CN113624982A - 一种睾酮的磁颗粒酶促化学发光免疫检测试剂 - Google Patents
一种睾酮的磁颗粒酶促化学发光免疫检测试剂 Download PDFInfo
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Abstract
本发明免疫分析技术领域,具体涉及一种睾酮的磁颗粒酶促化学发光免疫检测试剂。本发明公开了一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其组成成分:0.01‑0.05%的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液;所述睾酮释放试剂为双氢睾酮、达那唑、肝素钠、8‑苯胺‑1‑萘磺酸(ANS)、8‑苯胺‑1‑萘磺酸盐、三氯乙酸钠、2‑HydroxymethyleneEthisterone(CAS:2787‑02‑2)、炔孕酮(ethisterone,CAS:434‑03‑7)、2‑Hydroxymethyl‑1,2‑dehydroethisterone(CAS:60142‑08‑7)中的一种或多种组合。
Description
技术领域
本发明免疫分析技术领域,具体涉及一种睾酮的磁颗粒酶促化学发光免疫检测试剂。
背景技术
睾酮,又称睾丸素、睾丸酮或睾甾酮,是一种类固醇雌激素,由男性的睾丸或女性的卵巢分泌,肾上腺亦分泌少量睾酮。睾酮可促使副睾、输精管和精囊的生成,促使外生殖器官及第二性征的生长发育,影响精子的生成,不论男性或女性,它对健康都有着重要的影响。对于男性,睾酮水平的增高,常见于肾上腺皮质增生,肾上腺皮质肿瘤,睾丸肿瘤、男性性早熟等病症。而睾酮水平降低常见于隐睾症、无睾症、间质细胞发育不全、先天性曲细精管发育不全等原发性男性功能减退症,以及高泌乳素血症,皮质醇增多症等病症。对于女性,睾酮水平的异常常见于特发性多毛症、多囊卵巢综合征,松果体瘤,卵巢雄性化肿瘤等疾病。准确检测体内睾酮水平有助于这些疾病的辅助诊断和治疗。
血清中,睾酮主要与性激素结合蛋白、白蛋白和皮质醇结合蛋白结合,仅有2%的睾酮为游离状态。血清总睾酮的免疫检测需要将睾酮从性激素结合蛋白、白蛋白以及皮质醇结合蛋白上释放出来,以便于检测抗体识别。因此,检测试剂中的睾酮释放试剂的配方组成关系到睾酮的释放效果,直接影响到血清总睾酮免疫检测试剂的灵敏度和特异性。
发明内容
针对上述背景技术所提出的问题,本发明的目的是:旨在提供一种睾酮的磁颗粒酶促化学发光免疫检测试剂。
为实现上述技术目的,本发明采用的技术方案如下:
一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其组成成分:0.01-0.05%的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液;
所述睾酮释放试剂为双氢睾酮、达那唑、肝素钠、8-苯胺-1-萘磺酸(ANS)、8-苯胺-1-萘磺酸盐、三氯乙酸钠、2-HydroxymethyleneEthisterone(CAS:2787-02-2)、炔孕酮(ethisterone,CAS:434-03-7)、2-Hydroxymethyl-1,2-dehydroethisterone(CAS:60142-08-7)中的一种或多种组合。
作为本发明发一种优选方案,所述睾酮释放试剂中各个组分的浓度为:双氢睾酮的浓度为0.01-0.05mg/mL、达那唑的浓度为0.05-0.5mg/mL、肝素钠的浓度为2-50mg/mL、8-苯胺-1-萘磺酸(ANS)或8-苯胺-1-萘磺酸盐浓度为1-5mg/mL、三氯乙酸钠的浓度为1-25mg/mL,炔孕酮的浓度为0.05-0.25mg/mL、2-HydroxymethyleneEthisterone(CAS:2787-02-2)的浓度为0.01-0.5mg/mL、2-Hydroxymethyl-1,2-dehydroethisterone的浓度为0.01-0.60mg/mL;
所述睾酮释放试剂的配制方法为,
S1:按照组分配比取具有释放睾酮作用的化合物,用DMSO或DMF或纯水溶解,制得浓度为500mg/mL的母液。
S2:通过缓冲液稀释至对应浓度,制得睾酮释放试剂。
3、根据权利要求2所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述S2中,缓冲液为PBS缓冲液,码啉乙磺酸缓冲液(MES),Tris-HCL缓冲液、柠檬酸盐缓冲液、3-(N-吗啉基)-2-羟基丙磺酸(MOPSO)缓冲液,甘氨酸-盐酸缓冲液、乙酸-乙酸钠缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)、TRIS缓冲液中的一种。
作为本发明发一种优选方案,所述包被有睾酮抗原的超顺磁性微粒的制备方法为,
A1:取1mL的商品化的羧基磁珠(10mg/mL)至2.0mL的离心管中,将离心管置于磁分离器上进行磁分离,吸去上清;
A2:往离心管中加入1mL的标记缓冲液(0.1MMES,pH5.0),重悬磁珠,再置于磁分离器上进行磁分离,吸去上清;
A3:往离心管中加入1mL的标记缓冲液,并振荡混匀,从而重悬磁珠;
A4:再往重悬后的磁珠中加入100ul的睾酮抗原(睾酮-BSA耦合物),室温旋转混合30min;
A5:往磁珠及睾酮抗原的混合物中加入100ul的偶联剂EDC(10mg/mL,标记缓冲液配制),振荡混匀,室温旋转混合3h;
A6:反应完成后,将离心管置于磁分离器上进行磁分离,去上清后,加入1ml的磁珠清洗缓冲液(25mMTris-HCL,150mMNaCl,0.05%吐温20,pH7.2),振荡混匀后,再置于磁分离器上进行磁分离,再去上清。磁珠清洗重复3次;
A7:最后一步清洗完成,加入500ul磁珠保存缓冲液(50mMTris,150mMNaCl,1%BSA,0.05%PC300,pH7.2),重悬磁珠后冷藏保存。
作为本发明发一种优选方案,所述的碱性磷酸酶标记睾酮抗体的制备方法为,
B1:取适量的碱性磷酸酶溶于0.8ml纯水中,往碱性磷酸酶中加入0.2mL现配的0.1MNaIO4溶液,室温避光反应20min;
B2:将上述缓冲液加入到10KD超滤离心管中,4度超滤离心,并用pH9.5,0.2M的碳酸盐缓冲液进行缓冲液置换;
B3:往置换缓冲液后的碱性磷酸酶中加入一定量的睾酮抗体,室温避光反应3小时;
B4:加入0.1ml现配的4mg/mL的硼氢化钠溶液,渴匀后4度静置2小时;
B5:反应完成后用50kD超滤离心管进行超滤离心,并用50mMPH7.4的PBS缓冲液进行缓冲液置换,制得碱性磷酶标记睾酮抗体。
作为本发明发一种优选方案,所述的碱性磷酸酶发光底物含有50mMTris、0.5mMAPS-5、5mMMgCl2、2.5mMZnCl2、0.1%TritonX100、0.05%Proclin300,所述碱性磷酸酶发光底物的pH值为9.5±0.2。
采用本发明技术方案,
与现有技术相比,本发明检测试剂中的释放剂作为睾酮酶促化学发光免疫检测试剂的组成部份,能够快速、有效的将睾酮其结合蛋上解离出来,使得睾酮免疫检测试剂的灵敏度和特异性能够获得明显的提高。
附图说明
本发明可以通过附图给出的非限定性实施例进一步说明;
图1为无睾酮释放试剂的参考例的检测结果图;
图2实施例1检测结果图;
图3实施例2检测结果图;
图4实施例3检测结果图;
图5实施例4检测结果图;
图6实施例5检测结果图。
具体实施方式
为了使本领域的技术人员可以更好地理解本发明,下面结合附图和实施例对本发明技术方案进一步说明。
一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其组成成分:0.01-0.05%的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液;
所述睾酮释放试剂为双氢睾酮、达那唑、肝素钠、8-苯胺-1-萘磺酸(ANS)、8-苯胺-1-萘磺酸盐、三氯乙酸钠、2-HydroxymethyleneEthisterone(CAS:2787-02-2)、炔孕酮(ethisterone,CAS:434-03-7)、2-Hydroxymethyl-1,2-dehydroethisterone(CAS:60142-08-7)中的一种或多种组合
其中,包被有睾酮抗原的超顺磁性微粒的制备方法为,
A1:取1mL的商品化的羧基磁珠(10mg/mL)至2.0mL的离心管中,将离心管置于磁分离器上进行磁分离,吸去上清;
A2:往离心管中加入1mL的标记缓冲液(0.1MMES,pH5.0),重悬磁珠,再置于磁分离器上进行磁分离,吸去上清;
A3:往离心管中加入1mL的标记缓冲液,并振荡混匀,从而重悬磁珠;
A4:再往重悬后的磁珠中加入100ul的睾酮抗原(睾酮-BSA耦合物),室温旋转混合30min;
A5:往磁珠及睾酮抗原的混合物中加入100ul的偶联剂EDC(10mg/mL,标记缓冲液配制),振荡混匀,室温旋转混合3h;
A6:反应完成后,将离心管置于磁分离器上进行磁分离,去上清后,加入1ml的磁珠清洗缓冲液(25mMTris-HCL,150mMNaCl,0.05%吐温20,pH7.2),振荡混匀后,再置于磁分离器上进行磁分离,再去上清。磁珠清洗重复3次;
A7:最后一步清洗完成,加入500ul磁珠保存缓冲液(50mMTris,150mMNaCl,1%BSA,0.05%PC300,pH7.2),重悬磁珠后冷藏保存。
碱性磷酸酶标记睾酮抗体的制备方法为,
B1:取适量的碱性磷酸酶溶于0.8ml纯水中,往碱性磷酸酶中加入0.2mL现配的0.1MNaIO4溶液,室温避光反应20min;
B2:将上述缓冲液加入到10KD超滤离心管中,4度超滤离心,并用pH9.5,0.2M的碳酸盐缓冲液进行缓冲液置换;
B3:往置换缓冲液后的碱性磷酸酶中加入一定量的睾酮抗体,室温避光反应3小时;
B4:加入0.1ml现配的4mg/mL的硼氢化钠溶液,渴匀后4度静置2小时;
B5:反应完成后用50kD超滤离心管进行超滤离心,并用50mMPH7.4的PBS缓冲液进行缓冲液置换,制得碱性磷酶标记睾酮抗体。
碱性磷酸酶发光底物含有50mMTris、0.5mMAPS-5、5mMMgCl2、2.5mMZnCl2、0.1%TritonX100、0.05%Proclin300,所述碱性磷酸酶发光底物的pH值为9.5±0.2。
实施例1:一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其组成成分:0.01-0.05%的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液,
其中包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体和碱性磷酸酶发光底物按照上述方法制备;
睾酮释放试剂含0.02mg/mL双氢睾酮,0.1mg/mL达那唑,50mMPBS缓冲液,pH7.0。
实施例2:与实施例1不同之处在于睾酮释放试剂,睾酮释放试剂含3.0mg/mL三氯乙酸钠,0.1mg/mL炔孕酮,100mMTRIS缓冲液,pH7.4。
实施例3:与实施例1不同之处在于睾酮释放试剂,睾酮释放试剂含4.0mg/mL的肝素钠、0.03mg/mL双氢睾酮、2mg/mL的8-苯胺-1-萘磺酸(ANS),60mMTris-HCL缓冲液,调pH为7.2。
实施例4:与实施例1不同之处在于睾酮释放试剂,睾酮释放试剂含3.0mg/mL三氯乙酸钠、0.1mg/mL的2-HydroxymethyleneEthisterone(CAS:2787-02-2)、0.02mg/mL双氢睾酮,50mMMES缓冲液,调pH为6.0。
实施例5:与实施例1不同之处在于睾酮释放试剂,睾酮释放试剂含0.05mg/mL的双氢睾酮、0.1mg/mL的2-HydroxymethyleneEthisterone(CAS:2787-02-2)、2mg/mL的8-苯胺-1-萘磺酸(ANS),50mMHEPES缓冲液,调pH为8.0。
特异性:收取100临床样本,用罗氏睾酮试剂盒进行赋值后,将上述5种不同实施例得到的检测试剂对样本进行测试,测试结果与罗氏试剂测试结果进行比对,见图1-图6;
图1为无睾酮释放试剂的参考例的检测结果图,图2-图6分别为实施例1-实施例5的检测结果图,
图1的实验结果表明,直线方程为y=0.8952x+0.4372,相关系数为R2=0.7812;
图2的实验结果表明,直线方程为y=1.0358x+0.0112,相关系数为R2=0.9743;
图3的实验结果表明,直线方程为y=1.0031x+0.0063,相关系数为R2=0.9759;
图4的实验结果表明,直线方程为y=0.9749x+0.0700,相关系数为R2=0.9783;
图5的实验结果表明,直线方程为y=1.0001x+0.0319,相关系数为R2=0.9766;
图6的实验结果表明,直线方程为y=1.0453x+0.0146,相关系数为R2=0.9803;
通过图1与图2-图6的对比,我们发现,检测试剂能够快速、有效的将睾酮其结合蛋上解离出来,使得睾酮免疫检测试剂的灵敏度和特异性能够获得明显的提高。
上述实施例仅示例性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (6)
1.一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:其组成成分:0.01-0.05%的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液;
所述睾酮释放试剂为双氢睾酮、达那唑、肝素钠、8-苯胺-1-萘磺酸(ANS)、8-苯胺-1-萘磺酸盐、三氯乙酸钠、2-HydroxymethyleneEthisterone(CAS:2787-02-2)、炔孕酮(ethisterone,CAS:434-03-7)、2-Hydroxymethyl-1,2-dehydroethisterone(CAS:60142-08-7)中的一种或多种组合。
2.根据权利要求1所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述睾酮释放试剂中各个组分的浓度为:双氢睾酮的浓度为0.01-0.05mg/mL、达那唑的浓度为0.05-0.5mg/mL、肝素钠的浓度为2-50mg/mL、8-苯胺-1-萘磺酸(ANS)或8-苯胺-1-萘磺酸盐浓度为1-5mg/mL、三氯乙酸钠的浓度为1-25mg/mL,炔孕酮的浓度为0.05-0.25mg/mL、2-HydroxymethyleneEthisterone(CAS:2787-02-2)的浓度为0.01-0.5mg/mL、2-Hydroxymethyl-1,2-dehydroethisterone的浓度为0.01-0.60mg/mL;
所述睾酮释放试剂的配制方法为,
S1:按照组分配比取具有释放睾酮作用的化合物,用DMSO或DMF或纯水溶解,制得浓度为500mg/mL的母液,
S2:通过缓冲液稀释至对应浓度,制得睾酮释放试剂。
3.根据权利要求2所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述S2中,缓冲液为PBS缓冲液,码啉乙磺酸缓冲液(MES),Tris-HCL缓冲液、柠檬酸盐缓冲液、3-(N-吗啉基)-2-羟基丙磺酸(MOPSO)缓冲液,甘氨酸-盐酸缓冲液、乙酸-乙酸钠缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)、TRIS缓冲液中的一种。
4.根据权利要求1所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述包被有睾酮抗原的超顺磁性微粒的制备方法为,
A1:取1mL的商品化的羧基磁珠(10mg/mL)至2.0mL的离心管中,将离心管置于磁分离器上进行磁分离,吸去上清;
A2:往离心管中加入1mL的标记缓冲液(0.1M MES,pH5.0),重悬磁珠,再置于磁分离器上进行磁分离,吸去上清;
A3:往离心管中加入1mL的标记缓冲液,并振荡混匀,从而重悬磁珠;
A4:再往重悬后的磁珠中加入100ul的睾酮抗原(睾酮-BSA耦合物),室温旋转混合30min;
A5:往磁珠及睾酮抗原的混合物中加入100ul的偶联剂EDC(10mg/mL,标记缓冲液配制),振荡混匀,室温旋转混合3h;
A6:反应完成后,将离心管置于磁分离器上进行磁分离,去上清后,加入1ml的磁珠清洗缓冲液(25mM Tris-HCL,150mM NaCl,0.05%吐温20,pH7.2),振荡混匀后,再置于磁分离器上进行磁分离,再去上清。磁珠清洗重复3次;
A7:最后一步清洗完成,加入500ul磁珠保存缓冲液(50mM Tris,150mM NaCl,1%BSA,0.05%PC300,pH7.2),重悬磁珠后冷藏保存。
5.根据权利要求1所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述的碱性磷酸酶标记睾酮抗体的制备方法为,
B1:取适量的碱性磷酸酶溶于0.8ml纯水中,往碱性磷酸酶中加入0.2mL现配的0.1MNaIO4溶液,室温避光反应20min;
B2:将上述缓冲液加入到10KD超滤离心管中,4度超滤离心,并用pH9.5,0.2M的碳酸盐缓冲液进行缓冲液置换;
B3:往置换缓冲液后的碱性磷酸酶中加入一定量的睾酮抗体,室温避光反应3小时;
B4:加入0.1ml现配的4mg/mL的硼氢化钠溶液,渴匀后4度静置2小时;
B5:反应完成后用50kD超滤离心管进行超滤离心,并用50mM PH 7.4的PBS缓冲液进行缓冲液置换,制得碱性磷酶标记睾酮抗体。
6.根据权利要求1所述的一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其特征在于:所述的碱性磷酸酶发光底物含有50mM Tris、0.5mM APS-5、5mM MgCl2、2.5mM ZnCl2、0.1%TritonX100、0.05%Proclin 300,所述碱性磷酸酶发光底物的pH值为9.5±0.2。
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