黄人工抗体,其特征在于:所述人工抗体为人工抗原免疫动物,并经进一步后处理所得的单克隆抗体或多克隆抗体。
8.如权利要求7所述的斑蝥黄人工抗体,其特征在于:所述人工抗体为多克隆抗体。
9.如权利要求8所述斑蝥黄多克隆抗体的制备方法,其特征在于:包括以下步骤:
(1)将斑蝥黄人工抗原用生理盐水配置成0.5mg/mL的溶液,并与等体积的弗氏完全佐剂;将二者混合乳化,使成乳白色均匀的乳剂;
(3)初次免疫使用弗氏完全佐剂,加强免疫使用弗氏不完全佐剂,按多点式皮下注射的方式进行药。免疫动物包括新西兰大白兔、SD大鼠和昆明小鼠。当免疫动物为新西兰大白兔时免疫剂量为1-3mL/只;当免疫动物为SD大鼠时免疫剂量为0.2-1mL/只;当免疫动物为昆明小鼠时免疫剂量为0.1-0.5mL/只;免疫频率为10d一次,免疫周期在60-90d。
(4)收集免疫动物的抗血清,Protein G亲和层析法纯化后,将洗脱的多克隆抗体液置于4℃环境透析10-14h,冷冻干燥成粉末,低温保存。
10.使用斑蝥抗体,制备出一种斑蝥黄免疫层析检测卡,制备及其应用步骤如下:
(1)胶体金的制备:取约3mL的0.1-5%(W/V)四水合氯金酸溶液,加入100mL去离子水于圆底烧瓶中,油浴环境下加热至沸腾后,快速加入1-20mL 1%(W/V)的柠檬酸钠溶液,保持微沸状态加热5-30min,待体系颜色稳定为酒红色时停止加热,冷却即得。
(2)胶体金标记斑蝥黄抗体的制备:取胶体金100mL用0.1mol/L碳酸钾溶液调pH 7.0-10.0。边搅拌边加入斑蝥抗体,搅拌20min后再逐滴加入聚乙二醇20000(PEG20000),搅拌5-30min。反应完成后离心弃上清液,加入10mL PBS缓冲液(含0.4mg/mL PEG)清洗2次。将沉淀用含2%BSA的PBS缓冲液(pH=7.4)溶解,4℃保存备用。
(3)胶体金结合垫的制备:取制备好的胶体金标记抗体离心,取上清液涂于玻璃纤维膜上,室温下晾干,制成胶体金结合垫;
(4)免疫层析膜的包被:检测线(T)上包被适量的斑蝥黄抗体,线宽1.5-2.5mm;质控线(C)上包被适量的羊抗大鼠IgG,线宽1.5-2.5mm;
(5)免疫层析检测卡的组装:聚氯乙烯衬板作为支撑载体固定于免疫层析检测卡槽下壳体中,然后样品垫、胶体金结合垫、免疫层析膜和吸水滤纸依次排列连接于聚氯乙烯衬板上表面,再将检测卡槽上壳体与下壳体通过卡扣连接,就得到斑蝥黄检测卡。常温密封储存。
(6)检测方法及结果判读
取斑蝥黄免疫层析检测卡,将经处理的禽蛋样品液滴加在检测卡的样品池中,加样完成后将检测卡静置,等待5-30min后,观察检测线和质控线的颜色变化。如果C线显色明显,且T线无肉眼可见颜色,证明该样本为阳性样本,禽蛋中斑蝥黄含量在灵敏度下限以上;如果C线显色明显,且T线有肉眼可见颜色,证明该样本为阴性样本,禽蛋中斑蝥黄含量在灵敏度下限以下;如果C线不显色,表明该检测卡失效。
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