CN114354938A - 一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法 - Google Patents
一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法 Download PDFInfo
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- CN114354938A CN114354938A CN202210004392.3A CN202210004392A CN114354938A CN 114354938 A CN114354938 A CN 114354938A CN 202210004392 A CN202210004392 A CN 202210004392A CN 114354938 A CN114354938 A CN 114354938A
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- vanillylmandelic acid
- vanillylmandelic
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Abstract
本发明涉及一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法。香草扁桃酸溴化衍生物及合成方法均为针对性的新研究与设计,以新研发的香草扁桃酸溴化衍生物制备得到的香草扁桃酸溴化衍生物免疫原,以该香草扁桃酸溴化衍生物免疫原免疫实验动物制备得到的抗香草扁桃酸特异性抗体与30种常见激素及激素代谢物无任何交叉反应。使用上述抗香草扁桃酸特异性抗体与香草扁桃酸溴化衍生物酶标偶联物制备得到的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂的检测灵敏度达到ng/mL级别,能准确检测生物样本中微量香草扁桃酸的浓度,其检测的准确性、精密度、灵敏度与特异性都显著高于现有技术。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法。
背景技术
香草扁桃酸(Vanillymandelic Acid,VMA),其结构式如式Ⅳ所示:
式Ⅳ。
香草扁桃酸又称为3-甲氧基-4-羟基苦杏仁酸(DL-3-Methoxy-4-hydroxymandelic acid,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,尤其是能精确地测定生物样本中微量VMA含量的全自动化检测试剂。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检测试剂已成为本领域研究热点。
发明内容
本发明所要解决的技术问题是:针对现有技术的上述缺陷,提供一种使用方便、灵敏度高、特异性强的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法。应用该基于溴化衍生物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含量的全自动化测定,可以高通量、快速化、精确地测定生物样本中微量香草扁桃酸的含量,且具有操作简便、灵敏度高、特异性强、结果准确等优点,有效降低香草扁桃酸检测成本,有利于临床广泛推广使用。
本发明解决其技术问题所采用的技术方案是:提供一种基于溴化衍生物的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,包括R1试剂和R2试剂,所述R1试剂中包含抗香草扁桃酸特异性抗体和均相酶底物溶液;所述R2试剂包含香草扁桃酸溴化衍生物酶标偶联物和R2缓冲液。
所述的抗香草扁桃酸特异性抗体,由香草扁桃酸溴化衍生物免疫原免疫实验动物后产生,所述实验动物为兔、山羊、绵羊、大鼠、小鼠、豚鼠或马中的一种;优选地,所述实验动物为兔。
所述的香草扁桃酸溴化衍生物免疫原由香草扁桃酸溴化衍生物与载体连接而成,其结构式如下述式(Ⅰ)所示:
式(Ⅰ);
所述的载体为具有免疫原性的蛋白质或多肽,选自血清蛋白、卵清蛋白、丙种球蛋白、甲状腺球蛋白、血蓝蛋白或多聚赖氨酸中的一种;优选地,所述的载体为血清蛋白;更优选地,所述的载体为牛血清白蛋白。
所述的香草扁桃酸溴化衍生物酶标偶联物由香草扁桃酸溴化衍生物与人工诱变葡萄糖-6-磷酸脱氢酶(mG6PDH)连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ)。
式中mG6PDH为人工诱变葡萄糖-6-磷酸脱氢酶,所述人工诱变葡萄糖-6-磷酸脱氢酶的氨基酸序列为SEQ ID NO:1;
具体的,所述人工诱变葡萄糖-6-磷酸脱氢酶的氨基酸序列(SEQ ID NO:1)如下所示:
MGSAASLPPQATQVSFSKADFEELLHVVDTSQKNVKEALGKAGIALGEAPKQQPAPSAASICLEQEAWRSGKLSVTVLGATGCLAKKETFPALLDLFAHDYLQHEAVIVCFARKSMTTEDFRAYLSEALAKSHVCQCMPEVRDSIPAFLKKVHYCAGAYDSVEAMTELDKLLTVEEERFHLDDNDAPKKQSFRIFYFAIPPFVFQGAAKAIKEVAMAKDGSTRLIIEKPFGHDLTSAQELQECLSALFKEEEIYRMDHFLGYEICQNILSVRFGNQFLEPLMNNKHVAAVRVTLKECFGTEGRGGYFTKYGIIRDVIQNHLLQMLCLVAMERPGDMDWEADVRDRKVELLKAMEPFDPSSVVLGQYVGAKGKPGYLEDDSITEADRDMAKKCPTFCQMVVRINNERWQCVNFIVRAGKAIDECKCEIRVQFKEAECCGKLFGEQYPARNELVLRVSPGEAIYMKMSVKSPGLSKQLMQSEMDLSYSVRYPGIYCPSAYTRLILAGIRGQSESFVRCDELMRSWELFTPFLEKIADWQPKPYPYGSRGPAVACEALELYGYQKPEGYCWRPPHQPPALTRLMTR
所述的香草扁桃酸溴化衍生物,其结构式如下述式(Ⅲ)所示:
式(Ⅲ)。
本发明所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,制备方法包括以下步骤:
(S1.1)R1试剂的制备:将质量分数6.0%的氧化态的烟酰胺腺嘌呤二核苷酸、6.0%的葡萄糖-6-磷酸、0.3%的牛血清白蛋白、0.03%的NaN3的用浓度为60 mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗香草扁桃酸特异性抗体加入所述均相酶底物溶液中混匀,得到R1试剂,所述抗香草扁桃酸特异性抗体与均相酶底物溶液的体积比为1∶50~1∶5000。优选地,所述抗香草扁桃酸特异性抗体与均相酶底物溶液的体积比为1∶625。
(S1.2)R2试剂的制备:将质量分数0.3%的牛血清白蛋白、0.03%的NaN3用浓度为150 mmol/L、pH=8.0的Tris缓冲液溶解制成R2缓冲液,再将香草扁桃酸溴化衍生物酶标偶联物加入所述R2缓冲液中混匀,得到R2试剂,所述香草扁桃酸溴化衍生物酶标偶联物与R2缓冲液的体积比为1∶50~1∶5000。优选地,所述香草扁桃酸溴化衍生物酶标偶联物与R2缓冲液的体积比为1∶1250。
其中,所述抗香草扁桃酸特异性抗体的制备方法,包括以下步骤:
(S2.1)用浓度为10 mmol/L、pH=7.4的PBS缓冲液将香草扁桃酸溴化衍生物免疫原稀释至8.0 mg/ml,得到人工抗原溶液,然后将5.0 ml所述人工抗原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
(S2.2)1周后,将步骤(S2.1)中所述的人工抗原溶液用浓度为10 mmol/L、pH=7.4的PBS缓冲液稀释一倍,再将5.0 ml稀释后的人工抗原溶液与等量弗氏不完全佐剂混合,对上述实验动物进行注射;
(S2.3)之后每隔4周按步骤(S2.2)的方法对上述实验动物进行注射1次,共计注射5次,以加强免疫;
(S2.4)对加强免疫后的实验动物进行取血,分离纯化血液得到抗香草扁桃酸特异性抗体。
其中,所述香草扁桃酸溴化衍生物免疫原的制备方法,包括以下步骤:
(S3.1)将质量分数2.0%的载体溶解于10.0 mmol/L,pH=8.0的磷酸钠缓冲液中,得到载体溶液;
(S3.2)将质量分数1.0%的香草扁桃酸溴化衍生物与3.0%的二甲基甲酰胺、3.0%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应60~90分钟,得到香草扁桃酸溴化衍生物溶液;
(S3.3)将步骤(S3.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S3.1)中得到的载体溶液中,然后在0~4℃下搅拌反应12~24小时,得到偶联物溶液,在上述偶联物溶液中加入1.0 mmol/L的乙二胺四乙酸,再使用10.0 mmol/L、pH=7.0的HEPES缓冲液进行透析纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物免疫原溶液,在上述香草扁桃酸溴化衍生物免疫原溶液中加入质量分数0.05%的NaN3,于-20℃下储存。
其中,所述香草扁桃酸溴化衍生物酶标偶联物的制备方法,包括以下步骤:
(S4.1)将质量分数5.0%的规格为200KU的人工诱变葡萄糖-6-磷酸脱氢酶室温下溶解于含有50.0 mmol/L、pH=7.3的磷酸钠缓冲液中;然后加入1.0 mmol/L的乙二胺四乙酸;混合均匀后置于4℃下孵育10~20小时,得到人工诱变葡萄糖-6-磷酸脱氢酶溶液;
(S4.2)在无水状态下将质量分数1.5%的香草扁桃酸溴化衍生物与4.5%的二甲基甲酰胺、4.5%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应60~120分钟,得到香草扁桃酸溴化衍生物溶液;
(S4.3)将步骤(S4.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S4.1)中得到的人工诱变葡萄糖-6-磷酸脱氢酶溶液中,然后在0~4℃下搅拌反应12~24小时;将反应后的偶联产物通过G-50葡聚糖凝胶层析柱进行纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物酶标偶联物溶液,在上述香草扁桃酸溴化衍生物酶标偶联物溶液中加入质量分数0.05%的BSA和质量分数0.05%的NaN3,于0~4℃下储存。
其中,上述式(Ⅲ)所示的香草扁桃酸溴化衍生物的合成路线如下:
所述香草扁桃酸溴化衍生物的合成路线,具体制备步骤如下:
(S5.1)合成化合物3:
称取化合物1(10 g,51 mmol)溶解于DMF(100 ml)中,然后加入DIEA(20 ml,160mmol)、HATU(22 g,60 mmol)和化合物 2(9.6 g,60 mmol)制成反应混合物溶液,将此反应混合物溶液在室温下搅拌过夜;然后向此反应混合物溶液中加入纯化水(200mL),并进行过滤;将过滤所得的滤饼在真空中干燥,然后通过硅胶进行纯化,得到化合物3。
(S5.2)合成化合物4:
将化合物3(12g,35mmol)溶解于100mL的DCM中,然后添加 3,4-二氢-2H-吡喃(8.8g,105 mmol)和p-TosOH(0.3 g,1.7 mmol)制成反应混合物溶液,将此反应混合物溶液在室温下搅拌24小时;然后将溶剂浓缩得到粗产物,并将粗产物通过快速柱色谱法(SiO 2)进行纯化,得到化合物4。
(S5.3)合成化合物5:
在0°C下称取化合物4(5.1 g,10 mmol)溶解于DCM(50 mL)中,然后加入50 mL的TFA制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到化合物5。
(S5.4)合成化合物6:
在0℃下称取化合物5(4.1 g,10 mmol)溶解于DCM(50 mL)中,然后添加TEA(2 g,20 mmol)和溴乙酰溴(2.4 g,12 mmol)制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到化合物6。
(S5.5)合成香草扁桃酸溴化衍生物:
在0℃下称取化合物6(2.6 g,5 mmol)溶解于DCM(50ml)中,然后加入HCl/MeOH(2N,10 mL)制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到香草扁桃酸溴化衍生物。
本发明所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂的使用方法,包括以下步骤:
(S6.1)在全自动生化分析仪中加入待测样本、R1试剂,混匀,37℃温育3-5分钟;
(S6.2)加入R2试剂,混匀,37℃恒温5-10分钟后,340 nm波长进行检测,连续监测3分钟内的吸光度变化率,由全自动生化分析仪自动计算待测样本中香草扁桃酸的含量;
其中,所述的待测样本为生理样本;优选地,所述的生理样本为血清、血浆、尿液、唾液;更优选地,所述的生理样本为血清或血浆。所述R1试剂与R2试剂按1∶1~4∶1的体积比使用;优选地,按4∶1的体积比使用。
本发明的有益效果在于:
1、本发明所述的香草扁桃酸溴化衍生物及合成方法均为针对性的新研究与设计,现有技术中并不存在。
2、本发明以新研发的香草扁桃酸溴化衍生物制备得到的香草扁桃酸溴化衍生物免疫原,其免疫原性强于现有技术中制备的常规香草扁桃酸免疫原;以该香草扁桃酸溴化衍生物免疫原免疫实验动物制备得到的抗香草扁桃酸特异性抗体,其特异性、灵敏度与现有技术中制备的抗香草扁桃酸特异性抗体相比都有显著提高,并且与30种常见激素及激素代谢物无任何交叉反应。
3、本发明使用上述抗香草扁桃酸特异性抗体与香草扁桃酸溴化衍生物酶标偶联物制备得到的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂的检测灵敏度达到ng/mL级别,能准确检测生物样本中微量香草扁桃酸的浓度;该检测试剂使用方便,可以实现在全自动生化分析仪上对香草扁桃酸的高通量、快速化检测,其检测的准确性、精密度、灵敏度与特异性都显著高于现有技术;该检测试剂还能有效降低香草扁桃酸的检测成本,有利于临床推广使用。
附图说明
图1是实施例6中香草扁桃酸 ELISA检测方法的标准曲线;
图2是实施例7中香草扁桃酸均相酶免疫检测方法的标准曲线;
图3是实施例9中均相酶免疫法与高效液相色谱法检测香草扁桃酸的对比实验数据的散点图;
图4是式(Ⅲ)所示的香草扁桃酸溴化衍生物的1H NMR光谱扫描分析谱图;
图5是式(Ⅲ)所示的香草扁桃酸溴化衍生物的LC-MS分析谱图。
具体实施方式
下面结合附图以及具体实施方式,对本发明做进一步描述,这些附图均为简化的示意图,仅以示意方式说明本发明的基本结构,因此其仅显示与本发明有关的构成。除非特别指明,以下实施例中所用的试剂、仪器、设备、耗材均可从正规渠道商购买获得。
实施例1 香草扁桃酸溴化衍生物的合成
香草扁桃酸溴化衍生物的化学结构式如式(Ⅲ)所示:
式(Ⅲ)。
上述香草扁桃酸溴化衍生物的合成路线如下:
具体的合成步骤如下:
(1)合成化合物3:
称取化合物1(10 g,51 mmol)溶解于DMF(100 ml)中,然后加入DIEA(20 ml,160mmol)、HATU(22 g,60 mmol)和化合物 2(9.6 g,60 mmol)制成反应混合物溶液,将此反应混合物溶液在室温下搅拌过夜;然后向此反应混合物溶液中加入纯化水(200mL),并进行过滤;将过滤所得的滤饼在真空中干燥,然后通过硅胶进行纯化,得到白色固体化合物3(13g),产率:75%。
(2)合成化合物4:
将化合物3(12g,35mmol)溶解于100mL的DCM中,然后添加 3,4-二氢-2H-吡喃(8.8g,105 mmol)和p-TosOH(0.3 g,1.7 mmol)制成反应混合物溶液,将此反应混合物溶液在室温下搅拌24小时;然后将溶剂浓缩得到粗产物,并将粗产物通过快速柱色谱法(SiO 2)进行纯化,得到9 g化合物4,产率:51%。
(3)合成化合物5:
在0°C下称取化合物4(5.1 g,10 mmol)溶解于DCM(50 mL)中,然后加入50 mL的TFA制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到3.6 g化合物5,为白色固体,产率:88%。
(4)合成化合物6:
在0℃下称取化合物5(4.1 g,10 mmol)溶解于DCM(50 mL)中,然后添加TEA(2 g,20 mmol)和溴乙酰溴(2.4 g,12 mmol)制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到3.4g化合物6,为白色固体,产率:64%。
(5)合成香草扁桃酸溴化衍生物:
在0℃下称取化合物6(2.6 g,5 mmol)溶解于DCM(50ml)中,然后加入HCl/MeOH(2N,10 mL)制成反应混合物溶液;将此反应混合物溶液在室温下搅拌过夜,然后通过减压蒸发去除溶剂,并通过快速柱色谱法(SiO2)进行纯化,得到0.9 g呈黄色固体状的香草扁桃酸溴化衍生物,产率:50%。
对上述合成步骤制备得到的香草扁桃酸溴化衍生物进行结构鉴定,方法如下:(1)利用VARIAN MERCURY plus 400MHz对上述黄色固体化合物进行1H核磁共振光谱扫描,采用TMS作为内标;(2)LC-MS检测方法:仪器Agilent 1200A,色谱柱:C18;色谱柱尺寸:4.6 *50mm;流动相:B(ACN),A(0.05%FA in water);梯度(B%):as Acq.上述方法。
经鉴定,上述合成步骤制备得到的香草扁桃酸溴化衍生物具有式(Ⅲ)所示的化学结构。
实施例2 香草扁桃酸溴化衍生物免疫原的制备
本实施例中的香草扁桃酸溴化衍生物免疫原由式(Ⅲ)所示的香草扁桃酸溴化衍生物与牛血清白蛋白(BSA)连接而成,其结构式如下述式(Ⅰ)所示:
式(Ⅰ)。
该香草扁桃酸溴化衍生物免疫原的合成方法,具体步骤如下:
(S3.1)将质量分数2.0%的载体溶解于10.0 mmol/L,pH=8.0的磷酸钠缓冲液中,得到载体溶液;
(S3.2)将质量分数1.0%的香草扁桃酸溴化衍生物与3.0%的二甲基甲酰胺、3.0%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应75分钟,得到香草扁桃酸溴化衍生物溶液;
(S3.3)将步骤(S3.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S3.1)中得到的载体溶液中,然后在0~4℃下搅拌反应18小时,得到偶联物溶液,在上述偶联物溶液中加入1.0 mmol/L的乙二胺四乙酸,再使用10.0 mmol/L、pH=7.0的HEPES缓冲液进行透析纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物免疫原溶液,在上述香草扁桃酸溴化衍生物免疫原溶液中加入质量分数0.05%的NaN3,于-20℃下储存。
实施例3 抗香草扁桃酸特异性抗体的制备
将实施例2制备得到的香草扁桃酸溴化衍生物免疫原制成人工抗原溶液,采用多点皮下注射的方法接种实验动物兔,加强免疫后提取抗血清并纯化得到抗体,具体步骤如下:
(1)用浓度为10 mmol/L、pH=7.4的PBS缓冲液将香草扁桃酸溴化衍生物免疫原稀释至8.0 mg/ml,得到人工抗原溶液,然后将5.0 ml所述人工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)1周后,将步骤(1)中所述的人工抗原溶液用浓度为10 mmol/L、pH=7.4的PBS缓冲液稀释一倍,再将5.0 ml稀释后的人工抗原溶液与等量弗氏不完全佐剂混合,对上述实验动物兔进行注射;
(3)之后每隔4周按步骤(2)的方法对上述实验动物兔进行注射1次,共计注射5次,以加强免疫;
(4)对加强免疫后的实验动物兔进行取血,分离纯化血液得到抗香草扁桃酸特异性抗体。
实施例4 香草扁桃酸溴化衍生物酶标偶联物的制备
本实施例中的香草扁桃酸溴化衍生物酶标偶联物由式(Ⅲ)所示的香草扁桃酸溴化衍生物与人工诱变葡萄糖-6-磷酸脱氢酶(mG6PDH)连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ);
该香草扁桃酸溴化衍生物酶标偶联物的合成方法,具体步骤如下:
(S4.1)将质量分数5.0%的规格为200KU的人工诱变葡萄糖-6-磷酸脱氢酶室温下溶解于含有50.0 mmol/L、pH=7.3的磷酸钠缓冲液中;然后加入1.0 mmol/L的乙二胺四乙酸;混合均匀后置于4℃下孵育15小时,得到人工诱变葡萄糖-6-磷酸脱氢酶溶液;
(S4.2)在无水状态下将质量分数1.5%的香草扁桃酸溴化衍生物与4.5%的二甲基甲酰胺、4.5%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应90分钟,得到香草扁桃酸溴化衍生物溶液;
(S4.3)将步骤(S4.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S4.1)中得到的人工诱变葡萄糖-6-磷酸脱氢酶溶液中,然后在0~4℃下搅拌反应18小时;将反应后的偶联产物通过G-50葡聚糖凝胶层析柱进行纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物酶标偶联物溶液,在上述香草扁桃酸溴化衍生物酶标偶联物溶液中加入质量分数0.05%的BSA和质量分数0.05%的NaN3,于0~4℃下储存。
实施例5 基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂的制备
(1)R1试剂的制备:将质量分数6.0%的氧化态的烟酰胺腺嘌呤二核苷酸、6.0%的葡萄糖-6-磷酸、0.3%的牛血清白蛋白、0.03%的NaN3的用浓度为60 mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗香草扁桃酸特异性抗体加入所述均相酶底物溶液中混匀,得到R1试剂,所述抗香草扁桃酸特异性抗体与均相酶底物溶液的体积比为1∶625。
(2)R2试剂的制备:将质量分数0.3%的牛血清白蛋白、0.03%的NaN3用浓度为150mmol/L、pH=8.0的Tris缓冲液溶解制成R2缓冲液,再将香草扁桃酸溴化衍生物酶标偶联物加入所述R2缓冲液中混匀,得到R2试剂,所述香草扁桃酸溴化衍生物酶标偶联物与R2缓冲液的体积比为1∶1250。
实施例6 香草扁桃酸的ELISA检测
1.香草扁桃酸的ELISA检测标准曲线的制作
(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1 mg/mL的储存液。用ELISA 缓冲液将储存液依次稀释为80.00 ng/mL、40.00 ng/mL、20.00 ng/mL、10.00 ng/mL、5.00 ng/mL和0.00 ng/mL的标准溶液。其中,ELISA缓冲液含有50 mmol/L的Tris、145mmol/L的NaCl 以及质量百分比为0.25%的BSA。
(2) 利用香草扁桃酸的ELISA检测方法制作标准曲线
用PBS将实施例3中所制备的抗香草扁桃酸抗体稀释成1∶10000的终浓度溶液,100μL/孔包被在96孔酶联板上,4℃放置12-24 h;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200 μL/孔的体积百分比0.5%的BSA溶液,4 ℃封闭放置8-16 h。然后用PBS洗涤3次,加入20 μL /孔的标准品。再加入100 μL/孔工作浓度的HRP-香草扁桃酸偶联物;室温下孵育30 min后PBS洗板5次;然后每孔加入100 μL TMB 底物,室温孵育30min;再每孔加入100 μL 终止液(2 mol/L的硫酸),测定450 nm 的吸光值。根据各标准品所对应的450 nm 的吸光值定标,制作标准曲线,结果如图1所示。
2.待测样品中香草扁桃酸含量的检测
(1) 制备待测样品
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1 mg/mL的储存液,并将此储存液稀释于空白人造尿液中,至终浓度分别为0.00 ng/mL、2.50 ng/mL、15.00 ng/mL、75.00 ng/mL,分别形成空白、低、中、高浓度的尿液样本。空白人造尿液为不含香草扁桃酸的人造尿液。
(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检测结果
尿液样品 | 空白 | 低 | 中 | 高 |
样品浓度(ng/mL) | 0.00 | 2.50 | 15.00 | 75.00 |
测定1 | 0.00 | 2.51 | 15.19 | 76.25 |
测定2 | 0.00 | 2.55 | 14.96 | 75.07 |
测定3 | 0.00 | 2.56 | 15.05 | 74.83 |
平均值(ng/mL) | 0.00 | 2.54 | 15.07 | 75.38 |
回收率(%) | - | 101.6 | 100.5 | 100.5 |
由表1中结果可知:采用香草扁桃酸的ELISA检测方法测定不同浓度尿液样品中香草扁桃酸的回收率都较高,均在100%-102%之间,说明本发明制备的抗香草扁桃酸特异性抗体完全达到检测样本中微量香草扁桃酸含量的要求,并且检测结果的准确度较高。
实施例7 香草扁桃酸的均相酶免疫检测
1. 制作标准曲线
(1)设置迈瑞 BS480全自动生化分析仪反应参数(表2)。
(2)操作步骤为:先加入R1试剂,再加入标准品,最后加入R2试剂。加入R2试剂后,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R1试剂和R2试剂的体积比例,同时调整测光点,最后得出较理想的反应标准曲线图,如图1所示。
表2 迈瑞BS480全自动生化分析仪反应参数
2. 样本检测
通过本发明的基于溴化衍生物的香草扁桃酸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空白人造尿液中,至浓度分别为30.00 ng/mL、150.00 ng/mL、300.00 ng/mL,检测结果及数据分析见表3。
表3 样本测定值及精密度和回收率评估
血液样本 | 低 | 中 | 高 |
样本浓度(ng/mL) | 30.00 | 150.00 | 300.00 |
1 | 30.33 | 152.67 | 307.28 |
2 | 31.54 | 151.83 | 297.41 |
3 | 30.79 | 145.97 | 310.55 |
4 | 30.85 | 153.08 | 296.57 |
5 | 29.51 | 154.81 | 305.08 |
6 | 31.00 | 152.37 | 312.29 |
7 | 30.77 | 148.57 | 299.00 |
8 | 30.20 | 149.05 | 308.51 |
9 | 31.62 | 155.22 | 311.34 |
10 | 29.68 | 150.60 | 298.89 |
平均值(ng/mL) | 30.63 | 151.42 | 304.69 |
标准差(SD) | 0.70 | 2.90 | 6.17 |
精密度(CV%) | 2.29 | 1.92 | 2.03 |
回收率(%) | 102.10 | 100.95 | 101.56 |
检测结果表明:本发明制备的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂测定质控样本中微量香草扁桃酸含量的准确度高,回收率均在100%-103%之间;精密度高,CV均低于3%。
实施例8 常见激素干扰试验
选取30种常见激素及激素代谢物作为干扰物进行干扰试验,将干扰物浓度调整至10.00 ng/mL,采用实施例7所述香草扁桃酸的均相酶免疫检测方法进行测定,将待测干扰物与实施例5制备的R1试剂反应,再加入R2试剂;检测上述混合溶液的OD340吸光值,根据图2得到相应干扰物的检出值。30种常见激素及激素代谢物名称以及测定结果详见表4。
表4 常见激素干扰试验测定结果
序号 | 干扰物名称 | 检出值 (ng/mL) | 序号 | 干扰物名称 | 检出值 (ng/mL) |
1 | 皮质醇 (氢化可的松) | 0.00 | 2 | 醛固酮 | 0.00 |
3 | 雄烯二酮 | 0.00 | 4 | 雄甾酮 | 0.00 |
5 | 皮质脂酮 | 0.00 | 6 | 皮质酮 (可的松) | 0.00 |
7 | 去氧皮质酮 | 0.00 | 8 | 脱氢表雄酮 | 0.00 |
9 | 硫酸脱氢表雄酮 | 0.00 | 10 | 二氢睾酮 | 0.00 |
11 | 雌二醇 | 0.00 | 12 | 雌三醇 | 0.00 |
13 | 雌酮 | 0.00 | 14 | 本胆烷醇酮 | 0.00 |
15 | 17-羟孕烯醇酮 | 0.00 | 16 | 17-羟孕酮 | 0.00 |
17 | 孕烯醇酮 | 0.00 | 18 | 孕酮 | 0.00 |
19 | 睾酮 | 0.00 | 20 | 孕三醇 | 0.00 |
21 | 孕二醇 | 0.00 | 22 | 17α-羟基黄体酮 | 0.00 |
23 | 雄烯二酮 | 0.00 | 24 | 17-酮类固醇 | 0.00 |
25 | 17-羟皮质类固醇 | 0.00 | 26 | 肾上腺素 | 0.00 |
27 | 去甲肾上腺素 | 0.00 | 28 | 多巴胺 | 0.00 |
29 | 高香草酸 | 0.00 | 30 | 二羟基杏仁酸 | 0.00 |
测定结果显示:上述30种常见激素及激素代谢物使用香草扁桃酸的均相酶免疫检测方法进行测定的检出值均为0.00 ng/mL。由此可见,本发明制备的抗香草扁桃酸特异性抗体的特异性强,能与香草扁桃酸精确识别并结合,与常见干扰物无任何交叉反应。
实施例9 均相酶免疫法与高效液相色谱法检测香草扁桃酸的对比实验
使用实施例7所述香草扁桃酸的均相酶免疫检测方法与金标准高效液相色谱法对100例人体尿液样本分别进行测定,并作相关性分析,实验数据详见表5。
表 5 人体尿液样本测定结果对比数据
样本号 | 均相酶免疫法测定值(ng/mL) | 高效液相色谱法测定值(ng/mL) |
1 | 140.35 | 139.78 |
2 | 162.04 | 168.53 |
3 | 51.33 | 51.43 |
4 | 24.30 | 25.00 |
5 | 131.52 | 126.25 |
6 | 117.60 | 119.65 |
7 | 143.11 | 148.74 |
8 | 175.49 | 170.12 |
9 | 72.42 | 75.39 |
10 | 20.50 | 21.03 |
11 | 64.80 | 66.45 |
12 | 59.23 | 59.20 |
13 | 103.71 | 100.96 |
14 | 25.63 | 25.20 |
15 | 39.75 | 38.56 |
16 | 151.62 | 160.34 |
17 | 115.89 | 116.77 |
18 | 134.56 | 131.56 |
19 | 148.00 | 151.71 |
20 | 47.30 | 46.05 |
21 | 215.12 | 219.44 |
22 | 188.52 | 186.78 |
23 | 160.11 | 160.94 |
24 | 124.52 | 124.52 |
25 | 317.77 | 331.00 |
26 | 80.27 | 81.09 |
27 | 65.93 | 64.18 |
28 | 28.44 | 29.05 |
29 | 161.32 | 159.66 |
30 | 175.58 | 180.09 |
31 | 117.98 | 118.24 |
32 | 173.32 | 176.30 |
33 | 111.04 | 113.33 |
34 | 90.58 | 91.52 |
35 | 81.52 | 81.55 |
36 | 34.60 | 33.67 |
37 | 182.96 | 175.84 |
38 | 170.45 | 170.99 |
39 | 226.23 | 220.32 |
40 | 135.21 | 141.07 |
41 | 29.65 | 29.66 |
42 | 68.10 | 69.42 |
43 | 100.81 | 99.97 |
44 | 51.32 | 51.68 |
45 | 75.62 | 77.05 |
46 | 201.63 | 200.84 |
47 | 150.32 | 153.29 |
48 | 183.07 | 180.82 |
49 | 85.56 | 86.00 |
50 | 177.52 | 172.25 |
51 | 91.96 | 92.00 |
52 | 35.00 | 35.12 |
53 | 121.78 | 121.11 |
54 | 148.53 | 150.20 |
55 | 170.03 | 168.05 |
56 | 20.00 | 20.15 |
57 | 254.30 | 250.30 |
58 | 105.77 | 105.62 |
59 | 81.45 | 80.61 |
60 | 107.95 | 109.93 |
61 | 143.12 | 144.80 |
62 | 311.68 | 320.73 |
63 | 222.64 | 220.53 |
64 | 155.63 | 157.36 |
65 | 83.40 | 83.95 |
66 | 137.68 | 139.81 |
67 | 122.59 | 123.30 |
68 | 106.31 | 108.03 |
69 | 46.82 | 46.79 |
70 | 156.19 | 160.55 |
71 | 32.32 | 32.43 |
72 | 64.90 | 65.16 |
73 | 158.85 | 156.67 |
74 | 170.09 | 169.93 |
75 | 114.53 | 115.28 |
76 | 100.07 | 101.32 |
77 | 56.83 | 56.94 |
78 | 157.97 | 158.81 |
79 | 233.88 | 239.70 |
80 | 304.50 | 289.99 |
81 | 265.91 | 260.86 |
82 | 170.19 | 173.45 |
83 | 210.73 | 211.01 |
84 | 172.86 | 175.37 |
85 | 32.50 | 32.63 |
86 | 49.81 | 50.01 |
87 | 89.67 | 89.24 |
88 | 105.23 | 103.34 |
89 | 24.93 | 25.09 |
90 | 118.65 | 119.56 |
91 | 77.25 | 78.51 |
92 | 53.61 | 54.86 |
93 | 180.79 | 184.52 |
94 | 41.22 | 41.46 |
95 | 125.92 | 129.22 |
96 | 26.66 | 26.67 |
97 | 63.43 | 64.10 |
98 | 175.90 | 169.93 |
99 | 167.86 | 169.27 |
100 | 56.08 | 56.56 |
对上述实验数据作散点图,参见图3,得到的线性方程为:y = 1.0008x + 0.3399,相关系数R2 = 0.9975,表明采用本发明香草扁桃酸的均相酶免疫检测方法与金标准高效液相色谱法,检测人体尿液样本的实验数据具有较高的一致性,检测结果准确可靠。
以上述依据本发明的理想实施例为启示,通过上述的说明内容,相关技术人员完全可以在不偏离本项发明技术思想的范围内,进行多样的变更以及修改。本项发明的技术性范围并不局限于说明书上的内容,必须要根据权利要求范围来确定其技术性范围。
序列表
<110> 苏州博源医疗科技有限公司
<120> 一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂及其制备方法
<130> 2022.01.05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 583
<212> PRT
<213> 人工合成(Artificial Sequence)
<400> 1
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Claims (10)
1.一种基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,包括R1试剂和R2试剂,所述R1试剂中包含抗香草扁桃酸特异性抗体和均相酶底物溶液;所述R2试剂包含香草扁桃酸溴化衍生物酶标偶联物和R2缓冲液;
所述的抗香草扁桃酸特异性抗体,由香草扁桃酸溴化衍生物免疫原免疫实验动物后产生,所述实验动物为兔、山羊、绵羊、大鼠、小鼠、豚鼠或马中的一种;
所述的香草扁桃酸溴化衍生物免疫原由香草扁桃酸溴化衍生物与载体连接而成,其结构式如下述式(Ⅰ)所示:
式(Ⅰ);
所述的载体为具有免疫原性的蛋白质或多肽,选自血清蛋白、卵清蛋白、丙种球蛋白、甲状腺球蛋白、血蓝蛋白或多聚赖氨酸中的一种;
所述的香草扁桃酸溴化衍生物酶标偶联物由香草扁桃酸溴化衍生物与人工诱变葡萄糖-6-磷酸脱氢酶连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ);
式中mG6PDH为人工诱变葡萄糖-6-磷酸脱氢酶,所述人工诱变葡萄糖-6-磷酸脱氢酶的氨基酸序列为SEQ ID NO:1;
所述的香草扁桃酸溴化衍生物,其结构式如下述式(Ⅲ)所示:
式(Ⅲ)。
2.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述实验动物为兔。
3.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述的载体为血清蛋白。
4.根据权利要求3所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述的载体为牛血清白蛋白。
5.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,制备方法包括以下步骤:
(S1.1)R1试剂的制备:将质量分数6.0%的氧化态的烟酰胺腺嘌呤二核苷酸、6.0%的葡萄糖-6-磷酸、0.3%的牛血清白蛋白、0.03%的NaN3的用浓度为60 mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗香草扁桃酸特异性抗体加入所述均相酶底物溶液中混匀,得到R1试剂,所述抗香草扁桃酸特异性抗体与均相酶底物溶液的体积比为1∶50~1∶5000;
(S1.2)R2试剂的制备:将质量分数0.3%的牛血清白蛋白、0.03%的NaN3用浓度为150mmol/L、pH=8.0的Tris缓冲液溶解制成R2缓冲液,再将香草扁桃酸溴化衍生物酶标偶联物加入所述R2缓冲液中混匀,得到R2试剂,所述香草扁桃酸溴化衍生物酶标偶联物与R2缓冲液的体积比为1∶50~1∶5000。
6.根据权利要求5所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述抗香草扁桃酸特异性抗体与均相酶底物溶液的体积比为1∶625;所述香草扁桃酸溴化衍生物酶标偶联物与R2缓冲液的体积比为1∶1250。
7.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述抗香草扁桃酸特异性抗体的制备方法,包括以下步骤:
(S2.1)用浓度为10 mmol/L、pH=7.4的PBS缓冲液将香草扁桃酸溴化衍生物免疫原稀释至8.0 mg/ml,得到人工抗原溶液,然后将5.0 ml所述人工抗原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
(S2.2)1周后,将步骤(S2.1)中所述的人工抗原溶液用浓度为10 mmol/L、pH=7.4的PBS缓冲液稀释一倍,再将5.0 ml稀释后的人工抗原溶液与等量弗氏不完全佐剂混合,对上述实验动物进行注射;
(S2.3)之后每隔4周按步骤(S2.2)的方法对上述实验动物进行注射1次,共计注射5次,以加强免疫;
(S2.4)对加强免疫后的实验动物进行取血,分离纯化血液得到抗香草扁桃酸特异性抗体。
8.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述香草扁桃酸溴化衍生物免疫原的制备方法,包括以下步骤:
(S3.1)将质量分数2.0%的载体溶解于10.0 mmol/L,pH=8.0的磷酸钠缓冲液中,得到载体溶液;
(S3.2)将质量分数1.0%的香草扁桃酸溴化衍生物与3.0%的二甲基甲酰胺、3.0%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应60~90分钟,得到香草扁桃酸溴化衍生物溶液;
(S3.3)将步骤(S3.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S3.1)中得到的载体溶液中,然后在0~4℃下搅拌反应12~24小时,得到偶联物溶液,在上述偶联物溶液中加入1.0 mmol/L的乙二胺四乙酸,再使用10.0 mmol/L、pH=7.0的HEPES缓冲液进行透析纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物免疫原溶液,在上述香草扁桃酸溴化衍生物免疫原溶液中加入质量分数0.05%的NaN3,于-20℃下储存。
9.根据权利要求1所述的基于溴化衍生物的香草扁桃酸均相酶免疫检测试剂,其特征在于,所述香草扁桃酸溴化衍生物酶标偶联物的制备方法,包括以下步骤:
(S4.1)将质量分数5.0%的规格为200KU的人工诱变葡萄糖-6-磷酸脱氢酶室温下溶解于含有50.0 mmol/L、pH=7.3的磷酸钠缓冲液中,然后加入1.0 mmol/L的乙二胺四乙酸,混合均匀后置于4℃下孵育10~20小时,得到人工诱变葡萄糖-6-磷酸脱氢酶溶液;
(S4.2)在无水状态下将质量分数1.5%的香草扁桃酸溴化衍生物与4.5%的二甲基甲酰胺、4.5%的乙醇一起溶解于8.0 mmol/L、pH=7.5的磷酸钾缓冲液中得到混合溶液,将上述混合溶液在0~4℃下温和搅拌反应60~120分钟,得到香草扁桃酸溴化衍生物溶液;
(S4.3)将步骤(S4.2)中得到的香草扁桃酸溴化衍生物溶液缓慢滴加至步骤(S4.1)中得到的人工诱变葡萄糖-6-磷酸脱氢酶溶液中,然后在0~4℃下搅拌反应12~24小时;将反应后的偶联产物通过G-50葡聚糖凝胶层析柱进行纯化,纯化后所得的溶液即为香草扁桃酸溴化衍生物酶标偶联物溶液,在上述香草扁桃酸溴化衍生物酶标偶联物溶液中加入质量分数0.05%的BSA和质量分数0.05%的NaN3,于0~4℃下储存。
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