CN110054681B - 黄芪甲苷人工抗原及其人工抗体的制备方法和应用 - Google Patents
黄芪甲苷人工抗原及其人工抗体的制备方法和应用 Download PDFInfo
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- CN110054681B CN110054681B CN201910034969.3A CN201910034969A CN110054681B CN 110054681 B CN110054681 B CN 110054681B CN 201910034969 A CN201910034969 A CN 201910034969A CN 110054681 B CN110054681 B CN 110054681B
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Abstract
本发明报道了一种黄芪甲苷的人工抗原及其高特异性人工抗体的制备方法。首先用2,2,6,6‑四甲基哌啶‑1‑氧自由基(TEMPO)氧化法将黄芪甲苷分子中葡萄糖基上C6”‑CH2‑OH选择性氧化成‑COOH,然后再将所得的‑COOH与BSA上的氨基缩合得到人工抗原;最后用所得的人工抗原免疫动物成功制备出多克隆抗体,该抗体的效价大于1:128000,对黄芪甲苷的IC50为9.89μg/mL。进一步使用5个黄芪甲苷结构类似物测定了抗体的交叉反应率,结果表明,较之传统的高碘酸钠法制备的人工抗体,本发明采用的TEMPO法制备的人工抗体的特异性更高,可用于黄芪甲苷的ELISA分析。这一工作为黄芪甲苷及的高选择半抗原设计和人工抗体制备提供了一种新办法。
Description
技术领域
本发明涉及免疫分析领域,具体涉及一种黄芪甲苷人工抗原及其多克隆抗体及其制备方法和应用。
背景技术
苷类化合物是糖及其衍生物与另一非糖物质连接而成的一类化合物,广泛分布于植物之中。大量的皂苷、黄酮苷等糖苷类化合物已被现代药学研究证明为天然药物的有效成分,而广泛地应用于疾病治疗及保健预防领域。随着对天然糖苷的研究和应用的不断增多,对其方便、高效、灵敏的分析检测技术提出了更高的要求。但是,尽管过去几十年来以HPLC为代表的传统分析技术取得了巨大成就,但仍然存在一些缺点,如样品预处理复杂、分析时间长、仪器要求高等。免疫学检测技术具有检测速度快、费用低廉、仪器简单、灵敏度高和选择性强等优点,不但能够用于样品的定性筛选,还能进行定量测定,因此越来越多地应用于天然产物的分析研究。
免疫分析最重要的环节是制备被分析物的高特异性的人工抗体。由于绝大多数天然糖苷的分子量小(<1000Da),为半抗原而不能引起免疫反应。必须将半抗原(化合物本身或其衍生物)通过化学反应与载体蛋白共价结合,制成完全抗原(人工抗体)后才能有效地刺激机体产生特异性的抗体。半抗原设计的基本原则是尽量保持半抗原的原有分子特征结构以便能暴露在人工抗原的表面,使其能最大限度地被动物的免疫活性细胞所识别,从而刺激机体产生特异性免疫应答,产生对待测物具有高亲和力和高特异性的抗体。
对糖苷类化合物而言,最常用半抗原设计的方法是“高碘酸钠氧化法”,即先使用高碘酸钠将糖基氧化,开环转化为醛,然后与BSA的赖氨酸残基结合,脱水后形成希夫碱(如图1)。虽然采用这一方案成功合成了各种天然糖苷的人工抗原并应用于人工抗体的制备,如黄芪甲苷(Yu S.L.,et al.,Revista Brasileira De Farmacognosia,2014,24:282-287)、甘草苷(Fujii S.,et al.,Journal of Agricultural and Food Chemistry,2014,62:3377-3383)、大豆苷(Sakamoto S.,et al.,Food Chemistry,2015,169:127-133)、芍药苷(Zhao Y.,et al.,Planta Medica,2015,81:765-770)、三七皂苷R1(Limsuwanchote S.,et al.,Planta Medica,2014,80:337-342)和人参皂苷Re(Qu H.,et al.,Planta medica,2014,80:1143-1150)等。但是,由于糖基被高碘酸钠氧化破坏,化合物的结构特异性降低,从而使得相应的人工抗原的特异性降低。所得的抗体交叉反应率高,更适合于对一类化合物的分析,如大豆苷的单克隆抗体与染料木苷的交叉反应率高达82.35%(Sakamoto S.,etal.,Food Chemistry,2015,169:127-133)。到目前为止,尚无一个切实可行的方法来替代“高碘酸钠氧化”法。
黄芪甲苷是中药黄芪的主要活性成分之一。药理学研究表明,黄芪甲苷具有可改善心肺功能、增强机体免疫力、抗病毒、抗应激等多种功效(RenS,et al.Journal ofTraditional Chinese Medicine,2013,33:413-416)。黄芪甲苷的人工抗体制备现有技术路线也采用的是“高碘酸钠氧化”法(Yu S.L.,et al.,Revista Brasileira DeFarmacognosia,2014,24:282-287)。
发明内容
本发明的第一目的在于提供一种黄芪甲苷“2,2,6,6-四甲基哌啶-1-氧自由基(TEMPO)氧化”法的黄芪甲苷半抗原及其人工抗原的制备方法。这个方法不会打开黄芪甲苷分子中的糖环,最大程度的保留了黄芪甲苷的所有特征性基团(苷元和糖基),从而实现了高选择性。本发明的TEMPO氧化法与传统的NaIO4氧化法的对比见图1。
本发明的第二目的在于提供一种黄芪甲苷人工抗体的应用,即用人工抗体来检测待测样品中黄芪甲苷的含量。检测黄芪甲苷时样品前处理简单、检测迅速且选择性高,能够对样品中的黄芪甲苷进行有效检测。
为了实现本发明的上述目的,特采用以下技术方案:
一种黄芪甲苷人工抗原及其人工抗体的制备方法,其包括:
(1)取一定量的黄芪甲苷,加入到0.1~10倍质量的水中,再依次加入0.01~5倍质量的溴化钠、0.01~5倍质量的2,2,6,6-四甲基哌啶-1-氧自由基和0.01~5倍质量的次氯酸钠,在-10℃~100℃中反应10~600min,过滤,滤液用酸调pH至酸性,待固体析出后,浓缩,固液分离,固体用乙醇溶解、转移、浓缩至干,得黄芪甲苷半抗原;
(2)取一定量步骤(1)所得的黄芪甲苷半抗原,加入到0.1~10倍质量的二甲基甲酰胺溶液中,再依次加入0.2~2倍质量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺和0.2~2倍质量的N-羟基琥珀酰亚胺,室温避光搅拌1~4h后,将反应混合物5000rpm离心5min;
(3)在冰浴搅拌的状态下,将上述步骤(2)离心得到的上清液逐滴加入到5~20倍质量的载体蛋白溶液中,于4℃搅拌反应8~16h后,将反应混合物离心后装入透析袋,用PBS冰浴透析4~5天,即得黄芪甲苷人工抗原。
(4)将上述所得的黄芪甲苷人工抗原免疫动物、测定效价。
与现有技术相比,本发明的有益效果为:
使用TEMPO氧化法取代传统的的高碘酸钠氧化法制备黄芪甲苷半抗原,不会打开黄芪甲苷分子中的糖环,最大程度的保留了黄芪甲苷的所有特征性基团(苷元和糖基),免疫动物后产生对黄芪甲苷具有更高高亲和力和更高特异性的人工抗体。
附图说明
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为本发明技术路线与现有技术路线的对比图;
图2为黄芪甲苷人工抗原的UV表征图(A)和MALDI-TOF/MS表征图(B);
图3为黄芪甲苷人工抗原的TLC表征图(A)和SDS-PAGE表征图(B);
图4:黄芪甲苷间接竞争ELISA抑制曲线;
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
本实施方式提供一种黄芪甲苷人工抗原及其多克隆抗体及其制备方法,如图1所示,其主要包括:半抗原的制备、人工抗原的制备、免疫动物得到多克隆抗体。为凸显本发明较之现有技术的区别和有益效果,图1还对比了现有技术的制备方法。
实施例1
本实施例提供一种黄芪甲苷半抗原的制备方法,其包括:
取100mg黄芪甲苷,加入到500mL的水中,依次加入25mg溴化钠、5mg的TEMPO、1mL次氯酸钠,在0℃中反应120min,过滤,滤液用浓盐酸调pH 3,待固体析出后,浓缩至20mL,固液分离,固体用甲醇溶解转移,浓缩至干,即得黄芪甲苷半抗原。
实施例2
本实施例提供一种黄芪甲苷半抗原的制备方法,与实施例1相比不同之处在于调整了溶剂/试剂的比例和反应条件:
取100mg黄芪甲苷,加入到50mL的水中,依次加入25mg溴化钠、10mg的TEMPO、0.25mL次氯酸钠,在20℃中反应120min,过滤,滤液用浓盐酸调pH 2,待固体析出后,浓缩至10mL,固液分离,固体用乙醇溶解转移,浓缩至干,即得黄芪甲苷半抗原。
实施例3
本实施例提供一种黄芪甲苷人工抗原的制备方法,其包括:
(1)人工抗原的制备:取100mg黄芪甲苷半抗原,加入到50mL二甲基甲酰胺溶液中,再依次加入20mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺和20mg的N-羟基琥珀酰亚胺,室温避光搅拌2h后,将反应混合物5000rpm离心5min。在冰浴搅拌的状态下,将离心得到的上清液逐滴加入到1g的牛血清白蛋白溶液中,于4℃搅拌反应10h后,将反应混合物离心后装入透析袋,用PBS冰浴透析4天,即得黄芪甲苷人工抗原。
(2)抗原表征:分别配制1mg/mL的黄芪甲苷衍生物溶液、BSA溶液、人工抗原溶液,后用紫外分光光度计扫描它们在190nm-400nm范围内的光谱图,如图2A。用MALDI-TOF-MS测得半抗原与BSA的分子量分别为71754.909和66695.654,如图2B,计算得偶联率约为6:1。将3个溶液点样于硅胶板上,用氯仿:甲醇:水=13:7:2展开后,10%硫酸-乙醇溶液加热到108℃显色,结果如图3A,黄芪甲苷半抗原和人工抗原呈现棕红色斑点,Rf值分别为0.53和0;BSA不显色。用SDS-PAGE法进行蛋白分析,如图3B,人工抗原的电泳迁移率稍微小于BSA的电泳迁移率,表明人工抗原的分子量比BSA的分子量稍微大一点。上述一系列的表征结果,说明人工抗原制备成功,可用于下一步的动物免疫,制备人工抗体AbTEMPO。
实施例4
本实施例提供一种黄芪甲苷人工抗原的制备方法,与实施例3相比不同之处在于调整了溶剂/试剂的比例和反应条件:
取100mg黄芪甲苷半抗原,加入到500mL二甲基甲酰胺溶液中,再依次加入100mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺和100mg的N-羟基琥珀酰亚胺,室温避光搅拌2h后,将反应混合物5000rpm离心5min。在冰浴搅拌的状态下,将离心得到的上清液逐滴加入到2g的牛血清白蛋白溶液中,于4℃搅拌反应16h后,将反应混合物离心后装入透析袋,用PBS冰浴透析5天,即得黄芪甲苷人工抗原。
实施例5
本实施例提供一种黄芪甲苷多克隆抗体的制备方法,其包括:
(1)抗体的制备:将制备好的黄芪甲苷人工抗原作为免疫原,与弗氏完全佐剂1:1混合后对新西兰大白兔进行免疫;第一次免疫注射后,每隔2周注射一次,即加强免疫;从第四次免疫起,每次免疫后的第7天,采血测血清效价,达到理想效价后,再免疫一次,10天后取血,分离获得抗血清,即得黄芪甲苷多克隆人工抗体AbTEMPO。
(2)抗体的表征:
①抗体效价的测定:
首先采用方阵滴定法,确定多克隆抗体与包被原的最佳工作浓度。每横排分别加入100μL稀释2500,5000,10000倍的包被原溶液,包被酶标板。每竖列分别加入100μL稀释1000、2000、4000、8000、16000、32000、64000和128000倍的AbTEMPO溶液。使用HRP-山羊抗兔IgG进行间接ELISA测定。选择OD450nm值约1.0的包被原和AbTEMPO的组合作为最佳工作浓度。接着,用最佳工作浓度的100μL包被原溶液包被酶标板,并以5%的脱脂奶粉37℃封闭2h,洗涤三次待用。再分别加入100μL稀释1000、2000、4000、8000、16000、32000、64000和128000倍的AbTEMPO溶液,37℃孵育2h,洗涤三次。用PBS将HRP-山羊抗兔IgG稀释2000倍,每孔100μL,37℃孵育1h。洗涤三次后加100μL底物溶液,避光反应15min,加终止液50μL,测定OD450nm值。同时用阴性血清、PBS分别作阴性对照和空白对照。方阵滴定法测定不同稀释比例的人工抗体与包被原下检测到的OD450值,结果见表1:当包被原和AbTEMPO的稀释倍数组合为(5×103,1.6×104)与(5×103,3.2×104)时,对应的分OD450nm值分别为1.393和0.889,OD450nm值在1.0左右且降低明显(Δ0.504),因此我们选择包被原和AbTEMPO的最佳稀释倍数分别为最佳稀释倍数为5×103和2.4×104。间接ELISA试验检测抗体效价,如表2所示,结果显示两种人工抗原免疫兔子成功,都产生了特异性的抗体。但在同样的稀释倍数下,AbTEMPO的效价明显高于AbNaIO4(AbNaIO4为完全按照现有技术方案Yu S.L.,et al.,Revista BrasileiraDeFarmacognosia,2014,24:282-287制备的黄芪甲苷多克隆抗体)的效价,表明TEMPO氧化法制备的人工抗原的免疫原性更好,能够刺激动物机体产生更高效价的多克隆抗体。
表1:在不同稀释比例的人工抗体与包被原下检测到的OD450值
表2:两种抗体的效价测定结果
②两种抗体(AbTEMPO和AbNaIO4)的特异性对比结果
对AbTEMPO:以黄芪甲苷浓度的对数为横坐标,抑制率为纵坐标,绘制竞争抑制曲线,如图4所示。黄芪甲苷的浓度在3.125-25μg/mL范围内呈线性相关,其线性方程为y=91.419x-40.969(R2=0.9855)。通过线性方程计算得到AbTEMPO对黄芪甲苷的IC50为9.89μg/mL。用同样的方法计算得到AbNaIO4对黄芪甲苷的IC50为13.2μg/mL。结果表明AbTEMPO对黄芪甲苷的结合能力更强,灵敏度更高。
两种抗体对几种常见皂苷类化合物的交叉反应率测定结果如表3所示。两种抗体对黄芪甲苷类似物(黄芪皂苷I、环黄芪醇)交叉反应率较大,具有一定的识别能力;而对非黄芪甲苷类似物(人参皂苷Rg1、薯蓣皂苷、纤细皂苷)的交叉反应率均较小(<5%)。这说明通过TEMPO氧化和NaIO4氧化的两种人工抗原均刺激动物机体产生了对黄芪甲苷类化合物具有高选择性识别能力的特异性抗体。进一步,虽然黄芪皂苷I、环黄芪醇都是黄芪甲苷类似物,但两种抗体对其特异性也有明显差异:从表3可见,AbNaIO4与黄芪皂苷I、环黄芪醇的交叉反应率分别是AbTEMPO与二者的1.82倍、3.38倍。AbTEMPO表现出了更强的特异性。这说明对于糖苷类化合物而言,人工抗原合成时,糖环是否被破坏与抗体的特异性直接相关。
表3:两种人工抗体对黄芪甲苷及其结构类似物的交叉反应率
实施例6
本实施例提供一种黄芪甲苷多克隆抗体的制备方法,与实施例5相比不同之处在于调整了动物和免疫条件:
将制备好的黄芪甲苷人工抗原作为免疫原,与弗氏完全佐剂1:1混合后对豚鼠进行免疫;第一次免疫注射后,每隔2周注射一次,即加强免疫;从第四次免疫起,每次免疫后的第10天,采血测血清效价,达到理想效价后,再免疫一次,12天后取血,分离获得抗血清,即得黄芪甲苷多克隆人工抗体。
实施例7
本实施例提供一种黄芪甲苷多克隆抗体的应用,其包括:
使用AbTEMPO抗体测定黄芪多糖注射液中黄芪甲苷的含量:按照实施例5中“抗体的表征部分”的方法进行酶标板包被、试剂的添加、样品的测定等操作,通过外标法测得四川省七大洲动物药业有限公司、合肥中龙神力动物药业有限公司、江西鸿图动物药业有限公司和山西省芮城科龙兽药有限公司生产的黄芪多糖注射液中黄芪甲苷的含量分别为7.9μg/mL、12.43μg/mL、7.23μg/mL和4.54μg/mL。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
Claims (8)
2.根据权利要求1所述的一种黄芪甲苷人工抗原的制备方法,其特征在于,包括如下步骤:
(1)取一定量的黄芪甲苷,加入到0.1~10倍质量的水中,再依次加入0.01~5倍质量的溴化钠、0.01~5倍质量的2,2,6,6-四甲基哌啶-1-氧自由基(TEMPO)和0.01~5倍质量的次氯酸钠,在0℃~20℃中反应10~600min,过滤,滤液用酸调pH至酸性,待固体析出后,浓缩,固液分离,固体用乙醇溶解、转移、浓缩至干,得黄芪甲苷半抗原;
(2)取一定量步骤(1)所得的黄芪甲苷半抗原,加入到0.1~10倍质量的二甲基甲酰胺溶液中,再依次加入0.2~2倍质量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺和0.2~2倍质量的N-羟基琥珀酰亚胺,室温避光搅拌1~4h后,将反应混合物5000rpm离心5min;
(3)在冰浴搅拌的状态下,将上述步骤(2)离心得到的上清液逐滴加入到5~20倍质量的载体蛋白溶液中,于4℃搅拌反应8~16h后,将反应混合物离心后装入透析袋,用PBS冰浴透析4~5天,即得黄芪甲苷人工抗原。
3.根据权利要求2所述的一种黄芪甲苷人工抗原的制备方法,其特征在于,所述载体蛋白为牛血清白蛋白(BSA)。
4.根据权利要求1所述的一种黄芪甲苷人工抗原制备的黄芪甲苷人工抗体,其特征在于,用权利要求1所述的黄芪甲苷人工抗原去免疫动物,发生特异性免疫反应后所得的抗体。
5.根据权利要求4所述的一种黄芪甲苷人工抗原制备的黄芪甲苷人工抗体,其特征在于,所述黄芪甲苷人工抗体是多克隆抗体。
6.根据权利要求5所述的一种黄芪甲苷人工抗体的制备方法,其特征在于,包括如下步骤:
(1)将权利要求1中制备好的黄芪甲苷人工抗原作为免疫原,与弗氏完全佐剂后对动物进行注射;
(2)第一次免疫注射后,每隔2周注射一次,即加强免疫;
(3)从第四次免疫起,每次免疫后的第7-10天,采血测血清效价,达到理想效价后,再免疫一次,7-14天后取血,分离获得抗血清,即得黄芪甲苷多克隆人工抗体。
7.根据权利要求6所述的抗体的制备方法,其特征在于,所述动物为新西兰大白兔。
8.根据权利要求7所述的方法制备的黄芪甲苷多克隆人工抗体的应用,其特征在于,所述多克隆人工抗体用于黄芪甲苷、黄芪皂苷I、环黄芪醇化合物的免疫检测。
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