CN113848318B - 青环海蛇毒素竞争法胶体金/纳米花免疫层析检测卡 - Google Patents
青环海蛇毒素竞争法胶体金/纳米花免疫层析检测卡 Download PDFInfo
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Abstract
本发明涉及抗体工程与免疫检测领域,提供了一种青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡。本发明提供的青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡,检测时间10 min,胶体金免疫侧流层析检测卡的检测阈值(消除T线)达到25μg/mL,检测限(vLOD)达到0.1 ng/mL;纳米花免疫侧流层析检测卡的检测阈值(消除T线)达到25μg/mL,检测限(vLOD)达到0.01 ng/mL。首次利用纳米花材料与抗青环海蛇毒素SN311单克隆抗体结合,制备胶体金/纳米花免疫侧流层析检测卡,可实现对青环海蛇毒素SN311的高灵敏快速检测。
Description
技术领域
本发明属于抗体工程与免疫检测领域,具体涉及一种青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡。
背景技术
我国是海洋大国,在实现海洋资源可持续开发的过程中,沿海海域存在的有毒海洋生物势必对海洋作业人员的生命健康造成威胁,因此海洋生物伤的防治研究工作对生产、医疗工作、科学研究以及军队维护有重要的现实意义,所以必须尽快着手开展抗海洋生物伤的科学应用研究。
青环海蛇(HydrophiscyanocinctusDaudin)广泛分布在我国山东以南的沿海区域,其蛇毒毒性强烈,主要的成分为α-神经毒素和一些其他蛋白,起主要作用的为α-神经毒素。α-神经毒素能够结合在骨骼肌运动终板部位的烟碱型乙酰胆碱受体,从而阻断骨骼肌的神经肌肉接头传递,因此青环海蛇咬伤会出现肌肉麻痹,从而导致呼吸肌麻痹进而机体窒息死亡。由于青环海蛇蛇毒难以采集,对于青环海蛇毒素的研究及检测也相对滞后,此开展关于青环海蛇蛇毒检测方法的研究具有重要意义。
免疫胶体金层析技术的实现还得益于其载体免疫胶体金层析试纸条(immunochromatographicteststrips,ICTSs),一种通过硝酸纤维素膜将加入的待检测样品固定在能与样品发生特异性结合的受体处位置,随着复合物的随摸流的累积,胶体金在检测试纸条上显色,这个过程往往只需要几分钟时间,因此此技术大大降低了检测的时间成本,加之其操作简单,特异性强,灵敏度高,很快在科学研究中得到广泛应用。随着纳米材料技术的成熟,金属纳米粒子的使用也越来越广泛,金属纳米粒子具有有与纳米材料一样的光电性,以及其独特的生物相容性,自然而然受到免疫学的重视,在免疫学研究中的应用随着胶体金的广泛使用而更加深远。胶体金因为受离子层的静电作用在溶液中以胶体分散状态显现。胶体金纳米粒子作为标记材料,其制备工艺简单,稳定性强,粒子分散性好以及优质的光学特性,在免疫标记方法上具有独特的优势。金纳米花作为一种新型的高催化胶体金技术在近几年发展迅速,金纳米花是在胶体金基础上通过种子生长法放大胶体金粒子,行成的形似花状的纳米粒子,其具有更显著的光学消化系数和催化活性,一定程度上大大提高了标记检测的灵敏度。目前制备金纳米粒子的方法有物理合成法和化学合成法,但化学合成法中的液还原法也称为种子生长法因其操作简单而受到广大研究者的青睐。种子生长法是在胶体金颗粒的基础上利用还原氯金酸行成表面不规则的较大颗粒的金纳米花。实验中采用对苯二酚作还原剂,可以调控合成了不同粒径的纳米花。
发明内容
本发明的目的是提供一种快速和特异性较强的青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡,该检测卡能够实现对青环海蛇毒素SN311的快速灵敏检测。
为实现上述目的,本发明提供了一种青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡,检测卡包括样品垫、金标垫、硝酸纤维素膜、吸水垫和塑料卡壳;所述硝酸纤维素膜上设置有质控线(C线)和检测线(T线),所述质控线上喷有羊抗鼠的IgG抗体,所述检测线上包被有青环海蛇毒素SN311抗原;所述的金标垫上含有胶体金/纳米花标记的抗青环海蛇毒素SN311单克隆抗体。
所述金标垫中所含有胶体金/纳米花标记的抗青环海蛇毒素SN311单克隆抗体,是由胶体金/纳米花标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的。
所述胶体金的制备方法为:将1g HAuCl4溶于100mL ddH2O中,得到1wt%的氯金酸水溶液,然后按1mL/管平均分装于1.5mL无菌的EP管中,用锡箔纸密封后置于-20℃冰箱中保存备用;取1mL1wt%的氯金酸水溶液溶于100mL ddH2O中,混匀后加热煮沸,随后加入2mL1wt%的柠檬酸三钠溶液,继续加热煮沸7min,观察氯金酸水溶液的颜色逐渐稳定成酒红色,将其放置室温冷却后,用无菌ddH2O定容至100mL,即得到胶体金溶液。
所述纳米花金的制备方法为:取500μL直径18.4nm胶体金溶液加入100mL ddH2O中,然后加入750μL 1wt%的氯金酸水溶液混匀,再加入300μL 1wt%的柠檬酸三钠摇匀,最后加入1mL 0.03M的对苯二酚溶液,搅拌至溶液颜色变为蓝色即得到纳米花金溶液。
所述抗青环海蛇毒素SN311单克隆IgG抗体由保藏编号为CGMCC No.21012的抗海蛇蛇毒SN311单克隆抗体杂交瘤细胞株8B1分泌获得;所述抗海蛇蛇毒SN311单克隆抗体杂交瘤细胞株8B1已于2020年11月23日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏地址为北京市朝阳区北辰西路1号院3号。
所述胶体金标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液的制备方法为:取10mL胶体金溶液置冰水浴,搅拌,加入60μL 0.1M K2CO3,后缓慢滴加240μL 2.41mg/mL的抗青环海蛇毒素SN311单克隆IgG抗体,维持冰水浴搅拌1h,按终浓度加入1wt%的BSA,维持冰水浴,搅拌30min,按终浓度0.5wt%加入PEG 20000,继续搅拌30min,静置4℃平衡体系过夜,后置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5%BSA+0.5%Glycine+5%Trehalose+0.5%Tween20 in 0.01M pH7.4 PBS。
所述纳米花标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液的制备方法为:取10mL纳米花溶液置冰水浴,搅拌,缓慢滴加240μL 2.41mg/mL的抗青环海蛇毒素SN311单克隆IgG抗体,维持冰水浴搅拌1h,按终浓度1wt%加入BSA,维持冰水浴,搅拌30min,按终浓度0.5wt%加入PEG 20000,继续搅拌30min,静置4℃平衡体系过夜,后置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5%BSA+0.5%Glycine+5%Trehalose+0.5%Tween20 in 0.01MpH7.4 PBS。
所述检测线上包被的青环海蛇毒素SN311抗原,是由0.67mg/mL的青环海蛇毒素SN311蛋白稀释以0.01M pH7.4的PBS按1/10进行稀释在硝酸纤维膜上按照2μL/cm进行划线干燥后得到的。
所述质控线上包被的羊抗鼠IgG的抗体是由1mg/mL的羊抗鼠IgG的抗体溶液以0.01M pH7.4的PBS按1/8进行稀释在硝酸纤维膜上按照2μL/cm进行划线干燥后得到的。
上述一种胶体金/纳米花免疫侧流层析检测卡在青环海蛇毒素SN311检测中的应用。
本发明的有益效果在于:
本发明提供的青环海蛇毒素SN311竞争法胶体金/纳米花免疫侧流层析检测卡,检测时间仅为10min,胶体金免疫侧流层析检测卡的检测阈值(消除T线)达到25μg/mL,检测限(vLOD)达到0.1ng/mL;纳米花免疫侧流层析检测卡的检测阈值(消除T线)达到25μg/mL,检测限(vLOD)达到0.01ng/mL。
附图说明
图1是本发明单克隆抗体8B1的SDS-PAGE纯化结果鉴定。其中,泳道M为Marker;泳道1为未纯化的腹水;泳道2为纯化后的单克隆抗体。
图2是本发明小鼠腹水和纯化单克隆抗体效价测定结果。
图3是本发明胶体金胶体溶液数码照片图。
图4是本发明制备的胶体金溶液及胶体金免疫探针紫外可见全波长扫描图。
图5是本发明胶体金免疫侧流层析检测卡特异性分析。
图6是本发明胶体金免疫侧流层析检测卡灵敏度分析。
图7是本发明纳米花胶体溶液数码照片图。
图8是本发明制备的纳米花溶液及金纳米花免疫探针紫外可见全波长扫描图。
图9是本发明纳米花免疫侧流层析检测卡特异性分析。
图10是本发明纳米花免疫侧流层析检测卡灵敏度分析。
具体实施方式
以下实施实例用于说明本发明,但不用来限制本发明的范围。
实施例1本发明单克隆抗体8B1的纯化与鉴定
1)单克隆抗体腹水的制备
将处于对数生长期的抗海蛇蛇毒SN311单克隆抗体杂交瘤细胞株8B1按105~106cells/只注射到石蜡致敏过的9周龄Balb/c雌鼠腹腔,一周内持续观察小鼠,待小鼠腹部膨大且有紧张感时抽取腹水,置4℃,13000r/min离心20min,离心后分为三层(由下向上依次为聚集物,含有大量抗体的中间层,上层为脂质与其它结缔组织层),取中间层,于-80℃超低温冰箱保存。
2)单克隆抗体的纯化
采用辛酸-硫酸铵法粗提与Protein G亲和层析法精提相结合进行纯化,然后取10μL纯化后的抗体加入20μL 5×SDS-PAGE蛋白裂解液混匀,取3μL未纯化的腹水加入20μL 5×SDS-PAGE蛋白裂解液混匀,沸水煮沸10~15min使抗体完全变性后各取10μL点样后进行SDS-PAGE电泳。如图1所示,纯化后的抗体8B1在25kDa及50kDa处均有条带,分别对应IgG抗体的轻链和重链,而且几乎没有杂带,表明纯化效果良好,将抗体置-20℃冰箱中备用。
3)效价测定:采用常规非竞争酶联免疫吸附分析(ELISA)法测定腹水的效价及纯化后抗体的效价,结果如图2所示,纯化后的单克隆抗体8B1效价已经达到1:64000,表明经过纯化后抗体依然保持较高活性。
实施例2本发明胶体金免疫层析检测卡的制备与鉴定
1)胶体金(GNP)溶液的制备:将1g HAuCl4溶于100mL ddH2O中,得到1wt%的氯金酸水溶液,然后按1mL/管平均分装于1.5mL的无菌EP管中,用锡箔纸密封后置于-20℃冰箱中保存备用。取1mL1wt%的氯金酸水溶液溶于100mL ddH2O,混匀后加热煮沸,随后加入2mL1wt%的柠檬酸三钠溶液,继续加热煮沸7min。观察氯金酸水溶液的颜色逐渐稳定成酒红色,放置室温冷却后,用无菌ddH2O定容至100mL,即得到胶体金溶液,于4℃冰箱冷藏备用。
2)胶体金(GNP)的表征:所制胶体金胶体溶液为酒红色,未见浑浊,底部无沉淀,分散性良好,如图3所示;紫外可见光谱扫描,最大吸收峰约为520nm,峰形较宽,如图4所示。
3)胶体金免疫探针(GNP-mAb)的制备:
最适标记抗体量:取酶标条分别加入200μL胶体金(GNP)溶液,各加入0、1、2、3、4、5、6、7、8、9、10μL单克隆抗体8B1(2.41mg/mL),充分混匀后置于37℃恒温箱孵育30min;分别加入20μL 10%NaCl,混匀,5min后观测颜色变化趋于稳定的孔对应的体积,确定最适抗体标记量为4μL 2.41mg/mL mAb 8B1/200μL GNP(在放大体系中添加量为此处的120%)。
最适标记pH值:取酶标条分别加入200μL胶体金(GNP)溶液,各加入0、1、2、3、4、5、6、7、8μL 0.1M K2CO3,后每孔各加入上述确定的最适抗体抗体量,充分混匀后置于37℃恒温箱孵育30min;分别加入20μL 10wt%NaCl,5min后观测颜色最深且稳定的孔,确定最适标记pH为1μL 0.1M K2CO3/200μL GNP(在放大体系中添加量为此处的120%)。
金标抗体的纯化:取10mL胶体金(GNP)溶液置冰水浴,低速搅拌,用60μL 0.1MK2CO3调至最适标记pH,后缓慢滴加240μL mAb 8B1抗体(2.41mg/mL),维持冰水浴低速搅拌1h;按终浓度1wt%加入BSA,维持冰水浴,低速搅拌30min;按终浓度0.5wt%加入PEG20000,继续搅拌30min;静置4℃平衡体系过夜。置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5%BSA+0.5%Glycine+5%Trehalose+0.5%Tween20 in 1×PBS(pH7.4)。
实施例3本发明纳米花免疫层析检测卡的制备与鉴定
1)纳米花(AuNF)制备:取100mL ddH2O调pH值7.0,依次加入直径18.4nm胶体金溶液500μL,1wt%柠檬酸钠300μL,1wt%HAuCl4750μL,混合均匀;剧烈搅拌,一次性快速加入1.0mL 30mM对苯二酚,持续剧烈搅拌30min;置4℃保存。
2)纳米花(AuNF)的表征:所制金纳米花胶体为深蓝色,未见浑浊,底部无沉淀,分散性良好,如图7所示;紫外可见光谱扫描,最大吸收峰约为590nm,峰形较宽,如图8所示。
3)纳米花免疫探针(AuNF-mAb)的制备:
最适标记抗体量:取酶标条分别加入200μL纳米花(AuNF),各加入0、1、2、3、4、5、6、7、8、9、10μL单克隆抗体8B1(2.41mg/mL),充分混匀后置于37℃恒温箱孵育30min;分别加入20μL 10wt%NaCl,混匀,5min后观测颜色变化趋于稳定的孔对应的体积,确定最适抗体标记量为4μL 2.41mg/mL mAb 8B1/200μL GNP(在放大体系中添加量为此处的120%)。
最适标记pH值:取酶标条分别加入200μL纳米花(AuNF),各加入0、1、2、3、4、5、6、7、8μL 0.1M K2CO3,后每孔各加入上述确定的4μL最适抗体标记量,充分混匀后置于37℃恒温箱孵育30min;分别加入20μL 10wt%NaCl,5min后观测颜色最深且稳定的孔,确定最适标记pH为添加0μL 0.1M K2CO3/200μL GNP。
金标抗体的纯化:取10mL纳米花(AuNF)溶液置冰水浴,低速搅拌,缓慢滴加240μL8B1抗体(2.41mg/mL),维持冰水浴低速搅拌1h;按终浓度1wt%加入BSA,维持冰水浴,低速搅拌30min;按终浓度0.5wt%加入PEG 20000,继续搅拌30min;静置4℃平衡体系过夜。置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5%BSA+0.5%Glycine+5%Trehalose+0.5%Tween20 in 1×PBS(pH7.4)。
实施例4本发明胶体金免疫层析检测卡的检测应用
1)胶体金金标垫与样品垫的处理:样品垫采用玻纤GL-b02,免疫探针结合垫采用聚酯MA0280,金标垫和样品垫裁剪成4mm宽,20mm长的长条状,用封闭液(封闭液配方按质量百分比计为:1%BSA+0.5%PEG20000+0.5%Tween 20in 1×PBS(pH7.4))淹没封闭1h,然后置于37℃恒温箱干燥保存。
2)检测卡组装:硝酸纤维膜(Sartorius CN140,2.5cm×30cm)固定到卡式底板DB-6(6.0cm×30cm),检测抗原为青环海蛇毒素SN311(0.67mg/mL)以1×PBS(pH7.4)按1/10进行稀释在硝酸纤维膜上按照2μL/cm进行划线,得检测线(T线),羊抗小鼠IgG(GAMA,1mg/mL)以1×PBS(pH7.4)按1/8进行稀释在硝酸纤维膜上按照2μL/cm进行划线,得质控线(C线),置37℃恒温箱干燥,取5μL胶体金金标探针以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上,干燥,组装检测卡。
3)胶体金免疫层析检测卡特异性测定:以0.01M pH7.4的PBS将青环海蛇毒素SN311抗原(0.67mg/mL)稀释十倍、山羊抗小鼠IgG(GAMA,1mg/mL)稀释八倍在硝酸纤维膜上按照2μL/cm划线,同时取出金标垫,在每个金标垫上点上5μL胶体金金标探针(以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上),烘干备用。将10种不同的抗原(OVA,LF,KLH,BSA,OA,TTX,DA,CIT,STX,SN311)用0.01M PBS(pH7.4)稀释至终浓度为25μg/mL。后将干燥好的硝酸纤维膜裁成宽4mm,长60mm的长条,与干燥好的金标垫,剪好的吸水纸,处理好的样品垫一起组装成胶体金免疫层析检测卡,分别加入100μL稀释好的不同抗原,10min内观察显色情况。结果如图5所示:加入青环海蛇毒素SN311抗原的检测卡的T线完全消失,结果为阳性;而加入其它9种蛋白抗原时,检测卡T线均显色,结果显示为阴性。表明胶体金免疫层析检测卡只与青环海蛇毒素SN311抗原反应,与其它抗原包括牛奶中含有的乳蛋白和牛血清白蛋白均无反应,证明该免疫层析检测方法特异性强,无交叉反应。
4)胶体金免疫层析检测卡灵敏度测定:以0.01M pH7.4的PBS将青环海蛇毒素SN311抗原(0.67mg/mL)稀释十倍、山羊抗小鼠IgG(GAMA,1mg/mL)稀释八倍在硝酸纤维膜上按照2μL/cm划线,同时取出金标垫,在每个金标垫上点上5μL胶体金金标探针(以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上),烘干备用。用0.01M PBS(pH7.4)将青环海蛇毒素SN311稀释至终浓度为25μg/mL,10μg/mL,1μg/mL,0.1μg/mL,0.01μg/mL,0.001μg/mL,0.0001μg/mL和0μg/mL。后将干燥好的硝酸纤维膜裁成宽4mm,长60mm的长条,与干燥好的金标垫,剪好的吸水纸,处理好的样品垫一起组装成纳米花免疫层析检测卡,分别加入100μL稀释好的蛇毒SN311,10min内观察显色情况。实验结果如图6所示:在青环海蛇毒素SN311抗原浓度为25μg/mL时,肉眼观察T线颜色呈无色,T线消失,随着青环海蛇毒素SN311抗原浓度的降低,结合胶体金探针的能力逐渐减弱,抗原竞争逐渐削弱,肉眼观察到T线的颜色逐渐变深,当青环海蛇毒素SN311抗原浓度稀释至0.0001μg/mL时,T线及C线的显色与没有加入蛇毒SN311抗原所显示的一致,说明此时已经达到了胶体金免疫层析检测卡的下限。利用免疫色谱仪读取T线吸收值,以抗原稀释浓度为纵坐标,B/B0为横坐标,绘制柱状图,结果显示随T线浓度的上升,B/B0值越低,抑制率越强,进一步验证了上述所述的本实验制备的胶体金免疫层析试纸的最低检测限为0.1ng/mL。
实施例5本发明纳米花免疫层析检测卡的检测应用
1)纳米花金标垫与样品垫的处理:样品垫采用玻纤GL-b02,免疫探针结合垫采用聚酯MA0280,金标垫和样品垫裁剪成4mm宽,20mm长的长条状,用封闭液(封闭液配方按质量百分比计为:1%BSA+0.5%PEG20000+0.5%Tween 20in 1×PBS(pH7.4))淹没封闭1h,后干燥保存。
2)检测卡组装:硝酸纤维膜(Sartorius CN140,2.5cm×30cm)固定到卡式底板DB-6(6.0cm×30cm),检测抗原为青环海蛇毒素SN311(0.67mg/mL)以1×PBS(pH7.4)按1/10进行稀释在硝酸纤维膜上按照2μL/cm进行划线,得检测线(T线),羊抗小鼠IgG(GAMA,1mg/mL)以1×PBS(pH7.4)按1/8进行稀释在硝酸纤维膜上按照2μL/cm进行划线,得质控线(C线),置37℃恒温箱干燥,取5μL纳米花金标探针以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上,干燥,组装检测卡。
3)纳米花免疫层析检测卡特异性测定:以0.01M pH7.4的PBS将青环海蛇毒素SN311抗原(0.67mg/mL)稀释十倍、山羊抗小鼠IgG(GAMA,1mg/mL)稀释八倍在硝酸纤维膜上按照2μL/cm划线,同时取出金标垫,在每个金标垫上点上5μL纳米花金标探针(以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上),烘干备用。将9种不同的抗原(OVA,LF,KLH,BSA,OA,TTX,DA,CIT,SN311)用0.01M PBS(pH7.4)稀释至终浓度为25μg/mL。后将干燥好的硝酸纤维膜裁成宽4mm,长60mm的长条,与干燥好的金标垫,剪好的吸水纸,处理好的样品垫一起组装成金纳米花免疫层析检测卡,分别加入100μL稀释好的不同抗原,10min内观察显色情况。结果如图9所示:加入青环海蛇毒素SN311抗原的检测卡的T线完全消失,结果为阳性;而加入其它8种蛋白抗原时,检测卡T线均显色,结果显示为阴性。表明纳米花免疫层析检测卡只与青环海蛇毒素SN311抗原反应,与其它抗原包括牛奶中含有的乳蛋白和牛血清白蛋白均无反应,证明该免疫层析检测方法特异性强,无交叉反应。
4)纳米花免疫层析检测卡灵敏度测定:以0.01M pH7.4的PBS将青环海蛇毒素SN311抗原(0.67mg/mL)稀释十倍、山羊抗小鼠IgG(GAMA,1mg/mL)稀释八倍在硝酸纤维膜上按照2μL/cm划线,同时取出金标垫,在每个金标垫上点上5μL纳米花金标探针(以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂到金标垫上),烘干备用。用0.01M PBS(pH7.4)将青环海蛇毒素SN311稀释至终浓度为25μg/mL,10μg/mL,1μg/mL,0.1μg/mL,0.01μg/mL,0.001μg/mL,0.0001μg/mL,0.00001μg/mL和0μg/mL。后将干燥好的NC膜裁成宽4mm的长条,与干燥好的金标垫,剪好的吸水纸,处理好的样品垫一起组装成纳米花免疫层析检测卡,分别加入100μL稀释好的蛇毒SN311,10min内观察显色情况。实验结果如下图10所示:在青环海蛇毒素SN311抗原浓度为25μg/mL时,肉眼观察T线颜色呈无色,T线消失,随着青环海蛇毒素SN311抗原浓度的降低,结合纳米花探针的能力逐渐减弱,抗原竞争逐渐削弱,肉眼观察到T线的颜色逐渐变深,当青环海蛇毒素SN311抗原浓度稀释至0.00001μg/mL时,T线,C线的显色与没有加入蛇毒SN311抗原所显示的一致,说明此时已经达到了纳米花免疫层析检测卡的下限。利用免疫色谱仪读取T线吸收值,以抗原稀释浓度为纵坐标,B/B0为横坐标,绘制柱状图,结果显示随T线浓度的上升,B/B0值越低,抑制率越强,进一步验证了上述所述的本实验制备的纳米花免疫层析试纸的最低检测限为0.01ng/mL。
Claims (2)
1.一种青环海蛇毒素SN311竞争法胶体金或金纳米花免疫侧流层析检测卡,其特征在于:所述检测卡包括样品垫、金标垫、硝酸纤维素膜、吸水垫和塑料卡壳;所述的硝酸纤维素膜上设置有质控线和检测线,所述的质控线上喷有羊抗鼠的IgG抗体,所述的检测线上包被有青环海蛇毒素SN311抗原;所述的金标垫上含有胶体金或金纳米花标记的抗青环海蛇毒素SN311单克隆抗体;
所述金标垫中所含有胶体金或金纳米花标记的抗青环海蛇毒素SN311单克隆抗体,是由胶体金或金纳米花标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液以每100mm/s和2μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的;
所述胶体金的制备方法为:将1g HAuCl4溶于100mL ddH2O中,得到1wt%的氯金酸水溶液,然后按1mL/管平均分装于1.5mL无菌的EP管中,用锡箔纸密封后置于-20℃冰箱中保存备用;取1mL 1wt%的氯金酸水溶液溶于100mL ddH2O中,混匀后加热煮沸,随后加入2mL1wt%的柠檬酸三钠溶液,继续加热煮沸7min,观察氯金酸水溶液的颜色逐渐稳定成酒红色,将其放置室温冷却后,用无菌ddH2O定容至100mL,即得到胶体金溶液;
所述金纳米花的制备方法为:取500μL直径18.4nm胶体金溶液加入100mL ddH2O中,然后加入750μL 1wt%的氯金酸水溶液混匀,再加入300μL 1wt%的柠檬酸三钠摇匀,最后加入1mL0.03M的对苯二酚溶液,搅拌至溶液颜色变为蓝色即得到金纳米花溶液;
所述抗青环海蛇毒素SN311单克隆IgG抗体由保藏编号为CGMCC No.21012的抗海蛇蛇毒SN311单克隆抗体杂交瘤细胞株8B1分泌获得;所述抗海蛇蛇毒SN311单克隆抗体杂交瘤细胞株8B1已于2020年11月23日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏地址为北京市朝阳区北辰西路1号院3号;
所述胶体金标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液的制备方法为:取10mL胶体金溶液置冰水浴搅拌,加入60μL 0.1M K2CO3,后缓慢滴加240μL 2.41mg/mL的抗青环海蛇毒素SN311单克隆IgG抗体,维持冰水浴搅拌1h,按终浓度加入1wt%的BSA,维持冰水浴,搅拌30min,按终浓度0.5wt%加入PEG 20000,继续搅拌30min,静置4℃平衡体系过夜,置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5% BSA + 0.5% Glycine + 5% Trehalose + 0.5%Tween20溶于0.01M pH7.4 PBS;
所述金纳米花标记的抗青环海蛇毒素SN311单克隆IgG抗体溶液的制备方法为:取10mL金纳米花溶液置冰水浴搅拌,缓慢滴加240μL 2.41mg/mL的抗青环海蛇毒素SN311单克隆IgG抗体,维持冰水浴搅拌1h,按终浓度1wt%加入BSA,维持冰水浴,搅拌30min,按终浓度0.5wt%加入PEG 20000,继续搅拌30min,静置4℃平衡体系过夜,置4℃、1500r/min离心20min,弃沉淀,取上清置4℃、10000r/min离心5min,保留沉淀,按1/10体积重悬,重悬液配方按质量百分比计为:0.5% BSA + 0.5% Glycine + 5% Trehalose + 0.5% Tween20溶于0.01M pH7.4 PBS;
所述检测线上包被的青环海蛇毒素SN311抗原,是由0.67mg/mL的青环海蛇毒素SN311蛋白以0.01M pH7.4的PBS按1/10进行稀释,在硝酸纤维膜上按照2μL/cm进行划线干燥后得到的;
所述质控线上包被的羊抗鼠IgG的抗体是由1mg/mL的羊抗鼠IgG的抗体溶液以0.01MpH7.4的PBS按1/8进行稀释,在硝酸纤维膜上按照2μL/cm进行划线干燥后得到的。
2.权利要求1所述胶体金或金纳米花免疫侧流层析检测卡在青环海蛇毒素SN311检测中的应用。
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