CN110068685B - 一种点免疫印迹检测的检测装置及检测方法 - Google Patents
一种点免疫印迹检测的检测装置及检测方法 Download PDFInfo
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Abstract
本发明提供一种点免疫印迹检测的检测装置,包括负压吸引装置、孔板、连接孔板和负压吸引装置的软管及紧密贴合在孔板上端面的硝酸纤维膜,所述孔板包括上层托板、密封连接在上层托板下方的下层托盘及设置于上层托板、下层托盘之间的负压空腔,所述下层托盘一侧设置有一供软管套接的连接管,所述上层托板上具有70个通孔,所述硝酸纤维膜与孔板上端面的大小相同。本发明还提供一种点免疫印迹检测的检测方法,包括准备、加样、封闭、孵育一抗、孵育二抗、显影、分析的过程,本发明通过负压吸引装置进行负压吸引,有利于加样时样品的集中,避免样品扩散后交叉污染影响实验结果,实验结果更准确。
Description
技术领域
本发明属于免疫检测技术领域,具体涉及一种点免疫印迹检测的检测装置及检测方法。
背景技术
目前多用Western blot或者ELISA的方法来检测目的蛋白。Western blot法:将经过PAGE(聚丙烯酰胺凝胶电泳)分离出的蛋白质样品转移到PVDF膜(聚偏二氟乙烯膜)上,用目的蛋白对应的抗体结合蛋白抗原,再结合酶或者同位素标记过的二抗,以化学发光的方式检测目的蛋白的表达。ELISA法:受检样品(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应。用洗涤的方法把形成的抗原抗体复合物和其他物质分开。再加入酶标记的抗原或抗体,通过特异性反应而结合在固相载体上。此时固相载体上的酶量与标本中受检物质的量呈一定的比例,所以可根据呈色的深浅进行定性或定量分析。但两种方法操作复杂、样品需求量大,并且不能同时兼顾灵敏、特异、多样本检测等优点。
发明内容
本发明要解决的技术问题是提供一种点免疫印迹检测的检测装置及检测方法,以解决背景技术中所提出的问题。
为解决上述技术问题,本发明的实施例提供一种点免疫印迹检测的检测装置,其特征在于,包括负压吸引装置、孔板、连接孔板和负压吸引装置的软管及紧密贴合在孔板上端面的硝酸纤维膜,所述孔板包括上层托板、密封连接在上层托板下方的下层托盘及设置于上层托板、下层托盘之间的负压空腔,所述下层托盘一侧设置有一供软管套接的连接管,所述上层托板上具有70个通孔,所述硝酸纤维膜与孔板上端面的大小相同。
进一步的,所述上层托板的尺寸为8 cm×6 cm,所述通孔直径为2 mm。
进一步的,所述负压吸引装置选用手提式吸痰器,所述手提式吸痰器的负压值设定为0.06 MPa。
进一步的,70个所述通孔呈10×7设置,每两个通孔之间距离相等。
进一步的,所述孔板的上方可拆卸连接有一压板。
其中,所述压板包括矩形边框及转动连接在矩形边框四周的卡扣,所述下层托盘的四周设置有供卡扣卡接的卡块。
本发明还提供一种点免疫印迹检测的检测方法,其特征在于,包括以下步骤:
S1、准备:将手提式吸痰器通过软管和孔板相连接;将硝酸纤维膜剪至孔板上端面的上层托板大小,置于具有70个通孔的孔板上,打开手提式吸痰器,将手提式吸痰器的负压值设置为0.06 MPa,在硝酸纤维膜表面形成70个加样凹槽;
S2、加样:将70个加样凹槽分为对照品孔和样本孔,每个重复3次,对照品孔和样本孔分别加入对照品和各待测样本1μL;加样结束,将硝酸纤维膜自然晾干30 min;
S3、封闭:将硝酸纤维膜轻置于5%的脱脂奶粉溶液中,置于摇床上室温下封闭1.5h;然后,弃去封闭液,用TBS-T漂洗1-2次;其中,5%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5%;
S4、孵育一抗:用5% 牛血清白蛋白溶液按1:500比例稀释一抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上,37 ℃恒温孵育4 h;结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次,每次5 min;其中,5%牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5%;
S5、孵育二抗:用1%脱脂奶粉溶液按1:5000比例稀释二抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上室温孵育1.5 h;结束后将硝酸纤维膜放入TBS-T中洗涤3次,每次15 min;1%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1%;
S6、显影:将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置,将显影剂均匀滴加于硝酸纤维膜上,采用凝胶成像系统曝光显影并保存;
S7、分析:显影所得图像用Image J软件进行灰度值扫描分析;各样本目的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
进一步的,所述TBS-T溶液包括Tris、 NaCl、Tween-20 及ddH2O,其中,Tris的质量为 2.42 g,NaCl的质量为8.0 g,Tween-20的体积为1 mL,所述TBS-T溶液使用ddH2O将体积补充至1000ml。
进一步的,所述样本孔可根据待测样本的组数设置为多组样本孔。
本发明的上述技术方案的有益效果如下:
(1)本发明的点免疫印迹检测的检测装置,使硝酸纤维膜紧密贴合70孔板并在表面形成70个加样凹槽,利用这种负压吸引装置进行负压吸引,有利于加样时样品的集中,避免样品扩散后交叉污染影响实验结果,实验结果更准确。
(2)本发明点免疫印迹检测的检测装置及检测方法,进行点免疫印迹检测相比Western blot和ELISA的方法,点免疫印迹装置进行的免疫印迹检测不仅具有灵敏、特异、样品需要量少、简单、经济和快速的优点,同时还能一次进行多样本检测,适合大量人群的检测。
附图说明
图1为本发明中点免疫印迹检测的检测装置的结构示意图;
图2为本发明中孔板的俯视图;
图3为本发明中点免疫印迹检测的检测方法对胰腺癌小鼠组、胰腺炎小鼠组和正常对照组的血清FGB的相对浓度的检测结果图;
图4为本发明中点免疫印迹检测的检测方法检测胰腺癌小鼠组、胰腺炎小鼠组和正常对照组的血清FGB的相对浓度对比图。
附图标记说明:1、负压吸引装置;2、孔板;21、上层托板;22、下层托盘;23、连接管;24、通孔;25、负压空腔;3、软管;4、硝酸纤维膜;5、负压空腔。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
在本发明的描述中,需要说明的是,术语“中心”、“上”“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”、“前”、“后”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制。此外,术语“第一”、“第二”、“第三”仅用于描述目的,而不能理解为指示或暗示相对重要性。
在本发明的描述中,需要说明的是,除非另有明确的规定和限定,术语“安装”、“相连”、“连接”应作为广义理解,例如,可以是固定连接,也可以是可拆卸连接,或一体地连接;可以是机械连接,也可以是电连接;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通。对于本领域的普通技术人员而言,可以具体情况理解上述术语在本发明中的具体含义。
如图1和图2所示,一种点免疫印迹检测的检测装置,包括负压吸引装置1、孔板2、连接孔板2和负压吸引装置1的软管3及紧密贴合在孔板2上端面的硝酸纤维膜4,可行的,所述软管3可采用橡胶管或塑料软管,所述孔板2包括上层托板21、密封连接在上层托板21下方的下层托盘22及设置于上层托板21、下层托盘22之间的负压空腔25,所述下层托盘22一侧设置有一供软管3套接的连接管23,所述上层托板21上具有70个通孔24,所述硝酸纤维膜4与孔板2上端面的大小相同。使用时,用软管3紧密连接70孔板2的连接管23处和负压吸引装置1的接口;将硝酸纤维膜4置于70孔板2上,形成封闭的内腔,再打开负压吸引装置1进行抽气提供负压环境,使硝酸纤维膜紧4密贴合70孔板2并在表面形成70个加样凹槽,利用这种负压吸引装置1进行负压吸引,这种方式有利于加样时样品的集中,避免样品扩散交叉污染,影响实验结果。
在进一步的实施例中,所述上层托板21的尺寸为8 cm×6 cm,所述通孔24直径为2mm。
在进一步的实施例中,所述负压吸引装置1选用手提式吸痰器,所述手提式吸痰器的负压值设定为0.06 MPa。
在进一步的实施例中,70个所述通孔24呈10×7设置,每两个通孔24之间距离相等,可行的,所述通孔之间的距离为5.5cm-7cm。
在进一步的实施例中,所述孔板2的上方可拆卸连接有一压板。
其中,所述压板包括矩形边框及转动连接在矩形边框四周的卡扣,所述下层托盘22的四周设置有供卡扣卡接的卡块,这种卡块与卡扣的连接方式,可参照饭盒的连接方式,起到压紧硝酸纤维膜4即可。本发明的孔板上可拆卸连接有压板,通过压板将硝酸纤维膜4压在孔板上方,以保证硝酸纤维膜4与孔板紧密贴合。
在本发明中,点免疫印迹检测的原理是将待测样品吸附至硝酸纤维膜4(NC膜)上,固相载体以非共价键形式吸附蛋白质,且能保持多肽类型及其生物学活性不变。以硝酸纤维膜4上的蛋白质或多肽作为抗原,与目的蛋白相应的抗体发生免疫反应,再与辣根过氧化物酶(HRP)标记的二抗反应,经过放射自显影以检测目的蛋白的表达水平。
本发明还提供一种点免疫印迹检测的检测方法,包括以下步骤:
S1、准备:将手提式吸痰器通过软管3和孔板2相连接;将硝酸纤维膜4剪至具有70个通孔24的孔板3大小,置于具有70个通孔24的孔板2上,打开手提式吸痰器,将手提式吸痰器的负压值设置为0.06 MPa,在硝酸纤维膜4表面形成70个加样凹槽。
S2、加样:将70个加样凹槽分为对照品孔和样本孔,每个重复3次(即对照品孔和样本孔均具有3个复孔),对照品孔和样本孔分别加入对照品和各待测样本1μL;加样结束,将硝酸纤维膜自然晾干30 min。
S3、封闭:将硝酸纤维膜4轻置于5%的脱脂奶粉溶液中,置于摇床上室温下封闭1.5h;然后,弃去封闭液,用TBS-T漂洗1-2次;其中,5%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5%。
S4、孵育一抗:用5% 牛血清白蛋白溶液按1:500比例稀释一抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上,37 ℃恒温孵育4 h;结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次,每次5 min;其中,5%牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5%。
S5、孵育二抗:用1%脱脂奶粉溶液按1:5000比例稀释二抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上室温孵育1.5 h;结束后将硝酸纤维膜放入TBS-T中洗涤3次,每次15 min;1%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1%。在本专利中检测血清中FGB中使用的一抗为FGB鼠抗人单克隆抗体,购自Santa Cruz公司,货号:sc-271035;二抗为HRP标记的鼠抗人单克隆抗体,购自Santa Cruz公司,货号:sc-516102。
S6、显影:将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置,将显影剂均匀滴加于硝酸纤维膜上,采用凝胶成像系统曝光显影并保存;
S7、分析:显影所得图像用Image J软件进行灰度值扫描分析;各样本目的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
在进一步的实施例中,所述TBS-T溶液包括Tris、 NaCl、Tween-20 及ddH2O,其中,Tris的质量为 2.42 g,NaCl的质量为8.0 g,Tween-20的体积为1 mL,所述TBS-T溶液使用ddH2O将体积补充至1000ml,如下表1所示;
表1 TBS-T溶液的配方表
在进一步的实施例中,所述样本孔可根据待测样本的组数设置为多组样本孔。
在本实施例中,先采用质谱分析发现胰腺癌患者血清中FGB的丰度高于胰腺炎患者和正常人;然后,采用了点免疫印迹检测装置及其检测血清FGB的检测方法对前期的质谱分析结果进行验证。在本实施例中,分别对两组小鼠(每组7只)进行皮下注射胰腺癌细胞和腹腔注射雨蛙素,构建胰腺癌小鼠模型和胰腺炎小鼠模型,分别提取7只胰腺癌小鼠模型和7只胰腺炎小鼠模型的血清,制备成胰腺癌小鼠组和胰腺炎小鼠组。对照组小鼠(7只)不予处理,抽取对照组小鼠的血清,制备成正常对照组;然后采用点免疫印迹检测的检测方法对三组小鼠的血清FGB的相对浓度进行检测,检测结果如3所示;其中,a2~4列及a5~7列分别为3个不同浓度梯度的对照品,b、c、d列为胰腺癌小鼠组(b1、c1、d1为同一小鼠,b2、c2、d2为同一小鼠,以此类推,将7只胰腺癌小鼠的血清都滴入样品孔内),e、f、g列为胰腺炎小鼠组(e1、f1、g1为同一小鼠,e2、f2、g2为同一小鼠,以此类推,将7只胰腺炎小鼠的血清都滴入样品孔内),h、i、j列为正常对照品组(h1、i1、j1为同一小鼠,h2、i2、j2为同一小鼠,以此类推,将7只正常小鼠的血清均滴入样品孔内)。用Image J软件将图像反向后,统计各血清标本灰度值。各血清样本FGB相对浓度用待测样本灰度值/对照样本灰度值表示。各组FGB相对浓度统计见表2及图4所示。
注:*与胰腺炎组、正常对照组比较,P<0.05。QL-QU:四分位间距;Max-Min:最大值-最小值。
表2及图4的结果显示:FGB在胰腺癌小鼠血清中的相对浓度明显高于胰腺炎小鼠及正常对照组(P均<0.05),而胰腺炎组与正常对照组之间的相对浓度差异尚无统计学意义(P>0.05),结果与前期的质谱结果相一致,证实了点免疫印迹检测的检测装置及检测方法进行血清FGB检测的可行性。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种点免疫印迹检测的检测方法,其特征在于,包括以下步骤:
S1、准备:将手提式吸痰器通过软管和孔板相连接;将硝酸纤维膜剪至孔板上端面的上层托板大小,置于具有70个通孔的孔板上,打开手提式吸痰器,将手提式吸痰器的负压值设置为0.06MPa,在硝酸纤维膜表面形成70个加样凹槽;
S2、加样:将70个加样凹槽分为对照品孔和样本孔,每个重复3次,对照品孔和样本孔分别加入对照品和各待测样本1μL;加样结束,将硝酸纤维膜自然晾干30min;
S3、封闭:将硝酸纤维膜轻置于5%的脱脂奶粉溶液中,置于摇床上室温下封闭1.5h;然后,弃去封闭液,用TBS-T漂洗1-2次;其中,5%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5%;
S4、孵育一抗:用5%牛血清白蛋白溶液按1:500比例稀释一抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上,37℃恒温孵育4h;结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次,每次5min;其中,5%牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5%;
S5、孵育二抗:用1%脱脂奶粉溶液按1:5000比例稀释二抗,将硝酸纤维膜均匀的完全浸入其中,置于摇床上室温孵育1.5h;结束后将硝酸纤维膜放入TBS-T中洗涤3次,每次15min;1%脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1%;
S6、显影:将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置,将显影剂均匀滴加于硝酸纤维膜上,采用凝胶成像系统曝光显影并保存;
S7、分析:显影所得图像用Image J软件进行灰度值扫描分析;各样本目的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
2.根据权利要求1所述的一种点免疫印迹检测的检测方法,其特征在于,所述TBS-T溶液包括Tris、NaCl、Tween-20及ddH2O,其中,Tris的质量为2.42g,NaCl的质量为8.0g,Tween-20的体积为1mL,所述TBS-T溶液使用ddH2O将体积补充至1000ml。
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