CN109111494A - 雌二醇衍生物、免疫原、抗体、酶标偶联物、检测试剂及其制备方法 - Google Patents
雌二醇衍生物、免疫原、抗体、酶标偶联物、检测试剂及其制备方法 Download PDFInfo
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- CN109111494A CN109111494A CN201811003617.3A CN201811003617A CN109111494A CN 109111494 A CN109111494 A CN 109111494A CN 201811003617 A CN201811003617 A CN 201811003617A CN 109111494 A CN109111494 A CN 109111494A
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Abstract
本发明涉及一种用于生物样本中雌二醇检测的雌二醇衍生物及其制备方法,所述雌二醇衍生物的结构式如下述式(Ⅰ)所示:式(Ⅰ)。使用该雌二醇衍生物获得了高免疫原性的雌二醇免疫原与相应抗体以及雌二醇酶标偶联物,并制备出了雌二醇均相酶免疫检测试剂。该雌二醇免疫原特异性强、免疫原性高,制备出的抗雌二醇特异性抗体特异性强、效价高,能很好地识别并结合雌二醇,并且与常见的92种干扰物无任何交叉反应;含有上述抗雌二醇特异性抗体与雌二醇酶标偶联物的均相酶免疫检测试剂可以方便、快速、准确地确定生物样品中的雌二醇含量,并且可以在全自动生化分析仪上同时测定多个样品,实现雌二醇的高通量、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。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种雌二醇衍生物、免疫原、抗体、酶标偶联物、检测试剂及其制备方法。
背景技术
雌二醇(Estradiol,E2)的化学结构式如下述式(Ⅳ)所示:
式(Ⅳ)。
雌二醇为人体雌激素(estrogen)的主要组成部分,女性体内的E2主要由卵巢滤泡、黄体及妊娠时的胎盘产生,血液中70%的E2与性激素结合球蛋白(SHBG)结合,25%与血浆白蛋白结合,其余为游离型。男性体内的E2来源极少,主要由睾丸合成或为睾酮的代谢物。E2的合成呈周期性变化,能够促进女性生殖器官的形成及发育,促使第二性征的出现及维持,并与孕激素协同作用形成月经周期。此外,E2还具有广泛的代谢调节作用。E2含量异常升高可见于女性性早熟、双胎妊娠或多胎妊娠、子宫内膜癌、乳腺癌、卵巢癌(如卵巢颗粒层细胞瘤、卵巢性索间质细胞瘤、卵巢胚瘤、卵巢脂肪样细胞瘤等)、心脏病(如心肌梗塞、心绞痛、冠状动脉狭窄)以及其它疾病(系统性红斑狼疮、肝硬化、男性肥胖症等)。病理性E2含量降低的原因较多,如:卵巢发育不良、原发性卵巢衰竭、垂体性闭经或不孕、库兴氏综合征、阿狄森氏病、多囊卵巢综合征、席汉氏综合征、完全性或部分性葡萄胎、妊娠高血压综合征、异位妊娠、胎儿宫内死亡、肾功能不全、脑及垂体的局灶性病变等。因此,检测人体血液、尿液等生物样本中雌二醇的含量具有重要的临床意义。
目前,雌二醇检测常用的方法主要分为免疫学方法和仪器分析的方法,常用的雌二醇免疫学检测方法有:放射免疫分析法、酶联免疫分析法和化学发光免疫分析法等。这些方法都有其各自的缺陷,如放射免疫分析法具有严重的放射性污染;酶联免疫分析法只能进行手工操作,误差较大;化学发光免疫分析法需要配备价格昂贵的专用仪器等。常用的雌二醇仪器分析检测方法有:气相色谱法、气质联用法、液相色谱法、高效液相色谱法等,这些仪器分析方法虽然灵敏度较高,检测结果精密度高、准确性好,但是存在样品的前处理较繁琐、测定时间较长、需要配备专门的仪器设备,操作流程复杂,单个样品的检测时间较长,不能快速提供检测结果等缺陷,不适用于临床上大批量样品的检验。因此,研发一种灵敏度高、结果精确,且高通量、快速化,操作简便、成本低廉的雌二醇检测方法对临床诊断、食品卫生、环境激素检测等方面都具有重要意义。
发明内容
本发明要解决的技术问题是:提供一种用于生物样本中雌二醇检测的雌二醇衍生物及其制备方法,使用该雌二醇衍生物制备出免疫原性强的雌二醇免疫原,从而免疫实验动物得到高效价的抗雌二醇特异性抗体;同时,使用该雌二醇衍生物制备得到雌二醇酶标偶联物。利用该抗体和酶标偶联物制备的雌二醇均相酶免疫检测试剂可以实现在全自动生化分析仪上对雌二醇高通量、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,还能有效降低雌二醇检测成本,有利于临床推广使用。
本发明解决其技术问题所采用的技术方案是:
一、提供一种雌二醇衍生物,其结构式如下述式(Ⅰ)所示:
式(Ⅰ)。
二、提供一种上述的雌二醇衍生物的制备方法,包括以下步骤:
a.化合物3的合成
称取5.0 g(0.018 mol)的化合物1与10 g(0.048 mol)的化合物2共同溶解于100 mL的丙酮中,然后加入7.9 g(0.057 mol)的K2CO3,制成反应混合溶液,然后将此反应混合溶液加热回流12小时。将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到3.1 g白色固体状的化合物3。
b.雌二醇衍生物的合成
称取3.1 g(7.75 mmol)的化合物3,1.13 g(11.62 mmol)的化合物4,3.04 g(11.62mmol)的三苯基膦(PPh3),共同溶解于50 mL的四氢呋喃(THF)中,然后在0℃下加入2.35 g(11.62 mmol)的偶氮二甲酸二异丙酯(DIAD),制成反应混合溶液,然后将此反应混合溶液在室温下搅拌12小时。将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到1.1 g白色固体状的雌二醇衍生物。
三、提供一种雌二醇免疫原,由上述的雌二醇衍生物与载体连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ);
所述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、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。
四、提供一种上述的雌二醇免疫原的制备方法,包括以下步骤:
a.称取4.08 g磷酸二氢钾、6.39 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液A;
b.称取4.5 mg 蛋白质载体,4℃下溶解于4.5 ml上述缓冲溶液A中,制成载体溶液;
c.称取4.5 mg上述式(Ⅰ)所示的雌二醇衍生物,4℃下溶解于450μl上述缓冲溶液A中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述载体溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液A进行透析,透析后所得溶液即为雌二醇免疫原溶液,在雌二醇免疫原溶液中加入质量分数0.12%的NaN3,于-20℃下储存。
五、提供一种抗雌二醇特异性抗体,所述抗体由上述的雌二醇免疫原免疫实验动物后产生,所述抗体为完整抗体分子,或者为保留与雌二醇特异性结合能力的抗体片段或抗体衍生物,所述实验动物为兔、山羊、小鼠、绵羊、豚鼠或马中的一种,优选为兔。
六、提供一种上述的抗雌二醇特异性抗体的制备方法,包括以下步骤:
a.用PBS缓冲液将上述式(Ⅱ)所示的雌二醇免疫原稀释至3.0 mg/ml,得到抗原溶液,然后用3.0 ml所述抗原溶液与弗氏完全佐剂混合,对实验动物进行注射;
b. 2周后,再用3.0 ml相同的抗原溶液与弗氏不完全佐剂混合,对上述实验动物注射一次,之后每隔2周注射一次,共计注射6次;
c.对免疫后的实验动物取血,分离纯化得到效价为1:50000~1:80000的抗雌二醇特异性抗体。
七、提供一种雌二醇酶标偶联物,由上述的雌二醇衍生物与葡萄糖-6-磷酸脱氢酶连接而成,其结构式如下述式(Ⅲ)所示:
式(Ⅲ)。
八、提供一种上述的雌二醇酶标偶联物的制备方法,包括以下步骤:
a.称取1.635 g磷酸二氢钾、2.55 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液B;
b.称取4.5 mg葡萄糖-6-磷酸脱氢酶,4℃下溶解于4.5 ml上述缓冲溶液B中,制成葡萄糖-6-磷酸脱氢酶溶液;
c.称取4.5 mg上述式(Ⅰ)所示的雌二醇衍生物,4℃下溶解于450 μl上述缓冲溶液B中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述葡萄糖-6-磷酸脱氢酶溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液B进行透析,透析后所得溶液即为葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液,在葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液中加入质量分数0.75%的BSA和质量分数0.12%的NaN3,于2~8℃下储存。
九、提供一种雌二醇检测试剂,包括试剂A和试剂B,所述试剂A中包含上述的抗雌二醇特异性抗体和均相酶底物,所述均相酶底物由葡萄糖-6-磷酸、氧化态的烟酰胺腺嘌呤二核苷酸和Tris缓冲液配制而成;所述试剂B包含上述的雌二醇酶标偶联物和Tris缓冲液;使用时将所述试剂A与试剂B按1:1~4:1的体积比混合,优选按4∶1的体积比使用。
十、提供一种上述的雌二醇检测试剂的制备方法,包括以下步骤:
a.试剂A的制备:将7.5 g的氧化态的烟酰胺腺嘌呤二核苷酸、3.75 g的葡萄糖-6-磷酸用1.5 L、55 mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;再将上述抗雌二醇特异性抗体加入上述均相酶底物中,得到试剂A,所述抗雌二醇特异性抗体与均相酶底物的体积比为1:100~1:10000;优选地,所述抗雌二醇特异性抗体与均相酶底物的体积比为1: 500;
b.试剂B的制备:将上述的雌二醇酶标偶联物用120mM、pH=8.2的Tris缓冲液溶解,得到试剂B,所述雌二醇酶标偶联物与Tris缓冲液的体积比为1:100~1:10000;优选地,所述雌二醇酶标偶联物与Tris缓冲液的体积比为1: 2000。
本发明的有益效果是:通过反复的试验获得了一种全新的雌二醇衍生物,并使用该雌二醇衍生物获得了高免疫原性的雌二醇免疫原与相应抗体以及雌二醇酶标偶联物,并制备出了雌二醇均相酶免疫检测试剂。该雌二醇免疫原特异性强、免疫原性高,制备出的抗雌二醇特异性抗体特异性强、效价高,能很好地识别并结合雌二醇,并且与常见的92种干扰物无任何交叉反应;含有上述抗雌二醇特异性抗体与雌二醇酶标偶联物的均相酶免疫检测试剂可以方便、快速、准确地确定生物样品中的雌二醇含量,并且可以在全自动生化分析仪上同时测定多个样品,实现雌二醇的高通量、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是本发明的雌二醇ELISA检测反应曲线;
图2是本发明的雌二醇均相酶免疫检测反应曲线;
图3是本发明的雌二醇均相酶免疫法与高效液相色谱法相关性分析图。
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.雌二醇衍生物的合成
雌二醇衍生物的化学结构如式(Ⅰ)所示:
式(Ⅰ)。
上述雌二醇衍生物的合成路线及制备步骤如下:
具体的合成步骤如下:
a.化合物3的合成
称取5.0 g(0.018 mol)的化合物1与10 g(0.048 mol)的化合物2共同溶解于100 mL的丙酮中,然后加入7.9 g(0.057 mol)的K2CO3,制成反应混合溶液,然后将此反应混合溶液加热回流12小时。将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到3.1 g白色固体状的化合物3。
b.雌二醇衍生物的合成
称取3.1 g(7.75 mmol)的化合物3,1.13 g(11.62 mmol)的化合物4,3.04 g(11.62mmol)的三苯基膦(PPh3),共同溶解于50 mL的四氢呋喃(THF)中,然后在0℃下加入2.35 g(11.62 mmol)的偶氮二甲酸二异丙酯(DIAD),制成反应混合溶液,然后将此反应混合溶液在室温下搅拌12小时。将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到1.1 g白色固体状的雌二醇衍生物。
式(I)所示雌二醇衍生物的结构鉴定:利用Bruker Avance III plus 400 MHz和VARIAN MERCURY plus 300M对上述白色固体化合物进行核磁共振光谱扫描,采用TMS作为内标。结果如下:1H-NMR (400 MHz, CDCl3): δ 0.66 (s, 3H), 1.09-1.39 (m, 15H),1.44-1.51 (m, 2H), 1.57-1.67 (m, 3H), 1.77-1.89 (m, 3H), 2.06-2.12 (m, 1H),2.23-2.27 (m, 1H), 2.73-2.76 (m, 2H), 3.38-3.42 (m, 2H), 3.49-3.55 (m, 1H),3.85-3.89 (m, 2H), 4.50-4.52 (m, 1H), 6.55-6.60 (d, 1H), 6.63-6.66 (dd, 1H),7.00 (s, 2H), 7.12-7.15 (d, 1H);表征为结构式(I)所示的雌二醇衍生物。
实施例2. 雌二醇免疫原的合成
本实施例中的雌二醇免疫原由式(Ⅰ)所示的雌二醇衍生物与牛血清白蛋白(BSA)连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ)。
该雌二醇免疫原的合成方法具体步骤如下:
a.称取4.08 g磷酸二氢钾、6.39 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液A;
b.称取4.5 mg BSA,4℃下溶解于4.5 ml上述缓冲溶液A中,制成载体溶液;
c.称取4.5 mg上述式(Ⅰ)所示的雌二醇衍生物,4℃下溶解于450μl上述缓冲溶液A中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述载体溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液A进行透析,透析后所得溶液即为雌二醇免疫原溶液,在雌二醇免疫原溶液中加入质量分数0.12%的NaN3,于-20℃下储存。
实施例3.抗雌二醇特异性抗体的制备
将实施例2制备得到的雌二醇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:
a.用PBS缓冲液将上述式(Ⅱ)所示的雌二醇免疫原稀释至3.0 mg/ml,得到抗原溶液,然后用3.0 ml所述抗原溶液与弗氏完全佐剂混合,对实验动物进行注射;
b. 2周后,再用3.0 ml相同的抗原溶液与弗氏不完全佐剂混合,对上述实验动物注射一次,之后每隔2周注射一次,共计注射6次;
c.对免疫后的实验动物取血,分离纯化得到效价为1: 80000的抗雌二醇特异性抗体。
实施例4.雌二醇 ELISA检验
1.雌二醇 ELISA检测标准曲线的建立
(1) 标准品的制备
将雌二醇粉末(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200.00 pg/mL、80.00 pg/mL、40.00 pg/mL、20.00 pg/mL、10.00 pg/mL 和0.00 pg/mL 的标准溶液。其中,ELISA缓冲液含有50.0 mM Tris,145mMNaCl 和0.25%的BSA。
(2) 利用雌二醇的ELISA检验方法制备标准曲线
用PBS将实施例3中所制备的抗雌二醇抗体稀释成1 : 5000的终浓度溶液,100μL /孔包被在96孔酶联板上,4℃放置12-24h ;用PBS将上述包被有抗雌二醇抗体的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8-16 h。然后用PBS洗涤3次,加入20μL /孔的标准品。再加入100 μL /孔工作浓度的辣根过氧化物酶(HRP)-雌二醇偶联物;室温下孵育30min 后PBS 洗板5次;然后每孔加入100 μL TMB 底物,室温孵育30 min。再每孔加入100 μL 终止液(2 M 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,结果如附图1所示。
2.待测样品中雌二醇含量的检测
(1) 制作待测样品
制备方法:将雌二醇粉末(购于Sigma公司) 溶解于甲醇溶液制成1μg/mL 的储存液,并将此储存液稀释于不含雌二醇的空白人造血浆中,至终浓度分别为0.00,12.00,60.00,150.00 pg/mL,形成空白、低、中、高浓度的人造血浆样本。
(2) 测试方法
利用上述雌二醇的ELISA检验方法,将上述空白、低、中、高浓度的人造血浆样本代替标准品,测试上述空白、低、中、高浓度的人造血浆样本在450nm 的吸光值。
(3) 测试结果
对照图2中所示的雌二醇ELISA检验的标准曲线,计算每个样本中雌二醇含量,并对每个样本进行3个复孔测定,根据上述样本中雌二醇的实际含量计算回收率,结果如表1所示。
表1雌二醇的ELISA检测回收实验
血清样品 | 空白 | 低 | 中 | 高 |
样品浓度(pg/mL) | 0.00 | 12.00 | 60.00 | 150.00 |
测试1 | 0.00 | 12.63 | 59.25 | 152.33 |
测试2 | 0.00 | 12.27 | 62.00 | 153.70 |
测试3 | 0.01 | 12.41 | 61.96 | 147.20 |
平均值(pg/mL) | 0.00 | 12.44 | 61.07 | 151.08 |
回收率(%) | - | 103.64 | 101.78 | 100.72 |
由表1中结果可知:采用本发明雌二醇ELISA检测试剂测定不同浓度样品中的雌二醇回收率都较高,均>95%,说明本发明所述的抗雌二醇特异性抗体可以用于样本中雌二醇的检测,并且结果准确度高。
实施例5.雌二醇酶标偶联物的制备
本实施例中的雌二醇酶标偶联物由式(Ⅰ)所示的雌二醇衍生物与葡萄糖-6-磷酸脱氢酶(G6PDH)连接而成,其结构式如下述式(Ⅲ)所示:
式(Ⅲ);
该雌二醇酶标偶联物的合成方法具体步骤如下:
a.称取1.635 g磷酸二氢钾、2.55 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液B;
b.称取4.5 mg葡萄糖-6-磷酸脱氢酶,4℃下溶解于4.5 ml上述缓冲溶液B中,制成葡萄糖-6-磷酸脱氢酶溶液;
c.称取4.5 mg上述式(Ⅰ)所示的雌二醇衍生物,4℃下溶解于450 μl上述缓冲溶液B中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述葡萄糖-6-磷酸脱氢酶溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液B进行透析,透析后所得溶液即为葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液,在葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液中加入质量分数0.75%的BSA和质量分数0.12%的NaN3,于2~8℃下储存。
实施例6.雌二醇均相酶免疫检测试剂的制备
a.试剂A的制备:将7.5 g的氧化态的烟酰胺腺嘌呤二核苷酸、3.75 g的葡萄糖-6-磷酸用1.5 L、55 mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;再将上述抗雌二醇特异性抗体加入上述均相酶底物中,得到试剂A,所述抗雌二醇特异性抗体与均相酶底物的体积比为1:100~1:10000;本实施例中所述抗雌二醇特异性抗体与均相酶底物的体积比为1: 500;
b.试剂B的制备:将上述的雌二醇酶标偶联物用120mM、pH=8.2的Tris缓冲液溶解,得到试剂B,所述雌二醇酶标偶联物与Tris缓冲液的体积比为1:100~1:10000;本实施例中所述雌二醇酶标偶联物与Tris缓冲液的体积比为1: 2000。
实施例7. 雌二醇均相酶免疫检验及结果
1. 获得标准曲线:
(1)设置迈瑞 BS480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2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:将雌二醇标准品溶解于空白人造血浆中,至浓度分别为100.00,500.00,1200.00 pg/ml。检测数据及数据分析见表3。
表3 样品测定值及精密度和回收率评估
血液样品 | 低 | 中 | 高 |
样品浓度 (pg/ml) | 100.00 | 500.00 | 1200.00 |
1 | 102.53 | 507.09 | 1186.07 |
2 | 102.78 | 510.97 | 1193.14 |
3 | 98.87 | 503.03 | 1202.62 |
4 | 101.52 | 511.20 | 1208.74 |
5 | 101.00 | 496.18 | 1189.81 |
6 | 103.05 | 491.41 | 1220.39 |
7 | 100.60 | 502.39 | 1203.90 |
8 | 97.89 | 498.98 | 1195.28 |
9 | 102.24 | 500.85 | 1198.57 |
10 | 99.61 | 510.12 | 1211.69 |
平均值(pg/ml) | 101.01 | 503.22 | 1201.02 |
标准差(SD) | 1.758 | 6.664 | 10.592 |
精密度(CV%) | 1.74 | 1.32 | 0.88 |
回收率(%) | 101.01 | 100.64 | 100.09 |
检测结果:本发明的均相酶免疫检测试剂测定的准确度高,回收率均在95%-105%之间;精密度高,CV均低于5%。
实施例8.药物与激素干扰试验
选取62种常见药物与30种常见激素及激素代谢物进行干扰检测,调整浓度至1.00 ng/ml,采用实施例七的均相酶免疫方法进行测定:
1. 将待测干扰物与实施例六制备的试剂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,根据实施例七的标准曲线得到相应物质的浓度。
62种常见药物与30种常见激素及激素代谢物名称以及测定结果具体参见表4。
表4 常见干扰物测定结果
ID# | 化合物名称 | 等价于雌二醇的浓度 (pg/ml) | ID# | 化合物名称 | 等价于雌二醇的浓度 (pg/ml) |
1 | 阿司匹林 | 0.00 | 2 | 苯丙醇胺 | 0.00 |
3 | β-苯基乙胺 | 0.00 | 4 | 普鲁卡因酰胺 | 0.00 |
5 | 安非他命 | 0.00 | 6 | 普鲁卡因 | 0.00 |
7 | 氨苄青霉素 | 0.00 | 8 | 奎尼丁 | 0.00 |
9 | 甲氨二氮卓 | 0.00 | 10 | 佐美酸 | 0.00 |
11 | 氯丙嗪 | 0.00 | 12 | 苯肾上腺素 | 0.00 |
13 | 氯拉卓酸 | 0.00 | 14 | 桂皮酰艾克宁 | 0.00 |
15 | 二甲苯氧庚酸 | 0.00 | 16 | 芽子碱 | 0.00 |
17 | 非诺洛芬 | 0.00 | 18 | 地西洋 | 0.00 |
19 | 甲基苯丙胺 | 0.00 | 20 | 可替宁 | 0.00 |
21 | 龙胆酸 | 0.00 | 22 | 阿替洛尔 | 0.00 |
22 | 吉非贝齐 | 0.00 | 24 | 心得安 | 0.00 |
25 | 氢可酮 | 0.00 | 26 | 苯乙哌啶酮 | 0.00 |
27 | 布洛芬 | 0.00 | 28 | 苯基丁氮酮 | 0.00 |
29 | 丙咪嗪 | 0.00 | 30 | 麦角酸二乙基酰胺 | 0.00 |
31 | 二氨基二苯砜 | 0.00 | 32 | 大麻酚 | 0.00 |
33 | 萘普生 | 0.00 | 34 | 洛哌丁胺 | 0.00 |
35 | 氢氯噻嗪 | 0.00 | 36 | 异克舒令 | 0.00 |
37 | 哌替啶 | 0.00 | 38 | 苯基丙氨酸 | 0.00 |
39 | 烯丙羟吗啡酮 | 0.00 | 40 | 盐酸氟西汀 | 0.00 |
41 | 麻黄素 | 0.00 | 42 | 柳丁氨醇 | 0.00 |
43 | 烟酰胺 | 0.00 | 44 | 青霉素 | 0.00 |
45 | 甲胺呋硫 | 0.00 | 46 | 甲基二乙醇胺 | 0.00 |
47 | 异戊巴比妥 | 0.00 | 48 | 二亚甲基双氧苯丙胺 | 0.00 |
49 | 甲撑二氧苯丙胺 | 0.00 | 50 | 琥珀酸多西拉敏 | 0.00 |
51 | 四氢大麻酚 | 0.00 | 52 | 纳布啡 | 0.00 |
53 | 制霉菌素 | 0.00 | 54 | 去甲吗啡 | 0.00 |
55 | 乙酰吗啡 | 0.00 | 56 | 羟考酮 | 0.00 |
57 | 苄非他明 | 0.00 | 58 | 克他命 | 0.00 |
59 | 异丙嗪 | 0.00 | 60 | 苯海拉明 | 0.00 |
61 | 阿司帕坦 | 0.00 | 62 | 苯丁胺 | 0.00 |
63 | 皮质醇(氢化可的松) | 0.12 | 64 | 香草扁桃酸 | 0.00 |
65 | 雄烯二酮 | 0.21 | 66 | 雄甾酮 | 0.25 |
67 | 皮质脂酮 | 0.05 | 68 | 皮质酮(可的松) | 0.02 |
69 | 去氧皮质酮 | 0.03 | 70 | 脱氢表雄酮 | 0.07 |
71 | 硫酸脱氢表雄酮 | 0.07 | 72 | 二氢睾酮 | 0.19 |
73 | 醛固酮 | 0.03 | 74 | 雌三醇 | 0.36 |
75 | 雌酮 | 0.45 | 76 | 本胆烷醇酮 | 0.18 |
77 | 17-羟孕烯醇酮 | 0.00 | 78 | 17-羟孕酮 | 0.33 |
79 | 孕烯醇酮 | 0.01 | 80 | 孕酮 | 0.39 |
81 | 睾酮 | 0.17 | 82 | 孕三醇 | 0.21 |
83 | 孕二醇 | 0.25 | 84 | 17α-羟基黄体酮 | 0.15 |
85 | 雄烯二酮 | 0.06 | 86 | 17-酮类固醇 | 0.05 |
87 | 17-羟皮质类固醇 | 0.03 | 88 | 肾上腺素 | 0.00 |
89 | 去甲肾上腺素 | 0.00 | 90 | 多巴胺 | 0.00 |
91 | 高香草酸 | 0.00 | 92 | 二羟基杏仁酸 | 0.00 |
测定结果显示:上述62种常见药物与30种常见激素及激素代谢物等价于雌二醇的浓度均小于1.00 pg/ml。由此可见,本发明的抗体是抗雌二醇的特异性抗体,与常见干扰物无交叉反应。
实施例9.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(pg/ml) | 高效液相色谱法测定值(pg/ml) |
1 | 313.44 | 308.92 |
2 | 137.29 | 130.84 |
3 | 590.05 | 605.71 |
4 | 337.92 | 341.82 |
5 | 226.81 | 224.10 |
6 | 128.34 | 135.02 |
7 | 576.38 | 580.97 |
8 | 85.27 | 86.85 |
9 | 667.06 | 680.01 |
10 | 135.13 | 130.44 |
11 | 208.05 | 208.72 |
12 | 443.16 | 459.08 |
13 | 109.94 | 108.25 |
14 | 672.51 | 665.90 |
15 | 443.32 | 427.83 |
16 | 223.50 | 225.07 |
17 | 145.49 | 149.55 |
18 | 97.77 | 100.12 |
19 | 73.65 | 75.26 |
20 | 192.78 | 190.51 |
21 | 520.00 | 539.82 |
22 | 712.62 | 738.50 |
23 | 187.72 | 183.66 |
24 | 370.64 | 383.19 |
25 | 64.51 | 63.35 |
26 | 468.60 | 458.14 |
27 | 264.43 | 269.64 |
28 | 520.82 | 530.00 |
29 | 609.75 | 637.05 |
30 | 336.61 | 341.28 |
31 | 188.05 | 183.77 |
32 | 296.53 | 310.52 |
33 | 223.94 | 227.56 |
34 | 82.70 | 81.56 |
35 | 211.33 | 219.43 |
36 | 476.59 | 491.17 |
37 | 290.08 | 300.08 |
38 | 134.03 | 139.15 |
39 | 792.68 | 800.63 |
40 | 500.31 | 495.70 |
41 | 397.95 | 408.42 |
42 | 259.93 | 267.19 |
43 | 333.80 | 340.56 |
44 | 534.11 | 559.60 |
45 | 78.87 | 79.09 |
46 | 421.40 | 428.92 |
47 | 603.64 | 615.28 |
48 | 321.38 | 320.01 |
49 | 245.93 | 255.68 |
50 | 1003.26 | 984.52 |
51 | 539.77 | 535.24 |
52 | 177.66 | 175.83 |
53 | 348.28 | 333.35 |
54 | 209.23 | 211.77 |
55 | 185.20 | 187.96 |
56 | 371.21 | 382.30 |
57 | 638.27 | 621.96 |
58 | 382.05 | 390.05 |
59 | 729.90 | 746.18 |
60 | 375.88 | 393.02 |
61 | 289.41 | 291.11 |
62 | 199.52 | 200.19 |
63 | 89.00 | 89.34 |
64 | 531.57 | 547.13 |
65 | 383.26 | 386.72 |
66 | 528.22 | 507.58 |
67 | 222.91 | 221.00 |
68 | 730.85 | 746.06 |
69 | 438.97 | 433.12 |
70 | 183.00 | 173.65 |
71 | 229.34 | 231.43 |
72 | 265.87 | 276.37 |
73 | 190.62 | 200.88 |
74 | 538.45 | 550.63 |
75 | 66.10 | 66.69 |
76 | 560.24 | 586.31 |
77 | 288.16 | 277.35 |
78 | 405.69 | 412.79 |
79 | 187.02 | 189.82 |
80 | 92.99 | 97.50 |
81 | 263.38 | 269.21 |
82 | 300.10 | 290.54 |
83 | 106.52 | 109.66 |
84 | 270.00 | 293.18 |
85 | 245.56 | 259.03 |
86 | 427.24 | 438.25 |
87 | 734.33 | 740.99 |
88 | 321.35 | 335.91 |
89 | 150.06 | 153.65 |
90 | 333.52 | 339.02 |
91 | 522.41 | 500.98 |
92 | 62.62 | 64.12 |
93 | 305.81 | 311.70 |
94 | 507.16 | 513.52 |
95 | 325.70 | 308.87 |
96 | 198.65 | 206.09 |
97 | 120.34 | 128.10 |
98 | 85.52 | 89.72 |
99 | 257.20 | 265.99 |
100 | 26.66 | 26.61 |
对上述数据作图,参见图3,得到的线性方程为:y=1.0082x+ 1.2162,相关系数R2=0.9976,表明本发明的检测试剂测定雌二醇临床标本的准确度高。
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。
Claims (10)
1.一种雌二醇衍生物,其结构式如下述式(Ⅰ)所示:
式(Ⅰ)。
2.一种如权利要求1所述的雌二醇衍生物的制备方法,其特征在于,包括以下步骤:
a.化合物3的合成
称取5.0 g的化合物1与10 g的化合物2共同溶解于100 mL的丙酮中,然后加入7.9 g的K2CO3,制成反应混合溶液,然后将此反应混合溶液加热回流12小时;将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到3.1 g白色固体状的化合物3;
b.雌二醇衍生物的合成
称取3.1 g的化合物3,1.13 g的化合物4,3.04 g的三苯基膦,共同溶解于50 mL的四氢呋喃中,然后在0℃下加入2.35 g的偶氮二甲酸二异丙酯,制成反应混合溶液,然后将此反应混合溶液在室温下搅拌12小时;将反应结束后的溶液进行过滤,再将去除溶剂后得到的剩余物通过硅胶纯化柱进行纯化,得到1.1 g白色固体状的雌二醇衍生物。
3.一种雌二醇免疫原,其特征在于,由权利要求1或2所述的雌二醇衍生物与载体连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ);
所述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、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。
4.一种如权利要求3所述的雌二醇免疫原的制备方法,其特征在于,包括以下步骤:
a.称取4.08 g磷酸二氢钾、6.39 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液A;
b.称取4.5 mg蛋白质载体,4℃下溶解于4.5 ml上述缓冲溶液A中,制成载体溶液;
c.称取4.5 mg由权利要求1或2所述的雌二醇衍生物,4℃下溶解于450 μl上述缓冲溶液A中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述载体溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液A进行透析,透析后所得溶液即为雌二醇免疫原溶液,在雌二醇免疫原溶液中加入质量分数0.12%的NaN3,于-20℃下储存。
5.一种抗雌二醇特异性抗体,其特征在于,由权利要求3或4所述的雌二醇免疫原免疫实验动物后产生,所述抗体为完整抗体分子,或者为保留与雌二醇特异性结合能力的抗体片段或抗体衍生物,所述实验动物为兔、山羊、小鼠、绵羊、豚鼠或马中的一种,优选为兔。
6.一种如权利要求5所述的抗雌二醇特异性抗体的制备方法,其特征在于,包括以下步骤:
a.用PBS缓冲液将权利要求3或4所述的雌二醇免疫原稀释至3.0 mg/ml,得到抗原溶液,然后用3.0 ml所述抗原溶液与弗氏完全佐剂混合,对实验动物进行注射;
b. 2周后,再用3.0 ml相同的抗原溶液与弗氏不完全佐剂混合,对上述实验动物注射一次,之后每隔2周注射一次,共计注射6次;
c.对免疫后的实验动物取血,分离纯化得到效价为1:50000~1:80000的抗雌二醇特异性抗体。
7.一种雌二醇酶标偶联物,其特征在于,由权利要求1或2所述的雌二醇衍生物与葡萄糖-6-磷酸脱氢酶连接而成,其结构式如下述式(Ⅲ)所示:
式(Ⅲ)。
8.一种如权利要求7所述的雌二醇酶标偶联物的制备方法,其特征在于,包括以下步骤:
a.称取1.635 g磷酸二氢钾、2.55 g磷酸氢二钠、12.75 g氯化钠、1.425 g氯化镁,共同溶解于1.5 L去离子水中,调节pH至8.2,制成缓冲溶液B;
b.称取4.5 mg葡萄糖-6-磷酸脱氢酶,4℃下溶解于4.5 mL上述缓冲溶液B中,制成葡萄糖-6-磷酸脱氢酶溶液;
c.称取4.5 mg由权利要求1或2所述的雌二醇衍生物,4℃下溶解于450 μl上述缓冲溶液B中,制成雌二醇衍生物溶液;
d.当上述雌二醇衍生物溶液刚变澄清时,将其逐滴加入上述葡萄糖-6-磷酸脱氢酶溶液中,然后将此混合溶液在-2~-8℃下搅拌5小时;
e.将反应后的上述混合溶液用上述缓冲溶液B进行透析,透析后所得溶液即为葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液,在葡萄糖-6-磷酸脱氢酶-半抗原酶标偶联物溶液中加入质量分数0.75%的BSA和质量分数0.12%的NaN3,于2~8℃下储存。
9.一种雌二醇检测试剂,其特征在于,包括试剂A和试剂B,所述试剂A中包含权利要求5或6所述的抗雌二醇特异性抗体和均相酶底物,所述均相酶底物由葡萄糖-6-磷酸、氧化态的烟酰胺腺嘌呤二核苷酸和Tris缓冲液配制而成;所述试剂B包含权利要求7或8所述的雌二醇酶标偶联物和Tris缓冲液;使用时将所述试剂A与试剂B按1:1~4:1的体积比混合,优选按4∶1的体积比使用。
10.一种如权利要求9所述的雌二醇检测试剂的制备方法,其特征在于,包括以下步骤:
a.试剂A的制备:将7.5 g的氧化态的烟酰胺腺嘌呤二核苷酸、3.75 g的葡萄糖-6-磷酸用1.5 L、55 mM、pH=8.0的Tris缓冲液溶解制成均相酶底物;再将权利要求5或6所述的抗雌二醇特异性抗体加入所述均相酶底物中,得到试剂A,所述抗雌二醇特异性抗体与均相酶底物的体积比为1:100~1:10000;优选地,所述抗雌二醇特异性抗体与均相酶底物的体积比为1: 500;
b.试剂B的制备:将权利要求7或8所述的雌二醇酶标偶联物用120mM、pH=8.2的Tris缓冲液溶解,得到试剂B,所述雌二醇酶标偶联物与Tris缓冲液的体积比为1:100~1:10000;优选地,所述雌二醇酶标偶联物与Tris缓冲液的体积比为1: 2000。
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