CN105175530A - 一种香草扁桃酸免疫检测试剂及其制备方法 - Google Patents
一种香草扁桃酸免疫检测试剂及其制备方法 Download PDFInfo
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- CN105175530A CN105175530A CN201510496831.7A CN201510496831A CN105175530A CN 105175530 A CN105175530 A CN 105175530A CN 201510496831 A CN201510496831 A CN 201510496831A CN 105175530 A CN105175530 A CN 105175530A
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- vanilmandelic acid
- vanilmandelic
- acid
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Abstract
本发明公开了一种香草扁桃酸免疫原及其合成方法,以及由该免疫原获得的抗香草扁桃酸抗体、检测试剂及其制备方法。本发明制备的香草扁桃酸免疫原,免疫原性高,可以诱导得到高效价的抗香草扁桃酸特异性抗体,并且与常见的62种药物无任何交叉反应;由该抗体制备得到的香草扁桃酸检测试剂,可以精确快速地确定尿液等生物样品中的香草扁桃酸含量。与市场上现有的检测试剂比较,本发明检测试剂具有操作简便、灵敏度高、特异性强、结果准确等优点,还能有效降低香草扁桃酸检测成本,有利于临床大规模推广使用。
Description
技术领域
本发明属于生物技术领域,涉及香草扁桃酸免疫原、抗香草扁桃酸特异性抗体和香草扁桃酸检测试剂。
背景技术
香草扁桃酸(VanillymandelicAcid,VMA),其结构式如式(Ⅱ)所示:
式(Ⅱ)
香草扁桃酸又称为3-甲氧基-4-羟基苦杏仁酸(DL-3-Methoxy-4-hydroxymandelicacid,MHMA)或香草苦杏仁酸,是肾上腺髓质激素儿茶酚胺(CA)类物质的终末代谢产物,CA几乎全部在体内代谢,少部分的CA经单胺氧化酶作用,生成对-二羟杏仁酸,大部分CA在CA-O-甲基转移酶作用下转变为3-甲氧肾上腺素或去甲氧肾上腺素,最终代谢为3-甲氧基-4-羟基苦杏仁酸。体内的CA代谢非常迅速,其代谢产物多由尿液排出。临床上24h尿中VMA的含量升高见于嗜铬细胞瘤、神经母细胞瘤、原发性高血压、心肌梗塞、慢性肾功能不全、甲状腺机能亢进或减退、肾上腺皮质功能不全、交感神经生理功能异常等。因此,测定24h尿中VMA的排出量对于上述疾病的临床诊断具有重要意义。
经典的VMA检测方法主要有:可见光分光光度法,此方法操作过程繁琐,精确度差;气象色谱法,此法对仪器要求比较高,操作复杂;高相液相-电化学法,此方法易受仪器质量和工作环境的干扰,测定的灵敏度和专一性均需进一步提升;重氮法,此方法操作繁琐、可靠性低,回收率、重复性、稳定性还有待提高。以上测定方法都不能适应VMA临床检验的需要,目前市场上缺乏稳定性好、灵敏度高、特异性强的VMA检测试剂,尤其是质量好的自动化检验试剂。因此,研发一种质量达到临床检验要求、实用性强、性价比高,可应用于全自动生化分析仪的VMA检测试剂势在必行。
发明内容
本发明为了克服现有技术存在的缺陷,采用独特的香草扁桃酸制备免疫原性强的香草扁桃酸免疫原及其抗体,用该抗体制备的香草扁桃酸均相酶免疫检测试剂可以实现在全自动生化分析仪上对香草扁桃酸高通量、快速化的检测。该检测试剂具有操作简便、灵敏度高、特异性强、结果准确等优点,还能有效降低香草扁桃酸检测成本,有利于临床推广使用。
本发明的一个目的在于提供一种免疫原性强的香草扁桃酸免疫原。
本发明的另一个目的在于提供一种香草扁桃酸免疫原的制备方法。
本发明的又一个目的在于提供使用本发明香草扁桃酸免疫原制备得到的特异性强的抗香草扁桃酸特异性抗体。
本发明的再一个目的在于提供一种香草扁桃酸检测试剂及其制备方法。
本发明的香草扁桃酸免疫原,免疫原性高,可以诱导得到高效价的抗香草扁桃酸特异性抗体。该抗体特异性高,与香草扁桃酸的结合力强。由该抗体制备得到的香草扁桃酸检测试剂,可以快速、准确地确定样品中的香草扁桃酸含量。本发明是通过以下技术方案实现的:
一种香草扁桃酸免疫原,其结构式如式(Ⅰ)所示:
式(Ⅰ)
载体为具有免疫原性的蛋白质或多肽,优选为血清蛋白、血蓝蛋白和甲状腺球蛋白,更优选为血清白蛋白,进一步优选为牛血清白蛋白。
所述的香草扁桃酸免疫原由香草扁桃酸与上述载体连接而成,香草扁桃酸的化学结构如式(Ⅱ)所示:
式(Ⅱ)
该香草扁桃酸免疫原的具体合成途径和方法如下:
(1)将载体蛋白100~300mg溶解于25~75ml0.2M,pH8.5的磷酸缓冲液中;
(2)将如下化学品加入到小烧杯中搅拌溶解:100~300mg香草扁桃酸、1.75~5.25ml二甲基甲酰胺、1.75~5.25ml乙醇、3.5~10.5ml10mM,pH5.0的磷酸钾缓冲液、100~300mg1-乙基-3-(-3-二甲氨丙基)碳二亚胺、25~75mgN-羟基硫代琥珀酰亚胺,将这些化学品在室温下搅拌溶解反应30~60min;
(3)将溶解好的溶液滴加至载体蛋白溶液中,并在2~8℃下搅拌过夜,得到抗原;将合成好的抗原经过透析进行纯化,得到香草扁桃酸免疫原。
一种抗香草扁桃酸特异性抗体,由上述的香草扁桃酸免疫原免疫动物后生产得到。
所述的抗香草扁桃酸特异性抗体由上述制得的香草扁桃酸免疫原采用常规方法接种实验动物,加强免疫后取抗血清,具体步骤如下:
(1)用PBS将上述合成的BSA-香草扁桃酸免疫原稀释至0.1~3.0mg/ml,得到抗原溶液,然后用0.5~5.0ml抗原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
(2)2~3周后,再用0.5~5.0ml相同的抗原溶液与等量弗氏不完全佐剂对上述实验动物注射一次,之后每隔四周注射一次,共计注射3~6次;
(3)对上述实验动物取血,分离纯化得到效价为1:30000~1:50000的抗香草扁桃酸特异性抗体。
本发明的抗香草扁桃酸特异性抗体为完整的抗体分子,也包括保留与香草扁桃酸特异性结合能力的抗体片段或抗体衍生物。
本发明的抗体为采用单一的香草扁桃酸免疫原对动物加强免疫所获得的多克隆抗体,或者为免疫后经体细胞杂交获得的单克隆抗体;所述的实验动物为兔、山羊、小鼠、绵羊、豚鼠或马的一种,优选为兔。
本发明提供一种香草扁桃酸检测试剂,含有上述抗香草扁桃酸特异性抗体和指示试剂。
本发明指示试剂选自酶试剂、放射性同位素试剂、荧光试剂、发光试剂。优选的,指示试剂为酶试剂,由香草扁桃酸酶标偶联物和酶的底物所组成。
上述酶标偶联物为葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物;上述酶的底物为葡萄糖-6-磷酸。
香草扁桃酸均相酶免疫检测试剂在使用之前,为了避免指示试剂中的酶标偶联物和酶的底物发生反应,酶标偶联物和酶的底物是不混合的且分开放置,所以将酶的底物与上述抗香草扁桃酸特异性抗体混合在一起。因此,香草扁桃酸均相酶免疫检测试剂包括两类试剂:
(1)试剂A由抗香草扁桃酸特异性抗体和均相酶底物混合而成,具体制备步骤如下:
1)将2.018~8.072g(5.625~22.50mM)氧化态的烟酰胺腺嘌呤二核苷酸(NAD)、0.856~3.422g(5.625~22.50mM)葡萄糖-6-磷酸(G-6-P)用0.5~2L55mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;
2)将制备的抗香草扁桃酸特异性抗体加到上述均相酶底物中,抗体与均相酶底物的体积比为1:100~1:10000,优选为1:1000;
(2)试剂B由葡萄糖-6-磷酸脱氢酶-半抗原偶联物与Tris缓冲液混合而成,制备方法如下:
1)葡萄糖-6-磷酸脱氢酶(G6PDH)溶液的制备:
a.称取7.5~22.5mg规格为100KU的G6PDH,室温溶解于6~18mL含有72.6mg(0.05M)Tris、8mgMgCl2(3.3mM)和100mgNaCl的溶液中,该溶液pH=9.0;
b.加入112.5~337.5mg还原态的烟酰胺腺嘌呤二核苷酸(NADH),67.5~202.5mg葡萄糖-6-磷酸(G-6-P)以及0.375~1.125mL卡必醇;
c.逐滴加入1~3mL二甲基亚砜;
2)香草扁桃酸的激活:
a.在无水状态下称取5~15mg香草扁桃酸,溶解于300~900μLDMF中;
b.使上述溶液温度降到-2~-8℃;
c.加入1.5~4.5μL三丁胺;
d.加入0.75~2.25μL氯甲酸异丁酯;
e.-2~-8℃搅拌30~60分钟;
3)G6PDH与香草扁桃酸的连接:
a.将上述激活的香草扁桃酸溶液逐滴加入到上述溶解的G6PDH溶液中;
b.2-8℃搅拌过夜;
4)纯化产物:
通过G-25凝胶层析柱纯化连接产物,获得的最终产物即为葡萄糖-6-磷酸脱氢酶-半抗原偶联物,于2-8℃下储存。
5)将制备的葡萄糖-6-磷酸脱氢酶-半抗原偶联物加到120mM、pH=8.2的Tris缓冲液中,上述偶联物与Tris缓冲液的体积比为1:100~1:10000,优选为1:3000。
本发明的香草扁桃酸免疫原特异性强、免疫原性高,制备出的抗香草扁桃酸特异性抗体特异性强、效价高,并且与常见的62种药物无任何交叉反应;含有上述抗香草扁桃酸特异性抗体的均相酶免疫检测试剂可以方便、快速、准确地确定尿液等生物样品中的香草扁桃酸含量,并且可以在全自动生化分析仪上同时测定多个样品,实现香草扁桃酸的高通量快速化测定,准确度高,特异性强,精确度和检测效率相比之前都有了较大的提高,同时实现了检测过程的全自动化,对检测人员的要求不高,易于实现和推广使用。
附图说明
图1是香草扁桃酸的ELISA检测反应曲线;
图2是香草扁桃酸的均相酶免疫反应曲线;
图3是香草扁桃酸均相酶免疫相关性分析图。
具体实施方式
实施例一香草扁桃酸免疫原的合成
香草扁桃酸免疫原由牛血清白蛋白(BovineSerumAlbumin,BSA)与式(Ⅱ)所示的香草扁桃酸的基团连接而成,具体步骤如下:
1.将牛血清白蛋白200mg溶解于50ml0.2M,pH8.5的磷酸缓冲液中;
2.将如下化学品加入到小烧杯中搅拌溶解:200mg香草扁桃酸、3.5ml二甲基甲酰胺、3.5ml乙醇、7.0ml10mM,pH5.0的磷酸钾缓冲液、200mg1-乙基-3-(-3-二甲氨丙基)碳二亚胺、50mgN-羟基硫代琥珀酰亚胺,将这些化学品在室温下搅拌溶解反应30min;
3.将溶解好的溶液滴加至BSA溶液中,并在2~8℃下搅拌过夜,得到抗原;将合成好的抗原经过透析进行纯化,得到香草扁桃酸免疫原。
实施例二:抗香草扁桃酸特异性抗体的制备
将实施例一制备得到的香草扁桃酸免疫原采用常规方法接种实验动物兔,加强免疫后取抗血清,具体步骤如下:
1.用PBS将上述合成的香草扁桃酸免疫原稀释至1.0mg/ml,得到抗原溶液,然后用1.0ml抗原溶液与等量弗氏完全佐剂混合,对实验动物兔进行注射。
2.2~3周后,再用1.0ml相同的抗原溶液与等量弗氏不完全佐剂对上述实验动物兔注射一次,之后每隔四周注射一次,共计注射4次。
3.对步骤2的实验动物兔取血,分离纯化得到效价为1:30000~1:50000的抗香草扁桃酸特异性抗体。
实施例三:香草扁桃酸的ELISA检验
1.香草扁桃酸的ELISA检测标准曲线的建立
(1)标准品的制备
将香草扁桃酸粉末(购于Sigma公司)溶解于甲醇溶液,制备成1mg/ml的储存液。用ELISA缓冲液将储存液依次稀释为16.00mg/L、8.00mg/L、4.00mg/L、2.00mg/L、1.00mg/L和0.00mg/L的标准溶液。其中,ELISA缓冲液含有50.0mMTris,145mMNaCl和0.25%的BSA。
(2)利用香草扁桃酸的ELISA检验方法制备标准曲线
用PBS将实施例二中所制备的抗香草扁桃酸特异性抗体稀释成1:8000的终浓度溶液,100μL/孔包被在96孔酶联板上,4℃放置12-24h;用PBS将上述包被有抗香草扁桃酸抗体的96孔酶联板洗涤3次后,加入200μL/孔的0.5%的BSA溶液,4℃封闭放置8-16h。然后用PBS洗涤3次,加入20μL/孔的标准品。再加入100μL/孔工作浓度的HRP-香草扁桃酸偶联物;室温下孵育30min后PBS洗板5次;然后每孔加入100μLTMB底物,室温孵育30min。再每孔加入100μL终止液(2M硫酸)。测定450nm的吸光值。根据各标准品所对应的450nm的吸光值定标,制作标准曲线,结果如附图2所示。
2.待测样品中香草扁桃酸含量的检测
(1)制作待测样品
制备方法:将香草扁桃酸粉末(购于Sigma公司)溶解于甲醇溶液制成1000mg/L的储存液,并将此储存液稀释于空白尿液中,至终浓度分别为0.00,2.00,8.00,16.00mg/L,形成空白、低、中、高浓度的尿液样本。该空白尿液为不含香草扁桃酸的健康人尿液。
(2)测试方法
利用上述香草扁桃酸的ELISA检验方法,将上述空白、低、中、高浓度的尿液样本代替标准品,测试上述空白、低、中、高浓度的尿液样本在450nm的吸光值。
(3)测试结果
对照图1中所示的香草扁桃酸的ELISA检验的标准曲线,计算每个样本中香草扁桃酸含量,并对每个样本进行3个复孔测定,根据上述样本中香草扁桃酸的实际含量计算回收率,结果如表1所示。
表1香草扁桃酸的ELISA检测回收实验
尿液样品 | 空白 | 低 | 中 | 高 |
样品浓度(mg/L) | 0.00 | 2.00 | 8.00 | 16.00 |
测试1 | 0.02 | 2.19 | 8.17 | 16.32 |
测试2 | 0.04 | 2.06 | 7.95 | 15.85 |
测试3 | 0.01 | 1.96 | 8.20 | 16.09 |
平均值(mg/L) | 0.02 | 2.07 | 8.11 | 16.09 |
回收率(%) | - | 103.5 | 101.3 | 100.5 |
由表1中结果可知:采用本发明香草扁桃酸的ELISA检测试剂测定不同浓度样品中的香草扁桃酸回收率都较高,均>90%,说明本发明所述的抗香草扁桃酸特异性抗体可以用于样本中香草扁桃酸的检测,并且结果准确度高。
实施例四:葡萄糖-6-磷酸脱氢酶-半抗原偶联物的制备
1.葡萄糖-6-磷酸脱氢酶(G6PDH)溶液的制备:
(1)准确称取15mg规格为100KU的G6PDH,室温溶解于12mL含有72.6mg(0.05M)Tris、8mgMgCl2(3.3mM)和100mgNaCl的溶液中,该溶液pH=9.0,本步骤在烧杯C中进行。
(2)在上述烧杯C中加入225mg还原态的烟酰胺腺嘌呤二核苷酸(NADH),135mg葡萄糖-6-磷酸(G-6-P)以及0.75mL卡必醇(Carbitol)。
(3)在上述烧杯C中再逐滴加入2mL二甲基亚砜(dimethysulfoxide,DMSO)。
2.香草扁桃酸的激活:
(1)在无水状态下称取10mg上述香草扁桃酸,溶解于600μLDMF中。
(2)使上述溶液温度降到-2~-8℃。
(3)加入3μL三丁胺(tributylamine)。
(4)加入1.5μL氯甲酸异丁酯(isobutylchloroformate)。
(5)-2~-8℃搅拌30分钟。
3.G6PDH与香草扁桃酸的连接:
(1)将上述激活的香草扁桃酸溶液逐滴加入到上述溶解的G6PDH溶液中。
(2)2-8℃搅拌过夜。
4.纯化产物:
通过G-25凝胶层析柱纯化步骤3中的溶液,获得的最终产物为葡萄糖-6-磷酸脱氢酶-半抗原偶联物,于2-8℃下储存。
实施例五:香草扁桃酸均相酶免疫检测试剂的制备
1.试剂A的制备:将4.036g(11.25mM)氧化态的烟酰胺腺嘌呤二核苷酸(NAD)、1.711g(11.25mM)葡萄糖-6-磷酸(G-6-P)置于烧杯D中,用1L55mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;将上述制备的抗香草扁桃酸特异性抗体加到上述均相酶底物中,抗体与均相酶底物的体积比为1:1000。
2.试剂B的制备:将实施例四制备的葡萄糖-6-磷酸脱氢酶-半抗原偶联物加到120mM、pH=8.2的Tris缓冲液中,上述偶联物与Tris缓冲液的体积比为1:3000。
实施例六:香草扁桃酸均相酶免疫检验及结果
1.获得标准曲线:
(1)设置迈瑞BS-480全自动生化分析仪反应参数(见表2)。
(2)操作步骤为:先加试剂A,再加入标准品,最后加入试剂B。加入试剂B后,测定不同时间点的OD340吸光值,算出不同标准品浓度时的反应速率,实际操作过程中需不断调整试剂A和试剂B的体积比例,同时调整测光点,最后得出较理想的反应标准曲线图,如图3所示。
表2迈瑞BS-480全自动生化分析仪反应参数
2.样本检测:通过本发明的均相酶免疫检测试剂得到的标准曲线,重复测定低、中、高浓度质控样本10次,上述质控样本为:将香草扁桃酸标准品溶解于人尿液中,至浓度分别为2.00,8.00,16.00mg/L。检测数据及数据分析见表3。
表3样品测定及精密度和回收率评估
尿液样品 | 低 | 中 | 高 |
样品浓度 (mg/L) | 2.00 | 8.00 | 16.00 |
1 | 2.12 | 8.22 | 16.54 |
2 | 1.93 | 8.43 | 16.37 |
3 | 2.17 | 7.84 | 15.87 |
4 | 2.09 | 7.95 | 16.03 |
5 | 2.01 | 8.02 | 15.75 |
6 | 1.95 | 8.37 | 15.86 |
7 | 1.89 | 8.21 | 16.33 |
8 | 2.11 | 8.05 | 16.41 |
9 | 2.06 | 7.96 | 15.79 |
10 | 1.91 | 7.82 | 16.66 |
平均值(mg/L) | 2.024 | 8.087 | 16.161 |
标准差(SD) | 0.09 | 0.20 | 0.32 |
精密度(CV%) | 4.66% | 2.48% | 1.98% |
回收率 % | 101.2 | 101.1 | 101.0 |
检测结果:本发明的均相酶免疫检测试剂测定的准确度高,回收率达到95%-105%,精密度高,CV均低于5%。
实施例七:药物干扰试验
选取62种常见药物进行干扰检测,调整浓度至1.00mg/L,采用实施例六的均相酶免疫方法进行测定:
1.将待测干扰药物与实施例五制备的试剂A接触反应,再加入试剂B;
2.检测上述混合溶液的OD340吸光值,根据实施例六的标准曲线得到相应物质的浓度。
常见的62种药物名称以及测定结果具体参见表4。
表4常见干扰药物测定结果
ID# | 化合物名称 | 等价于香草扁桃酸的浓度 (mg/L) | ID# | 化合物名称 | 等价于香草扁桃酸的浓度 (mg/L) |
1 | 阿司匹林 | 0.0 | 32 | 苯丙醇胺 | 0.0 |
2 | β-苯基乙胺 | 0.0 | 33 | 普鲁卡因酰胺 | 0.0 |
3 | 安非他命 | 0.0 | 34 | 普鲁卡因 | 0.0 |
4 | 氨苄青霉素 | 0.0 | 35 | 奎尼丁 | 0.0 |
5 | 甲氨二氮卓 | 0.0 | 36 | 佐美酸 | 0.0 |
6 | 氯丙嗪 | 0.0 | 37 | 苯肾上腺素 | 0.0 |
7 | 氯拉卓酸 | 0.0 | 38 | 桂皮酰艾克宁 | 0.0 |
8 | 二甲苯氧庚酸 | 0.0 | 39 | 芽子碱 | 0.0 |
9 | 非诺洛芬 | 0.0 | 40 | 地西洋 | 0.0 |
10 | 甲基苯丙胺 | 0.0 | 41 | 可替宁 | 0.0 |
11 | 龙胆酸 | 0.0 | 42 | 阿替洛尔 | 0.0 |
12 | 吉非贝齐 | 0.0 | 43 | 心得安 | 0.0 |
13 | 氢可酮 | 0.0 | 44 | 苯乙哌啶酮 | 0.0 |
14 | 布洛芬 | 0.0 | 45 | 苯基丁氮酮 | 0.0 |
15 | 丙咪嗪 | 0.0 | 46 | 麦角酸二乙基酰胺 | 0.0 |
16 | 二氨基二苯砜 | 0.0 | 47 | 大麻酚 | 0.0 |
17 | 萘普生 | 0.0 | 48 | 洛哌丁胺 | 0.0 |
18 | 氢氯噻嗪 | 0.0 | 49 | 异克舒令 | 0.0 |
19 | 哌替啶 | 0.0 | 50 | 苯基丙氨酸 | 0.0 |
20 | 烯丙羟吗啡酮 | 0.0 | 51 | 盐酸氟西汀 | 0.0 |
21 | 麻黄素 | 0.0 | 52 | 柳丁氨醇 | 0.0 |
22 | 烟酰胺 | 0.0 | 53 | 青霉素 | 0.0 |
23 | 甲胺呋硫 | 0.0 | 54 | 甲基二乙醇胺 | 0.0 |
24 | 异戊巴比妥 | 0.0 | 55 | 二亚甲基双氧苯丙胺 | 0.0 |
25 | 甲撑二氧苯丙胺 | 0.0 | 56 | 琥珀酸多西拉敏 | 0.0 |
26 | 四氢大麻酚 | 0.0 | 57 | 纳布啡 | 0.0 |
27 | 制霉菌素 | 0.0 | 58 | 去甲吗啡 | 0.0 |
28 | 乙酰吗啡 | 0.0 | 59 | 羟考酮 | 0.0 |
29 | 苄非他明 | 0.0 | 60 | 克他命 | 0.0 |
30 | 异丙嗪 | 0.0 | 61 | 苯海拉明 | 0.0 |
31 | 阿司帕坦 | 0.0 | 62 | 苯丁胺 | 0.0 |
测定结果显示:上述62种常见药物等价于香草扁桃酸的浓度均小于0.01mg/L。由此可见,本发明的抗体是抗香草扁桃酸的特异性抗体,与其它药物无交叉反应。
实施例八:相关性分析
对100例临床标本分别使用高效液相色谱法和本发明的均相酶免疫试剂进行相关性分析,测定的数据参见表5。
表5临床样本测定值
样本号 | 均相酶免疫法测定值(mg/L) | 高效液相色谱法测定值(mg/L) |
1 | 4.32 | 4.11 |
2 | 6.09 | 5.98 |
3 | 15.32 | 15.49 |
4 | 12.38 | 12.32 |
5 | 9.65 | 9.23 |
6 | 1.06 | 1.02 |
7 | 4.66 | 4.85 |
8 | 7.94 | 7.85 |
9 | 9.24 | 9.13 |
10 | 12.05 | 11.84 |
11 | 16.08 | 15.75 |
12 | 15.23 | 15.10 |
13 | 0.97 | 1.03 |
14 | 12.36 | 12.25 |
15 | 13.36 | 13.24 |
16 | 5.26 | 5.30 |
17 | 1.98 | 1.81 |
18 | 4.69 | 4.58 |
19 | 4.33 | 4.54 |
20 | 14.05 | 13.56 |
21 | 5.21 | 5.01 |
22 | 8.25 | 8.17 |
23 | 6.22 | 6.05 |
24 | 4.25 | 4.26 |
25 | 7.55 | 7.21 |
26 | 10.23 | 11.56 |
27 | 15.23 | 14.98 |
28 | 12.22 | 11.99 |
29 | 6.23 | 6.01 |
30 | 5.25 | 6.05 |
31 | 7.89 | 7.85 |
32 | 7.23 | 7.15 |
33 | 9.22 | 9.41 |
34 | 0.55 | 0.59 |
35 | 8.25 | 8.03 |
36 | 4.06 | 3.96 |
37 | 8.79 | 8.56 |
38 | 7.22 | 7.15 |
39 | 6.23 | 6.04 |
40 | 3.12 | 3.09 |
41 | 12.56 | 12.48 |
42 | 16.01 | 15.87 |
43 | 11.18 | 10.95 |
44 | 11.23 | 11.08 |
45 | 15.23 | 15.19 |
46 | 2.36 | 2.35 |
47 | 15.23 | 15.20 |
48 | 8.79 | 8.59 |
49 | 10.22 | 10.31 |
50 | 7.25 | 7.03 |
51 | 9.79 | 9.56 |
52 | 15.02 | 14.92 |
53 | 2.87 | 2.69 |
54 | 4.35 | 4.16 |
55 | 7.22 | 7.01 |
56 | 12.03 | 11.76 |
57 | 14.23 | 13.96 |
58 | 11.22 | 10.98 |
59 | 1.96 | 1.35 |
60 | 0.89 | 1.02 |
61 | 4.21 | 3.88 |
62 | 1.56 | 1.23 |
63 | 12.03 | 12.11 |
64 | 5.36 | 5.21 |
65 | 10.03 | 9.87 |
66 | 7.96 | 8.05 |
67 | 2.33 | 2.49 |
68 | 6.13 | 6.34 |
69 | 14.28 | 14.03 |
70 | 16.01 | 15.78 |
71 | 12.23 | 11.96 |
72 | 14.47 | 14.25 |
73 | 5.38 | 5.69 |
74 | 7.29 | 7.15 |
75 | 4.35 | 4.41 |
76 | 0.99 | 0.96 |
77 | 6.33 | 6.23 |
78 | 5.79 | 5.86 |
79 | 13.22 | 13.41 |
80 | 14.05 | 14.21 |
81 | 9.55 | 9.46 |
82 | 7.58 | 7.51 |
83 | 0.99 | 1.06 |
84 | 7.68 | 7.89 |
85 | 12.35 | 12.42 |
86 | 14.22 | 14.02 |
87 | 8.99 | 8.79 |
88 | 15.23 | 15.48 |
89 | 4.39 | 4.42 |
90 | 8.56 | 8.49 |
91 | 7.22 | 7.36 |
92 | 5.23 | 5.16 |
93 | 8.97 | 8.67 |
94 | 10.88 | 10.96 |
95 | 2.58 | 2.34 |
96 | 12.55 | 12.14 |
97 | 16.34 | 16.09 |
98 | 5.79 | 5.62 |
99 | 7.69 | 7.52 |
100 | 12.33 | 12.68 |
对上述数据作图,参见图3,得到的线性方程为:y=0.9946x-0.0266,相关系数R2=0.997,表明本发明的检测试剂测定香草扁桃酸临床标本的准确度高。
需要说明的是,以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所做的等效结构或等效流程变换,或直接或间接运用在其他相关技术领域,均同理包括在本发明的专利保护范围内。
Claims (8)
1.一种香草扁桃酸免疫原,其结构式如式(Ⅰ)所示:
式(Ⅰ)
载体为具有免疫原性的蛋白质或多肽,选自血清蛋白、血蓝蛋白或甲状腺球蛋白中的一种,优选为血清蛋白,更优选为牛血清白蛋白。
2.一种如权利要求1所述的香草扁桃酸免疫原的制备方法,其特征在于包含以下步骤:
(1)将载体蛋白100~300g溶解于25~75ml0.2M,pH8.5的磷酸缓冲液中;
(2)将如下化学品加入到小烧杯中搅拌溶解:100~300mg香草扁桃酸、1.75~5.25ml二甲基甲酰胺、1.75~5.25ml乙醇、3.5~10.5ml10mM,pH5.0的磷酸钾缓冲液、100~300mg1-乙基-3-(-3-二甲氨丙基)碳二亚胺、25~75mgN-羟基硫代琥珀酰亚胺,将上述化学品在室温下搅拌溶解反应30~60min;
(3)将溶解好的溶液滴加至载体蛋白溶液中,并在2~8℃下搅拌过夜,得到抗原;将合成好的抗原经过透析进行纯化,得到香草扁桃酸免疫原。
3.一种抗香草扁桃酸特异性抗体,由权利要求1-2任意一项所述的香草扁桃酸免疫原免疫实验动物后产生的完整抗体分子,或者为保留与香草扁桃酸特异性结合能力的抗体片段或抗体衍生物。
4.根据权利要求3所述的一种抗香草扁桃酸特异性抗体,其特征在于所述的完整的抗体分子、抗体片段或抗体衍生物,为采用单一的香草扁桃酸免疫原对动物加强免疫所获得的多克隆抗体,或者为免疫后经体细胞杂交获得的单克隆抗体;所述的实验动物为兔、山羊、小鼠、绵羊、豚鼠或马的一种,优选为兔。
5.一种如权利要求3-4中任一项所述的抗香草扁桃酸特异性抗体的制备方法,其特征在于包含以下步骤:
(1)用PBS将香草扁桃酸免疫原稀释至0.1~3.0mg/ml,得到抗原溶液,然后用0.5~5.0ml抗原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
(2)2~3周后,再用0.5~5.0ml相同的抗原溶液与等量弗氏不完全佐剂对上述实验动物注射一次,之后每隔四周注射一次,共计注射3~6次;
(3)对步骤(2)的实验动物取血,分离纯化得到效价为1:30000~1:50000的抗香草扁桃酸特异性抗体。
6.一种香草扁桃酸检测试剂,含有权利要求3-5所述的抗香草扁桃酸特异性抗体和指示试剂,所述的指示试剂选自酶试剂、放射性同位素试剂、荧光试剂或发光试剂中的一种,优选为酶试剂;所述的酶试剂由香草扁桃酸酶标偶联物和酶的底物组成,酶标偶联物优选为葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物,酶的底物优选为葡萄糖-6-磷酸。
7.一种如权利要求6所述的香草扁桃酸检测试剂的制备方法,其特征在于包含以下步骤:
(1)试剂A:将2.018~8.072g、5.625~22.50mM氧化态的烟酰胺腺嘌呤二核苷酸和0.856~3.422g、5.625~22.50mM葡萄糖-6-磷酸用0.5~2L55mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;将权利要求4-6中任一项所述的抗香草扁桃酸特异性抗体加到上述均相酶底物中,抗香草扁桃酸特异性抗体与均相酶底物的体积比为1:100~1:10000;
(2)试剂B:将香草扁桃酸酶标偶联物加到120mM、pH=8.2的Tris缓冲液中,香草扁桃酸酶标偶联物与Tris缓冲液的体积比为1:100~1:10000;
所述的抗香草扁桃酸特异性抗体与均相酶底物的体积比优选为1:1000;
所述的香草扁桃酸酶标偶联物与Tris缓冲液的体积比优选为1:3000。
8.根据权利要求6-7任一项所述的香草扁桃酸检测试剂,其特征在于所述的香草扁桃酸酶标偶联物的制备方法包含以下步骤:
(1)葡萄糖-6-磷酸脱氢酶溶液的制备:称取7.5~22.5mg规格为100KU的葡萄糖-6-磷酸脱氢酶,室温溶解于6~18mL含有72.6mg0.05MTris、8mg3.3mMMgCl2和100mgNaCl的溶液中,pH=9.0;在溶液中加入112.5~337.5mg还原态的烟酰胺腺嘌呤二核苷酸、67.5~202.5mg葡萄糖-6-磷酸以及0.375~1.125mL卡必醇;再逐滴加入1~3mL二甲基亚砜;
(2)香草扁桃酸的激活:在无水状态下称取5~15mg香草扁桃酸,溶解于300~900μL二甲基甲酰胺中;使上述溶液温度降到-2~-8℃;加入1.5~4.5μL三丁胺;加入0.75~2.25μL氯甲酸异丁酯;-2~-8℃搅拌30~60分钟;
(3)葡萄糖-6-磷酸脱氢酶与香草扁桃酸的连接:将步骤(2)激活的香草扁桃酸溶液逐滴加入到步骤(1)溶解的葡萄糖-6-磷酸脱氢酶溶液中;2-8℃搅拌过夜;
(4)纯化产物:通过G-25凝胶层析柱纯化连接产物,获得的最终产物为葡萄糖-6-磷酸脱氢酶-半抗原偶联物,于2-8℃下储存。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106596917A (zh) * | 2016-12-19 | 2017-04-26 | 苏州博源医疗科技有限公司 | 高香草酸均相酶免疫检测试剂、制备方法及检测方法 |
CN107353200A (zh) * | 2017-06-27 | 2017-11-17 | 苏州博源医疗科技有限公司 | 一种香草扁桃酸衍生物、其合成方法及一种香草扁桃酸免疫原、其制备方法及其应用 |
CN109529794A (zh) * | 2018-12-27 | 2019-03-29 | 中国人民解放军第四军医大学 | 光学纯扁桃酸衍生物-纤维素手性固定相、制备方法及应用 |
CN114956997A (zh) * | 2022-03-30 | 2022-08-30 | 北京丹大生物技术有限公司 | 香草扁桃酸半抗原衍生物及其合成方法,香草扁桃酸抗原及其制备方法,抗体及试剂盒 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0450095A1 (en) * | 1989-10-19 | 1991-10-09 | Yamasa Shoyu Kabushiki Kaisha (Yamasa Corporation) | Method of assaying d-vanillylmandelic acid, and reagent and kit therefor |
CN104530222A (zh) * | 2014-12-20 | 2015-04-22 | 苏州博源医疗科技有限公司 | 紫杉醇免疫原、抗紫杉醇特异性抗体和紫杉醇检测试剂 |
CN104774256A (zh) * | 2015-05-11 | 2015-07-15 | 苏州博源医疗科技有限公司 | 儿茶酚胺免疫原、衍生物及合成方法、特异性抗体和检测试剂及制备方法 |
CN104807923A (zh) * | 2014-01-23 | 2015-07-29 | 杭州纽贝生物科技有限公司 | 一种人尿中检测香草扁桃酸的方法 |
-
2015
- 2015-08-14 CN CN201510496831.7A patent/CN105175530A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0450095A1 (en) * | 1989-10-19 | 1991-10-09 | Yamasa Shoyu Kabushiki Kaisha (Yamasa Corporation) | Method of assaying d-vanillylmandelic acid, and reagent and kit therefor |
CN104807923A (zh) * | 2014-01-23 | 2015-07-29 | 杭州纽贝生物科技有限公司 | 一种人尿中检测香草扁桃酸的方法 |
CN104530222A (zh) * | 2014-12-20 | 2015-04-22 | 苏州博源医疗科技有限公司 | 紫杉醇免疫原、抗紫杉醇特异性抗体和紫杉醇检测试剂 |
CN104774256A (zh) * | 2015-05-11 | 2015-07-15 | 苏州博源医疗科技有限公司 | 儿茶酚胺免疫原、衍生物及合成方法、特异性抗体和检测试剂及制备方法 |
Non-Patent Citations (1)
Title |
---|
CÉCILE GAUCHET等: "The use of enzyme immunoassays for the detection of abzymatic activities. Application to an enantioselective thioacetal hydrolysis activity", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106596917A (zh) * | 2016-12-19 | 2017-04-26 | 苏州博源医疗科技有限公司 | 高香草酸均相酶免疫检测试剂、制备方法及检测方法 |
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