CN111289741A - 一种偶联cfp-10特异性单抗的纳米磁珠及其制备方法与应用 - Google Patents
一种偶联cfp-10特异性单抗的纳米磁珠及其制备方法与应用 Download PDFInfo
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Abstract
本发明适用于结核菌临床辅助检测技术领域,提供了一种偶联CFP‑10特异性单抗的纳米磁珠及其制备方法与应用。本发明所述的偶联CFP‑10特异性单抗的纳米磁珠由CFP‑10特异性单抗与纳米磁珠偶联获得;所述CFP‑10特异性单抗所识别的抗原在RD1区,所述纳米磁珠为含NHS功能基团的纳米磁珠。本发明提供的含NHS(琥珀酰亚胺)功能基团的超顺磁性纳米磁珠微球偶联CFP‑10单抗及建立的富集捕获结核分枝杆菌,能增加结核分枝杆菌的阳性检出率及特异性,提高结核的临床诊断。
Description
技术领域
本发明属于结核菌临床辅助检测技术领域,尤其涉及一种偶联CFP-10特异性单抗的纳米磁珠及其制备方法与应用。
背景技术
结核病是由结核分枝杆菌感染引起的传染病,主要通过呼吸道传播,随着病情不断恶化而导致全身多系统疾病,属于严重损害人类健康的慢性传染病。据世界卫生组织(WHO)统计报道,2016年全球新发现的结核病例约1040万,中国新发病例占全球的8.6%,成为排名世界第三的结核病高负担国家。因此,对于结核病疫情的防控刻不容缓,早期诊断和及时规范治疗显得尤为重要。
目前对于致病结核分枝杆菌的检测方法有很多,主要包括细菌学检测技术、免疫学检测技术和分子生物学检测技术等,但结核病的诊断仍主要依赖于传统的结核分枝杆菌培养和涂片抗酸染色。由于培养法其培养周期较长,一般需要2-8周,不能满足结核病早期临床诊断,因此结核病(TB)的诊断主要依赖于涂片抗酸染色法,尤其是在中低收入国家。涂片抗酸染色镜检观察法是细菌学检测方法中常用的方法,但是由于敏感性低,对细菌的数量要求较高,只有当痰液中含5000-10000个菌/ml才能检测到抗酸菌,因此阳性检出率较低。而且由于其他杆菌如放线菌和红线菌都具有部分抗酸染色特性,造成该方法的特异性较差。由此可以看出,对标本中致病结核分枝杆菌通过特异性标志物进行富集,对提高细菌学检查的阳性率和特异性至关重要。
培养滤液蛋白10(CFP-10)是由结核分枝杆菌的RD1区基因Rv3874编码,在体内和早期分泌抗原靶蛋白6(ESAT-6)形成1∶1异二聚体复合物发挥生物学功能,激发强烈的特异性T细胞应答。由于CFP-10所在的RD1区只存在于结核分枝杆菌和牛分枝杆菌中,而其他分枝杆菌及BCG中均缺失该区序列,因此成为结核病诊断研究的热点。
发明内容
为解决结核诊断中因结核分枝杆菌浓度要求导致阳性检出率低以及特异性的问题,本发明提供了一种偶联CFP-10特异性单抗的纳米磁珠。
本发明的另一目的在于提供上述偶联CFP-10特异性单抗的纳米磁珠的制备方法。
本发明的再一目的在于提供上述偶联CFP-10特异性单抗的纳米磁珠的应用。所述偶联CFP-10特异性单抗的纳米磁珠应用于富集捕获结核分枝杆菌进行抗酸染色及免疫荧光检测,以便满足临床的需求。
本发明的目的通过下述技术方案实现:
一种偶联CFP-10特异性单抗的纳米磁珠,由CFP-10特异性单抗与纳米磁珠偶联获得;所述CFP-10特异性单抗所识别的抗原在RD1区,所述纳米磁珠为含NHS(琥珀酰亚胺)功能基团的纳米磁珠。
所述RD1区,只存在于结核分枝杆菌和牛分枝杆菌中,而其他分枝杆菌及BCG中均缺失该RD1区;本发明所用的识别抗原RD1区的CFP-10特异性单抗具有特异性,特异识别结核分枝杆菌。所述CFP-10特异性单抗,通过制备基因克隆表达结核分枝杆菌RD1区的CFP-10抗原,免疫小鼠制备获得CFP-10特异性单抗。
所述含NHS(琥珀酰亚胺)功能基团的纳米磁珠优选采用上海医脉赛生物科技有限公司的含NHS(琥珀酰亚胺)功能基团的纳米磁珠(NHS磁珠,目录号:FE06002)。
上述偶联CFP-10特异性单抗的纳米磁珠的制备方法,包括以下步骤:
取含2.5mg的纳米磁珠50ul于EP管中;洗涤缓冲液洗涤磁珠,磁场吸附,弃上清;用5ml偶联缓冲液重悬磁珠,在5ml偶联缓冲液重悬磁珠中加入5ml浓度为0.5mg/ml的CFP-10单抗,置于旋转混合仪4℃震荡过夜;磁场分离磁珠,(移除含蛋白的上清液紫外分光光度计测定蛋白浓度);加入封闭液缓冲液重悬磁珠置于旋转混合仪室温混合4小时;磁场吸附,弃上清;封闭液缓冲液洗涤偶联蛋白的磁珠,磁场吸附,弃上清;用1ml封闭缓冲液重悬磁珠,于2-8℃储存。
所述洗涤缓冲液为磷酸盐缓冲液(PBS),pH 7.4;
所述偶联缓冲液为150mM NaCl,0.01%Tween-20,50mM MES,pH7.0;
所述封闭液缓冲液为150mM NaCl,100mM Tris-HCl,pH 7.0。
所述CFP-10单抗的浓度为0.5mg/ml。
上述制备方法获得的偶联CFP-10特异性单抗的纳米磁珠的检测方法采用紫外分光光度计直接检测法或双抗夹心ELISA法中的一种。
所述紫外分光光度计直接检测法,采用直接法紫外分光光度计测未偶联抗体的浓度即可知道偶联上抗体的浓度。
所述双抗夹心ELISA法,用HRP标记CFP-10单抗,通过酶底物显色来检测抗体偶联到磁珠上与否及其活性和免疫荧光进行验证是否将单抗偶联到磁珠上。
上述偶联CFP-10特异性单抗的纳米磁珠,在富集结核分枝杆菌中应用。
本发明采用抗酸染色镜检及免疫荧光来验证偶联CFP-10单抗的磁珠是否富集捕获到结核分枝杆菌。
上述偶联CFP-10特异性单抗的纳米磁珠,通过富集结核分枝杆菌,在抗酸染色检测中应用。
上述偶联CFP-10特异性单抗的纳米磁珠的应用,还体现在用于制备抗酸染色检测试剂。
本发明利用的NHS磁珠是含NHS(琥珀酰亚胺)功能基团的超顺磁性纳米磁珠微球,可与CFP-10抗体的伯胺进行偶联反应形成稳定的酰胺键。偶联后的抗体磁珠当在达到某一条件时,使得其能有效的辨识分枝杆菌并与之结合。随后再通过一个外加磁场作用,使结合上分枝杆菌的磁珠沉降下来,从而达到了分离和富集结核分枝杆菌的作用。
本发明相对于现有技术具有如下的优点及效果:
1.基于含NHS(琥珀酰亚胺)功能基团的纳米磁珠偶联CFP-10特异性的单克隆抗体,与现有商品化内部含有化学聚合物、对分枝杆菌细胞表面上的脂阿拉伯甘露聚糖和分枝菌酸具有特异性和亲和力的磁珠相比:现有商品化磁珠虽然一定程度上能富集捕获到TB,但捕获的分枝杆菌有的不是结核分枝杆菌,缺乏特异性;本发明偶联上CFP-10特异性单抗的磁珠能特异性富集结核分枝杆菌,提高结核细菌学检测的阳性率。因此基于本发明偶联上CFP-10特异性单抗的磁珠在结核细菌学检测中更高的特异性。
2.通过本发明的研究,确定了一种纳米磁珠偶联CFP-10特异性单抗;并且摸索出制备的偶联单抗磁珠用于富集捕获的用量及反应条件。基于本发明的纳米磁珠偶联CFP-10特异性单抗建立了抗酸染色检测技术。由于本发明偶联抗体的磁珠能富集结核分枝杆菌,且偶联的为CFP-10特异性单抗,因此在结核诊断检测中能特异性的提高结核分枝杆菌的阳性检出率,以满足临床要求。
附图说明
图1显示偶联CFP-10特异性单抗的磁珠进行双抗夹心ELISA法鉴定结果图。
图2显示偶联CFP-10特异性单抗的磁珠进行免疫荧光检测鉴定的免疫荧光鉴定图;其中:A、对照组的磁珠明场图,×40;B、对照组的磁珠荧光图,×40;C、实验组的磁珠明场图,×40;D、实验组的磁珠荧光图,×40。
图3显示偶联CFP-10特异性单抗的磁珠富集捕获结核分枝杆菌抗酸染色的鉴定结果图。其中:A、总菌数50000个的磁珠偶联CFP-10单抗富集抗酸染色,×100油镜;B、总菌数50000个的直接涂片抗酸染色,×100油镜;C、总菌数3000个的磁珠偶联CFP-10单抗富集抗酸染色,×100油镜;D、总菌数3000个的直接涂片抗酸染色,×100油镜;E、总菌数300个的磁珠偶联CFP-10单抗富集抗酸染色,×100油镜;F、总菌数300个的直接涂片抗酸染色,×100油镜。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1偶联CFP-10特异性单抗的磁珠的制备及鉴定
1、偶联CFP-10特异性单抗的纳米磁珠的制备方法,具体步骤如下:
取含2.5mg的纳米磁珠50ul于EP管中;洗涤缓冲液洗涤磁珠,磁场吸附,弃上清;用5ml偶联缓冲液重悬磁珠,在5ml偶联缓冲液重悬磁珠中加入5mlCFP-10单抗(0.5mg/ml,总抗体为2.5mg),置于旋转混合仪4℃震荡过夜;磁场分离磁珠,(移除含蛋白的上清液紫外分光光度计测定蛋白浓度);加入封闭液缓冲液重悬磁珠置于旋转混合仪室温混合4小时;磁场吸附,弃上清;封闭液缓冲液洗涤偶联蛋白的磁珠,磁场吸附,弃上清;用1ml封闭缓冲液重悬磁珠,于2-8℃储存。
所述洗涤缓冲液为磷酸盐缓冲液(PBS),pH 7.4;
所述偶联缓冲液为150mM NaCl,0.01%Tween-20,50mM MES,pH7.0;
所述封闭液缓冲液为150mM NaCl,100mM Tris-HCl,pH 7.0。
所述CFP-10单抗的浓度为0.5mg/ml。
所述CFP-10特异性单抗所识别的抗原在RD1区,所述纳米磁珠为含NHS(琥珀酰亚胺)功能基团的纳米磁珠。
所述RD1区,只存在于结核分枝杆菌和牛分枝杆菌中,而其他分枝杆菌及BCG中均缺失该RD1区;本发明所用的识别抗原RD1区的CFP-10特异性单抗具有特异性,特异识别结核分枝杆菌。所述CFP-10特异性单抗,通过制备基因克隆表达结核分枝杆菌RD1区的CFP-10抗原,免疫小鼠制备获得CFP-10特异性单抗。
所述含NHS(琥珀酰亚胺)功能基团的纳米磁珠优选采用上海医脉赛生物科技有限公司的含NHS(琥珀酰亚胺)功能基团的纳米磁珠(NHS磁珠,目录号:FE06002)。
2、偶联CFP-10特异性单抗的磁珠的鉴定
(1)双抗夹心ELISA法
偶联CFP-10单抗的磁珠PBS 10倍稀释,100ul/孔加入;调整CFP-10抗原浓度为1ug/ml、0.5ug/ml、0.25ug/ml、0.125ug/ml、62.5ng/ml、6.25ng/ml、每孔100ul加入;室温震荡孵育1小时,磁珠洗板机洗涤5次;酶稀液稀释HRP标记的CFP-10单抗,100ul/孔加入;室温震荡孵育30min,磁珠洗板机洗涤5次;显色液A与显色液B(1∶1)混合100ul/孔,37℃避光10min;硫酸终止液50ul/孔终止反应;采用酶标仪双波长450nm/630nm测定各孔的吸光值。其ELISA鉴定结果曲线如图1所示,双抗体夹心法中偶联抗体检测最低浓度为2.5ng/ml的抗原,通过计算得出磁珠上偶联CFP-10单抗1.7mg。
(2)免疫荧光法
取10ul偶联抗体的磁珠,及等量未偶联抗体的空磁珠均匀地涂在具有粘附性玻片上自然晾干。分别设置实验组:偶联抗体的磁珠+二抗;阴性对照组:空磁珠+二抗;空白对照组:PBS+二抗。二抗(山羊抗小鼠标记FITC)1:200用PBS稀释后加100ul覆盖在磁珠上,避光孵育1小时。PBS洗涤后,50%甘油封片,荧光显微镜下观察,结果如图2所示,免疫荧光实验显示偶联上抗体的磁珠与FITC标记的二抗孵育后可见荧光,而对照无荧光。
实施例2偶联CFP-10单抗的磁珠富集捕获结核分枝杆菌抗酸染色的鉴定
结核分枝杆菌通过超声波细胞破碎机将聚集的团块分散开,通过紫外分光光度计测得吸光度,计算出结核分枝杆菌浓度。将已知浓度的结核分枝杆菌稀释成高、中、低三个不同浓度,分成实验组与对照组,使得每组含菌数量相同,分别为上万个(50000个)、几千个(3000个)和几百个(300个)。对照组直接进行萋尼法抗酸染色及加空磁珠进行萋尼法抗酸染色,实验组偶联抗体的磁珠富集结核分枝杆菌后进行萋尼法抗酸染色。具体为实验将稀释后的结核分杆菌菌液及适量偶联抗体磁珠室温震荡反应一小时,磁场分离后涂片;对照组用等量菌液和磁珠,加空磁珠的同样室温振荡反应一小时后涂片,不加磁珠的直接涂片。待三组片子都自然干燥后加石碳酸品红溶液100ul/张在60℃烤片机中染色一小时,自来水浸泡1-2min,3%盐酸水溶液脱色至淡粉色,蒸馏水浸泡10-20min,0.1%美蓝溶液复染30s,95%乙醇分色5-10s,无水乙醇脱水1min,二甲苯透明1min,中性树脂封片,100×倍油镜观察,鉴定结果图如图3所示,在含不同数量菌悬液的三组实验中,与对照组相比,实验组进行的抗酸染色结果显示图片上的结核分枝杆菌均明显多于对照组,而且结核分枝杆菌多富集在磁珠周围,说明磁珠偶联CFP-10特异性单抗能有效富集结核分枝杆菌。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种偶联CFP-10特异性单抗的纳米磁珠,其特征在于:由CFP-10特异性单抗与纳米磁珠偶联获得;所述CFP-10特异性单抗所识别的抗原在RD1区,所述纳米磁珠为含NHS功能基团的纳米磁珠。
2.权利要求1所述的偶联CFP-10特异性单抗的纳米磁珠的制备方法,其特征在于:包括以下步骤:
取含2.5mg的纳米磁珠50ul于EP管中;洗涤缓冲液洗涤磁珠,磁场吸附,弃上清;用5ml偶联缓冲液重悬磁珠,在5ml偶联缓冲液重悬磁珠中加入5ml浓度为0.5mg/ml的CFP-10单抗,置于旋转混合仪4℃震荡过夜;磁场分离磁珠;加入封闭液缓冲液重悬磁珠置于旋转混合仪室温混合4小时;磁场吸附,弃上清;封闭液缓冲液洗涤偶联蛋白的磁珠,磁场吸附,弃上清;用1ml封闭缓冲液重悬磁珠,于2-8℃储存。
3.如权利要求2所述的偶联CFP-10特异性单抗的纳米磁珠的制备方法,其特征在于:
所述洗涤缓冲液为磷酸盐缓冲液,pH 7.4;
所述偶联缓冲液为150mM NaCl,0.01%Tween-20,50mM MES,pH7.0;
所述封闭液缓冲液为150mM NaCl,100mM Tris-HCl,pH 7.0。
4.如权利要求2所述的偶联CFP-10特异性单抗的纳米磁珠的制备方法,其特征在于:制备获得的偶联CFP-10特异性单抗的纳米磁珠的检测采用紫外分光光度计直接检测法或双抗夹心ELISA法中的一种。
5.如权利要求4所述的偶联CFP-10特异性单抗的纳米磁珠的制备方法,其特征在于:所述紫外分光光度计直接检测法,采用直接法紫外分光光度计测未偶联抗体的浓度即可知道偶联上抗体的浓度。
6.如权利要求4所述的偶联CFP-10特异性单抗的纳米磁珠的制备方法,其特征在于:所述双抗夹心ELISA法,用HRP标记CFP-10单抗,通过酶底物显色来检测抗体偶联到磁珠上与否及其活性和免疫荧光进行验证是否将单抗偶联到磁珠上。
7.权利要求1所述的偶联CFP-10特异性单抗的纳米磁珠在富集结核分枝杆菌中应用。
8.权利要求1所述的偶联CFP-10特异性单抗的纳米磁珠在抗酸染色检测中应用。
9.权利要求1所述的偶联CFP-10特异性单抗的纳米磁珠的应用,其特征在于:用于制备抗酸染色检测试剂。
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