CN116514907B - 一种快速检测真菌毒素腾毒素的免疫层析试纸条 - Google Patents
一种快速检测真菌毒素腾毒素的免疫层析试纸条 Download PDFInfo
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- CN116514907B CN116514907B CN202310390538.7A CN202310390538A CN116514907B CN 116514907 B CN116514907 B CN 116514907B CN 202310390538 A CN202310390538 A CN 202310390538A CN 116514907 B CN116514907 B CN 116514907B
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Classifications
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Abstract
本发明公开了一种快速检测真菌毒素腾毒素的免疫层析试纸条,其是利用结构如式(Ⅰ)所示的半抗原制备的免疫层析试纸条,本发明采用胶体金免疫层析技术开发出可快速定性或半定量检测样品中腾毒素残留的免疫层析试纸条,操作简单,经济实用,适合于现场检测,可广泛应用于谷物中腾毒素污染的检测及快速筛查。
Description
技术领域
本发明涉及食品安全监测和免疫分析检测技术领域,具体地说,涉及一种快速检测真菌毒素腾毒素的免疫层析试纸条。
背景技术
腾毒素(Tentoxin,Ten)是由链格孢霉(Alternaria)产生的具有明显毒理学意义的一种霉菌毒素,具有环四肽结构。链格孢霉是一类广泛分布于泥土和各种农作物的真菌,能够通过田间侵染、贮存接触等途径广泛污染谷物和饲料,可以产生70多种毒素,对人和动物具有诱变性、致癌性和致畸性等慢性或急性毒性作用。TEN能够降低细胞内腺苷三磷酸(ATP)酶活性,抑制光合磷酸化,导致植物萎黄病。体外实验表明,TEN通过细胞色素P450-3A作用形成羟基化和脱甲基代谢产物。但是,TEN对哺乳动物的毒性试验尚未见报道。
Ten主要存在于谷物、番茄、柑橘、樱桃等农作物和水果中,对小麦的污染非常普遍,检出率高,检出量大,世界各地均有报道。2016年欧洲食品***(EFSA)已经就食品中链格孢霉属毒素对人的健康风险发布了科学意见,TEN作为链格孢霉产生的代表性毒素,其最大日摄入量阈值初步设为1500ng/kg bw/d。2016~2018年国家食品污染物监测显示小麦及其制品中Ten的污染严重,检出率超过90%,最大检出值超过200μg/kg。目前,TEN的检测主要采用传统的大型仪器检测方法,包括高效液相色谱法(HPLC-UV)、液相色谱-质谱联用法(LC-MS)和液相色谱-串联质谱法(LC-MS/MS)等。这些方法均须在实验室条件下操作,样品前处理繁琐费时,还需配备昂贵的仪器设备,检测成本较高、耗时长、操作复杂,在实际应用过程中有很大的限制性,难以满足大量样品和现场样品快速检测的需要。基于胶体金标记抗体与抗原特异性结合反应的免疫层析试纸条的检测结果肉眼可见,不需要大型仪器设备,检测成本低,分析时间短,从而可实现对各种真菌毒素的定性、在线、快速检测,但目前尚未见利用免疫层析试纸条检测腾毒素的相关报道。
发明内容
本发明的目的是提供腾毒素半抗原及其制备方法与应用。
本发明的另一目的是提供一种快速检测腾毒素的免疫层析试纸条及其应用。
为了实现本发明目的,本发明的腾毒素半抗原,其结构如式(Ⅰ)所示:
本发明还提供腾毒素全抗原,由所述腾毒素半抗原与载体蛋白偶联后得到的,即半抗原的羧基与载体蛋白的游离氨基发生缩合反应。
其中,所述载体蛋白为牛血清白蛋白、卵清白蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白;优选牛血清白蛋白(BSA)、卵清白蛋白(OVA)。
所述腾毒素全抗原制备的特异性抗体,包括多克隆抗体和单克隆抗体。其中,所述腾毒素单克隆抗体是以腾毒素半抗原-载体蛋白偶联物(如腾毒素半抗原-BSA偶联物)作为免疫原,通过免疫试验动物,结合杂交瘤细胞制备技术获得的;优选的,所述单克隆抗体的效价为1:810000。
本发明的腾毒素半抗原可通过如下方法制备:取无水三氯化铝0.39g,加入100mL1,2-二氯乙烷和0.5g琥珀酸酐,搅拌30min;再加入0.414g腾毒素的1,2-二氯乙烷溶液10mL,室温搅拌1h,在搅拌下缓慢加入200mL纯水,搅拌后静置;分去水相,有机相再次水洗,无水硫酸钠干燥,蒸干,上硅胶小柱,用体积比为10:1的二氯甲烷-甲醇混合溶液洗脱分离,即得半抗原产物。腾毒素半抗原合成路线见图1。
本发明还提供由所述腾毒素半抗原,或所述腾毒素全抗原,或所述特异性抗体制备的腾毒素检测试剂或试剂盒。
本发明还提供所述腾毒素半抗原,或所述腾毒素全抗原,或所述特异性抗体,或所述检测试剂或试剂盒在腾毒素的免疫分析检测中的应用。
本发明还提供所述腾毒素半抗原,或所述腾毒素全抗原,或所述特异性抗体,或所述检测试剂在制备腾毒素的胶体金检测试纸条中的应用。
本发明还提供一种快速检测腾毒素的免疫层析试纸条(图2),包括样品垫、结合物释放垫、反应膜、吸水垫和底板,所述反应膜上设有检测线和质控线,所述样品垫、结合物释放垫、反应膜和吸水垫依次粘贴在底板上。优选的,所述结合物释放垫1/3~1/2被覆盖于样品垫下。
所述底板可为PVC底板或其他硬质不吸水的材料;所述样品垫可为吸滤纸或滤油纸;所述结合物释放垫可为玻璃棉或聚酯材料;所述吸水垫为吸水纸;所述反应膜可为硝酸纤维素膜或醋酸纤维素膜。
其中,所述结合物释放垫上包被有腾毒素特异性抗体-胶体金标记物,所述腾毒素特异性抗体可以是多克隆抗体或单克隆抗体;所述检测线包被有所述腾毒素全抗原(如腾毒素半抗原-OVA偶联物),所述质控线包被有羊抗鼠二抗。
所述腾毒素特异性抗体-胶体金标记物的制备方法包括以下步骤:特异性抗体与胶体金溶液混合后,加入10% BSA至BSA在该溶液中的终浓度为1%后,进行静置、离心得到沉淀,用复溶缓冲液洗涤、重悬所得沉淀;
优选的,所用复溶缓冲液为含BSA的质量分数为0.1%~0.5%、蔗糖的质量分数为2%~4%、pH 7.2的0.02mol/L磷酸盐缓冲液。
本发明还提供所述免疫层析试纸条的制备方法,包括如下步骤:
1)制备喷涂有腾毒素单克隆抗体-胶体金标记物的结合物释放垫;
2)制备具有包被有腾毒素半抗原-载体蛋白偶联物的检测线和包被有羊抗鼠二抗的质控线的反应膜;
3)将步骤1)和2)制备好的结合物释放垫、反应膜与样品垫、吸水垫和底板组装成试纸条。
具体地,所述免疫层析试纸条的制备方法如下:
1)利用傅克反应,在三氯化铝的催化下,在腾毒素苯环上进行酰基化反应,引入琥珀酸酐间隔臂,制备腾毒素半抗原;
2)将腾毒素半抗原与载体蛋白偶联,制备腾毒素半抗原-载体蛋白偶联物;
3)用腾毒素半抗原-载体蛋白偶联物免疫小鼠,将小鼠脾细胞和小鼠骨髓瘤细胞通过融合、筛选,得到分泌腾毒素单克隆抗体的杂交瘤细胞株;
4)分别将腾毒素半抗原-载体蛋白偶联物和羊抗鼠二抗包被于反应膜的检测线(T)和质控线(C)上;
5)用柠檬酸三钠与氯金酸反应制备胶体金;
6)将制备的腾毒素单克隆抗体加入到制备的胶体金中,得到腾毒素单克隆抗体-胶体金标记物;
7)将腾毒素单克隆抗体-胶体金标记物喷涂在结合物释放垫上,37℃烘干2h后取出,置于干燥环境中保存备用;
8)将样品垫用含1% BSA、pH 7.2的0.02mol/L磷酸盐缓冲液浸泡2h,37℃烘干2h备用;
9)在底板上按顺序粘贴上样品垫、结合物释放垫、反应膜、吸水垫,结合物释放垫从起始端有1/3区域被样品垫覆盖。最后切成3.95mm宽的小条,装在特制的塑料制卡壳中,以铝箔袋密封,4~30℃条件下保存12个月。结合物释放垫的1/3被样品垫覆盖可以延长检测结果观察时间,使样品垫将检测液体充分吸收并与金标抗体充分反应,从而减少误差。
本发明进一步提供利用所述免疫层析试纸条检测谷物中腾毒素的方法,将待测样本粉碎,称取(1.0±0.05)g至15mL聚苯乙烯离心管中,加入10mL 80%的甲醇,涡旋振荡3min,3000r/min室温离心5min,上清液即为待检样本液;用微量移液器吸取100μL待检样本液垂直滴加于试纸条的样品垫上,液体流动开始计时,反应10min,根据检测线和质控线的显色判定结果。
本发明的腾毒素快速检测试纸条采用高度特异性的抗体抗原反应及免疫层析分析技术,将腾毒素特异性抗体-胶体金标记物固定于结合物释放垫上,样品中的腾毒素在流动过程中与结合物释放垫上的腾毒素特异性抗体-胶体金标记物结合,形成腾毒素-抗体-胶体金标记物。样本中的腾毒素与反应膜检测线上的腾毒素半抗原-载体蛋白偶联物竞争结合腾毒素特异性抗体-胶体金标记物,根据检测线红色条带深浅来判断待测样品液中是否含有腾毒素。
检测时,样品经处理后滴入样品垫,当腾毒素在样品中的浓度低于检测限或为零时,特异性抗体-胶体金标记物在层析过程中会与固定在反应膜上的腾毒素半抗原-载体蛋白偶联物结合,在检测线(T)和质控线(C)上各出现一条红色条带,且T线显色比C线显色深或与C线显色一致;如果腾毒素在样品中的浓度等于或高于检测限,特异性抗体-胶体金标记物会与腾毒素全部结合,从而在T线处因为竞争反应不会与腾毒素半抗原-载体蛋白偶联物结合而不出现红色条带或比C线显色浅。如图3所示。
阴性:当质控线(C)显示出红色条带,检测线(T)同时也显示出红色条带,且(T)线颜色接近或深于(C)线时,判为阴性。
阳性:当质控线(C)显示出红色条带,而检测线(T)不显色或(T)线颜色浅于(C)线时,判为阳性。
无效:当质控线(C)不显示出红色条带,则无论检测线(T)显示出红色条带与否,该试纸条均判为无效。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(1)本发明从设计腾毒素半抗原结构出发,制备免疫原和包被原,进而筛选高专一性、高亲和性的腾毒素单克隆抗体,特异性好,开发的胶体金免疫层析试纸条可直接用于腾毒素的检测,实际应用价值大。
(2)本发明的检测谷物中腾毒素的胶体金免疫层析试纸条,能够一步实现检测,结果准确,无需洗涤和标准参照,能够对单个或批量样本实时快速定性或半定量检测,检测灵敏度高,检测限达到10μg/kg。所述试纸条对其他链格孢霉毒素无交叉反应。
(5)本发明的检测谷物中腾毒素的胶体金免疫层析试纸条,具有灵敏度高、特异性强、成本低、操作简单、检测时间短、不受检测设备限制、适合各种单位使用、储存简单、保质期长的优点,可在现场大批量样本中腾毒素的快速检测及筛查中发挥重要作用,能够更好地辅助我国食品企业及政府职能监管部门等开展相关检测工作。
附图说明
图1为腾毒素半抗原合成图。
图2为试纸条剖面结构示意图,图中:1、样品垫;2、结合物释放垫;3、反应膜;4、吸水垫;5、检测线;6、质控线;7、底板。
图3为试纸条检测结果判定图。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解,这些实施例仅用于说明本发明,而不用来限制本发明的范围。
实施例1快速检测腾毒素的免疫层析试纸条的制备
该试纸条的制备方法主要包括以下步骤:
1)制备喷涂有腾毒素单克隆抗体-胶体金标记物的结合物释放垫;
2)制备具有包被有腾毒素半抗原-载体蛋白偶联物的检测线和包被有羊抗鼠二抗的质控线的反应膜;
3)将步骤1)和2)制备好的结合物释放垫、反应膜与样品垫、吸水垫和底板组装成试纸条。
下面分步详细叙述:
1、腾毒素半抗原的合成(合成路线见附图1)
取无水三氯化铝0.39g,加入100mL 1,2-二氯乙烷和0.5g琥珀酸酐,搅拌30min;再加入0.414g腾毒素的1,2-二氯乙烷溶液10mL,室温搅拌1h,在搅拌下缓慢加入200mL纯水,搅拌后静置;分去水相,有机相再次水洗,无水硫酸钠干燥,蒸干,上硅胶小柱,用体积比为10:1的二氯甲烷-甲醇混合溶液洗脱分离,即得半抗原产物0.24g,收率47%。
2、免疫原的制备
取腾毒素半抗原19mg,加1mL N,N-二甲基甲酰胺(DMF)溶解澄清,加N-羟基琥珀酰亚胺(NHS)9.26mg、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)15.3mg,充分溶解混匀,室温反应2h,得到半抗原活化液A液;取牛血清白蛋白(BSA)50mg,加0.1mol/L pH8.0的PB缓冲液6mL溶解,得到B液;将A液逐滴滴加到B液中,室温反应4h,停止反应,用0.02mol/LPBS缓冲液透析纯化3天,每天换液3次,离心分装,得到腾毒素半抗原-BSA偶联物,即为免疫原。
3、包被原的制备
取腾毒素半抗原11mg,加1mL DMF溶解澄清,加NHS 5.2mg、EDC 8.2mg,充分溶解混匀,室温反应2h,得到半抗原活化液A液;取卵清白蛋白(OVA)100mg,加0.1mol/L pH9.1的CB缓冲液6mL溶解,得到B液;将A液逐滴滴加到B液中,室温反应4h,停止反应,用0.02mol/LPBS缓冲液透析纯化3天,每天换液3次,离心分装,得到腾毒素半抗原-OVA偶联物,即为包被原。
4、腾毒素单克隆抗体的制备
(1)动物免疫
将步骤2得到的免疫原注入Balb/c小鼠体内,免疫剂量为150μg/只,使其产生抗血清。
(2)细胞融合和克隆化
取免疫Balb/c小鼠脾细胞,按8:1(数量配比)比例与SP2/0骨髓瘤细胞融合,采用间接竞争ELISA测定细胞上清液,筛选阳性孔。利用有限稀释法对阳性孔进行克隆化,直到得到稳定分泌单克隆抗体的杂交瘤细胞株。
(3)细胞冻存和复苏
将杂交瘤细胞用冻存液制成1×106个/mL的细胞悬液,在液氮中长期保存。复苏时取出冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养。
(4)单克隆抗体的制备与纯化
增量培养法:将杂交瘤细胞置于细胞培养基中,在37℃条件下进行培养,用辛酸-饱和硫酸铵法将得到的培养液进行纯化,得到单克隆抗体,-20℃保存。
所述细胞培养基为向RPMI1640培养基中添加小牛血清和碳酸氢钠,使小牛血清在细胞培养基中的终浓度为20%(质量分数),碳酸氢钠在细胞培养基中的终浓度为0.2%(质量分数);所述细胞培养基的pH为7.4。
(5)抗体效价的测定
在酶标板的各孔中分别包被100μL稀释到一定浓度的包被原,4℃包被过夜;清洗液洗涤1次后,每孔加入封闭液150μL,37℃孵育2h后甩干;每孔分别加入用抗体稀释液稀释的抗体100μL,37℃孵育0.5h;清洗液洗涤3次后,每孔分别加入用酶标二抗稀释液稀释2500倍的酶标二抗,37℃孵育0.5h;清洗液洗涤3次后,再加入显色液(50μL底物液A液,50μL底物液B液),室温避光反应15min;加入50μL终止液终止反应;用酶标仪测定吸光度,吸收波长为450/630nm。本发明制备的腾毒素单克隆抗体的效价可达到1:810000。
5、腾毒素单克隆抗体-胶体金标记物的制备
(1)胶体金的制备
用双蒸去离子水将1%氯金酸稀释成0.01%(质量分数),取100mL置于锥形瓶中,用恒温电磁搅拌器加热至沸腾,在持续高温、持续搅拌下加入1.5mL 1%柠檬酸三钠,继续匀速搅拌加热至溶液呈透亮的红色时停止,冷却至室温后用去离子水恢复到原体积,4℃保存。制备好的胶体金外观纯净、透亮、无沉淀和漂浮物,在日光下观察颜色为酒红色。
(2)腾毒素单克隆抗体-胶体金标记物的制备
在磁力搅拌下,用0.2mol/L碳酸钾溶液调节胶体金的pH值至7.2,按每毫升胶体金溶液中加入30μg抗体的标准向胶体金溶液中加入上述腾毒素单克隆抗体,继续搅拌混匀30min;静置10min后加入10% BSA,使其在胶体金溶液中的终浓度为1%,静置10min。12000r/min、4℃离心40min,弃上清液,沉淀用复溶缓冲液洗涤两次,用体积为初始胶体金体积1/10的复溶缓冲液将沉淀重悬,置4℃备用。
复溶缓冲液:含BSA的质量分数为0.2%、蔗糖的质量分数为3%、pH 7.2的0.02mol/L磷酸盐缓冲液。
6、结合物释放垫的制备
将结合物释放垫浸泡于含0.5% BSA、5%蔗糖、pH 7.4的0.02mol/L磷酸盐缓冲液中,均匀浸湿2h,37℃烘干备用。用Bio dot划膜仪将制备好的腾毒素单克隆抗体-胶体金标记物均匀喷涂在结合物释放垫上,每1cm结合物释放垫喷涂0.01mL腾毒素单克隆抗体-胶体金标记物后,置于37℃环境(湿度<20%)中2h后取出,置于干燥环境(湿度<20%)中保存备用。
7、样品垫的制备
将样品垫用含1% BSA、pH为7.2的0.2mol/L磷酸盐缓冲液浸泡2h,37℃烘干2h备用。
8、反应膜的制备
将腾毒素半抗原-OVA偶联物包被到反应膜上构成检测线,将羊抗鼠二抗包被在反应膜上构成质控线。
包被过程:用0.01mol/L、pH 7.2的磷酸缓冲液将腾毒素半抗原-OVA偶联物稀释到1mg/mL,用Bio dot划膜仪将其包被于硝酸纤维素膜上的检测线(T线),包被量为1.0μL/cm;用0.01mol/L、pH 7.2的磷酸盐缓冲液将羊抗鼠二抗稀释到200μg/mL,用Bio dot划膜仪将其包被于硝酸纤维素膜上的质控线(C线),包被量为1.0μL/cm。将包被好的反应膜置于37℃条件下干燥16h,备用。
9、试纸条的组装
根据附图2所示试纸条剖面结构,将样品垫(1)、结合物释放垫(2)、反应膜(3)、吸水垫(4)依次按顺序粘贴在PVC底板(7)上;结合物释放垫从起始端有1/3区域被样品垫覆盖,结合物释放垫的末端与反应膜的始端相连,反应膜的末端与吸水垫的始端相连,样品垫的始端与PVC底板的始端对齐,吸水垫的末端与PVC底板的末端对齐;所述反应膜上有检测线(5)和质控线(6),检测线(T线)和质控线(C线)均为与所述试纸条的长相垂直的条状带;检测线位于靠近结合物释放垫的末端的一侧;质控线位于远离结合物释放垫的末端的一侧;将试纸用机器切成3.95mm宽的小条,装在特制的塑料制卡壳中,以铝箔袋密封,保存在4℃下备用。
实施例2谷物中腾毒素的检测
1、样本前处理
将待测样本粉碎,称取(1.0±0.05)g至15mL聚苯乙烯离心管中,加入10mL 80%的甲醇,涡旋振荡3min,3000r/min室温离心5min,上清液即为待检样本液。
2、用试纸条检测
用微量移液器吸取100μL待检样本液垂直滴加于试纸条的样品垫上,液体流动开始计时,反应10min,判定结果。
3、分析检测结果
阴性(-):T线显色比C线显色深或与C线显色一致,表示样品中腾毒素浓度低于检测限,如图3a、3b。
阳性(+):T线显色比C线显色浅或T线不显色,表示样品中腾毒素浓度等于或高于检测限,如图3c、3d。
无效:未出现C线,表明不正确的操作过程或试纸条已变质失效,如图3e、3f。
实施例3免疫层析试纸条的灵敏度、准确性、特异性分析
1、检测限试验
取空白小麦、玉米样本,在其中分别添加腾毒素至终浓度为2.5μg/kg、5μg/kg、10μg/kg、20μg/kg,取试纸条进行检测,每个样本重复测定三次。
用试纸条检测小麦、玉米样本时,当其中无腾毒素和其添加浓度为2.5μg/kg、5μg/kg时,试纸条上显示出T线显色比C线显色深或与C线显色一致,呈阴性;当其中腾毒素添加浓度为10μg/kg、20μg/kg时,试纸条上显示出T线显色比C线显色浅或T线不显色,呈阳性,表明本试纸条对谷物中腾毒素的检测限为10μg/kg。
2、假阳性率、假阴性率试验
取空白小麦、玉米样本及添加腾毒素至终浓度为10μg/kg的阳性小麦、玉米样本各20份,用3个批次生产的试纸条分别进行检测,计算其阴阳性率。
结果表明:用3个批次生产的试纸条检测阳性小麦、玉米样本时,结果全为阳性,可知阳性符合率为100%,假阴性率为0;检测空白小麦、玉米样本时,结果全为阴性,可知阴性符合率为100%,假阳性率为0。说明本发明的检测腾毒素的试纸条可以对谷物中腾毒素残留进行快速准确检测。
3、特异性试验
用本试纸条检测1000μg/kg交链格孢酚、交链孢酚单甲醚、交链孢霉烯、细格菌素、细交链格孢酮酸等其他链格孢霉毒素时,试纸条上显示出T线显色比C线显色深,呈阴性,说明本试纸条对其他链格孢霉毒素无交叉反应,特异性良好。
尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.腾毒素半抗原,其特征在于,其结构如式(Ⅰ)所示:
式(Ⅰ)。
2.腾毒素全抗原,其特征在于,由权利要求1所述腾毒素半抗原与载体蛋白偶联后得到的;其中,所述载体蛋白为牛血清白蛋白、卵清白蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白。
3. 权利要求1所述腾毒素半抗原的制备方法,其特征在于,取无水三氯化铝0.39g,加入100mL 1,2-二氯乙烷和0.5g琥珀酸酐,搅拌30min;再加入0.414g腾毒素的1,2-二氯乙烷溶液10mL,室温搅拌1h,在搅拌下缓慢加入200mL纯水,搅拌后静置;分去水相,有机相再次水洗,无水硫酸钠干燥,蒸干,上硅胶小柱,用体积比为10:1的二氯甲烷-甲醇混合溶液洗脱分离,即得半抗原产物。
4.由权利要求1所述腾毒素半抗原,或权利要求2所述腾毒素全抗原制备的腾毒素检测试剂或试剂盒。
5.权利要求1所述腾毒素半抗原,或权利要求2所述腾毒素全抗原,或权利要求4所述检测试剂或试剂盒在腾毒素的免疫分析检测中的应用。
6.权利要求1所述腾毒素半抗原,或权利要求2所述腾毒素全抗原,或权利要求4所述检测试剂在制备腾毒素的胶体金检测试纸条中的应用。
7.一种快速检测真菌毒素腾毒素的免疫层析试纸条,其特征在于,包括样品垫、结合物释放垫、反应膜、吸水垫和底板,所述反应膜上设有检测线和质控线,所述样品垫、结合物释放垫、反应膜和吸水垫依次粘贴在底板上;其中,所述结合物释放垫上包被有腾毒素特异性抗体-胶体金标记物;所述检测线包被有权利要求2所述腾毒素全抗原,所述质控线包被有羊抗鼠二抗。
8. 根据权利要求7所述的试纸条,其特征在于,所述腾毒素特异性抗体-胶体金标记物的制备方法包括以下步骤:特异性抗体与胶体金溶液混合后,加入10%牛血清白蛋白至牛血清白蛋白在该溶液中的终浓度为1%后,进行静置、离心得到沉淀,用复溶缓冲液洗涤、重悬所得沉淀;所用复溶缓冲液为含牛血清白蛋白的质量分数为0.1%~0.5%、蔗糖的质量分数为2%~4%、pH 7.2的0.02mol/L磷酸盐缓冲液。
9. 利用权利要求7或8所述试纸条检测腾毒素的方法,其特征在于,将待测样本粉碎,称取1.0 g±0.05g至15mL聚苯乙烯离心管中,加入10mL 80%的甲醇,涡旋振荡3min,3000r/min室温离心5min,上清液即为待检样本液;用微量移液器吸取100μL待检样本液垂直滴加于试纸条的样品垫上,液体流动开始计时,反应10min,根据检测线和质控线的显色判定结果。
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