CN110456087B - 一种舍曲林检测试剂及其制备和使用方法 - Google Patents
一种舍曲林检测试剂及其制备和使用方法 Download PDFInfo
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- CN110456087B CN110456087B CN201910767565.5A CN201910767565A CN110456087B CN 110456087 B CN110456087 B CN 110456087B CN 201910767565 A CN201910767565 A CN 201910767565A CN 110456087 B CN110456087 B CN 110456087B
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- sertraline
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Abstract
本发明涉及一种舍曲林检测试剂及其制备和使用方法。通过反复试验获得了一种全新的舍曲林马来酰亚胺衍生物,并使用该舍曲林马来酰亚胺衍生物制备得到高免疫原性的舍曲林免疫原,进而免疫实验动物得到高效价的抗舍曲林特异性抗体;同时,使用该舍曲林马来酰亚胺衍生物制备得到舍曲林酶标偶联物。含有上述抗舍曲林特异性抗体与舍曲林酶标偶联物的舍曲林检测试剂可以实现在全自动生化分析仪上对舍曲林含量的自动化测定,可以高通量、快速化、精确地测定生物样本中舍曲林的含量,且具有操作简便、灵敏度高、特异性强、结果准确等优点,有效降低舍曲林检测成本,有利于临床广泛推广使用。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种舍曲林检测试剂及其制备和使用方法。
背景技术
舍曲林(Sertraline),其化学结构式如式(Ⅳ)所示:
式(Ⅳ)
舍曲林是一种强效和选择性的神经元5-羟色胺再摄取抑制剂,在临床剂量下,舍曲林阻断人体血小板对5-羟色胺的摄取,对去甲肾上腺素和多巴胺仅有微弱影响。体外研究显示,舍曲林对肾上腺素能受体(α1,α2,β)、胆碱能受体、GABA 受体、多巴胺能受体、组胺受体、5-羟色胺能受体(5HT1A, 5HT1B, 5HT2)或苯二氮卓受体没有明显的亲和力。对上述受体的拮抗作用被认为与其它精神疾病用药的镇静作用、抗胆碱作用和心脏毒性相关。舍曲林对单胺氧化酶没有抑制作用。舍曲林的血浆蛋白结合率为98%,主要通过肝脏代谢。舍曲林用药过量症状包括因5-羟色胺引起的不良反应,如嗜睡、胃肠不适、心动过速、震颤、激动和头晕等,舍曲林没有特效的解毒剂。因此,应对任何服用舍曲林的患者进行药物浓度监测,并对用药过量的患者给予积极治疗。
目前,检测舍曲林的常用方法为气相色谱-质谱法(GC-MS)、高效液相色谱法(HPLC)和酶联免疫分析法(ELISA)等,但是这些方法操作复杂,在临床大规模推广应用方面均具有一定的局限性。目前市场上缺乏稳定性好、灵敏度高、特异性强的舍曲林检测试剂,尤其是质量好的高通量自动化检验试剂。因此,研发一种质量达到临床要求、实用性强、性价比高,可应用于全自动生化分析仪的舍曲林检测试剂已成为国内外体外诊断试剂行业的热点。本发明可以实现在全自动生化分析仪上对舍曲林的高通量、快速化检测,且具有操作简便、灵敏度高、特异性强、结果准确等优点,能有效满足国内日益增长的临床检测需求。
发明内容
本发明要解决的技术问题是:针对现有技术的上述缺陷,提供一种操作简便、灵敏度高、特异性强的舍曲林检测试剂及其制备和使用方法。应用该检测试剂可以实现在全自动生化分析仪上对舍曲林含量的自动化测定,可以高通量、快速化、精确地测定生物样本中舍曲林的含量,且具有操作简便、灵敏度高、特异性强、结果准确等优点,有效降低舍曲林检测成本,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:提供一种舍曲林检测试剂,包括试剂R1和试剂R2,所述试剂R1中包含抗舍曲林特异性抗体和均相酶底物溶液;所述试剂R2包含舍曲林酶标偶联物和R2缓冲液。
本发明所述的舍曲林检测试剂,制备方法包括以下步骤:
A.试剂R1的制备:将质量分数3.5%的氧化态的烟酰胺腺嘌呤二核苷酸、3.5%的葡萄糖-6-磷酸、0.12%的甲基化牛血清白蛋白、0.03%的NaN3的用75 mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗舍曲林特异性抗体加入所述均相酶底物溶液中混匀,得到试剂R1,所述抗舍曲林特异性抗体与均相酶底物溶液的体积比为1:100~1:10000;优选地,所述抗舍曲林特异性抗体与均相酶底物溶液的体积比为1:900。
B.试剂R2的制备:将质量分数0.12%的甲基化牛血清白蛋白、0.03%的NaN3用150mmol/L、pH=8.5的Tris缓冲液溶解制成R2缓冲液,再将舍曲林酶标偶联物加入所述R2缓冲液中混匀,得到试剂R2,所述舍曲林酶标偶联物与R2缓冲液的体积比为1:100~1:10000;优选地,所述舍曲林酶标偶联物与R2缓冲液的体积比为1:3600。
本发明所述的舍曲林均相酶免疫检测试剂,所述的抗舍曲林特异性抗体,由舍曲林免疫原免疫实验动物后产生,所述抗体为完整抗体分子,或者为保留与舍曲林特异性结合能力的抗体片段或抗体衍生物,所述实验动物为山羊、绵羊、小鼠、豚鼠、兔或马中的一种,优选为山羊。
所述抗舍曲林特异性抗体的制备方法,包括以下步骤:
a.用PBS缓冲液将舍曲林免疫原稀释至2.5 mg/ml,得到免疫原溶液,然后用2.5ml所述免疫原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
b.2周后,再用2.5 ml相同的免疫原溶液与等量弗氏不完全佐剂混合,对上述实验动物注射一次,之后每隔4周注射一次,共计注射5次;
c.对免疫后的实验动物取血,分离纯化得到抗舍曲林特异性抗体。
本发明所述的舍曲林均相酶免疫检测试剂,其中,所述舍曲林免疫原由舍曲林马来酰亚胺衍生物与载体连接而成,其结构式如下述式(Ⅰ)所示:
式(Ⅰ);
所述载体为具有免疫原性的甲基化蛋白质或多肽,选自甲基化血清蛋白、甲基化卵清蛋白、甲基化血蓝蛋白或甲基化甲状腺球蛋白中的一种,优选为甲基化血清蛋白,更优选为甲基化牛血清白蛋白。
所述舍曲林免疫原的制备方法,包括以下步骤:
(1)将质量分数1.25%的载体蛋白溶解于0.3 mmol/L,pH=8.0的磷酸缓冲液中,得到载体蛋白溶液;
(2)将质量分数0.8%的舍曲林马来酰亚胺衍生物、7.5%的二甲基甲酰胺、7.5%的乙醇溶解于20 mmol/L,pH=5.2的磷酸钾缓冲液中,再加入质量分数0.8%的1-乙基-3-(-3-二甲氨丙基)碳二亚胺,将上述化学物质在0℃下搅拌反应120分钟;
(3)将步骤(2)中溶解好的溶液缓慢滴加至步骤(1)中的载体蛋白溶液中,并在-8℃下搅拌48小时,得到偶联物溶液,将反应后的偶联物溶液进行透析纯化,纯化后所得溶液即为舍曲林免疫原溶液,在舍曲林免疫原溶液中加入质量分数0.08%的NaN3,于-20℃下储存。
本发明所述的舍曲林均相酶免疫检测试剂,其中,所述的舍曲林酶标偶联物由舍曲林马来酰亚胺衍生物与葡萄糖-6-磷酸脱氢酶连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ)。
所述舍曲林酶标偶联物的制备方法,包括以下步骤:
①葡萄糖-6-磷酸脱氢酶溶液的制备:将质量分数5.0%的规格为300KU的葡萄糖-6-磷酸脱氢酶,室温下溶解于含有0.08 mol/L Tris、6.0 mmol/L MgCl2和3.3 mmol/LNaCl,pH=9.2的溶液中;在此溶液中加入质量分数12.5%的还原态的烟酰胺腺嘌呤二核苷酸、7.5%的葡萄糖-6-磷酸以及1.25%的卡必醇;加热到37℃,再缓慢加入质量分数0.75%的二甲基亚砜,摇匀后静置60秒;
②舍曲林马来酰亚胺衍生物的激活:在无水状态下将质量分数0.8%的舍曲林马来酰亚胺衍生物,溶解于7.5%的二甲基甲酰胺中;将此溶液温度降到-8℃;然后加入2.25%的三丁胺、2.75%的氯甲酸异丁酯、1.75%的碳二亚胺;0.75%的N-羟基硫代琥珀酰亚胺;0℃搅拌120分钟;
③葡萄糖-6-磷酸脱氢酶与舍曲林马来酰亚胺衍生物的连接:将步骤②中激活的温度为0℃的舍曲林马来酰亚胺衍生物溶液逐滴加入到步骤①中溶解的温度为37℃的葡萄糖-6-磷酸脱氢酶溶液中;-4℃搅拌48小时;
④纯化产物:将反应后的连接产物通过G-25葡聚糖凝胶层析柱进行纯化,纯化后所得溶液即为舍曲林酶标偶联物溶液,在舍曲林酶标偶联物溶液中加入质量分数0.8%的BSA和质量分数0.08%的NaN3,于-20℃下储存。
本发明所述的舍曲林均相酶免疫检测试剂,其中,所述的舍曲林马来酰亚胺衍生物,其结构式如下述式(Ⅲ)所示:
式(Ⅲ);
上述式(Ⅲ)所示的舍曲林马来酰亚胺衍生物的合成路线如下:
本发明所述的舍曲林均相酶免疫检测试剂,其中,所述的舍曲林马来酰亚胺衍生物的合成路线,具体制备步骤如下:
(a)化合物2的合成:称取7.8 g化合物1溶解于100 ml三氟乙酸中,然后加入7ml三氟甲磺酸和2.3 g硝酸钾,制成反应混合液,室温下搅拌1.5 小时,在此反应混合液中加入100 ml纯化水,然后用100ml乙酸乙酯萃取2次,将萃取得到的有机物用200ml卤水冲洗,用MgSO4干燥后进行过滤,再通过减压的方法除去溶剂,最后将得到的粗产物经硅胶纯化得到化合物2,
(b)化合物3的合成:将15 g化合物2溶解于300 mL乙醇中,然后加入21 g铁粉混合,将以上混合物回流加热16小时,而后将反应物冷却并进行过滤,将收集到的滤液浓缩得到粗品,再用SiO2快速色谱分析柱对粗品进行纯化,得到化合物3,
(c)化合物4的合成:将13.7 g化合物3加入26 mL甲酸中溶解,然后在0℃下加入由25 mL甲酸和49 mL乙酸酐配制的溶液,将此混合物在室温下搅拌2小时,再加入冰水,并用二氯甲烷进行萃取,萃取得到的有机物用1mol/L的氢氧化钠水溶液冲洗,再用卤水洗涤,用Na2SO4进行干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0-95:5的二氯甲烷:甲醇溶处理,得到化合物4,
(d)化合物5的合成:将10 g化合物4加入285 ml丙酮中溶解,然后加入57 ml浓硫酸和285 ml纯化水组成的混合物,并放入冰水浴中,再加入2.2 g亚硝酸钠,将此混合物在冰水浴中搅拌0.5小时,然后加热至室温,将上述混合物在75℃下加热反应2小时,在冰水浴中冷却,再用高浓度的氢氧化铵水溶液终止反应,将反应混合物用乙酸乙酯萃取3次,萃取得到的有机物用纯化水冲洗,用Na2SO4干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0的二氯甲烷:甲醇到95:5的二氯甲烷:甲醇处理,得到化合物5,
(e)化合物6的合成:将3.5 g化合物5加入50 ml二甲基甲酰胺中溶解,然后在0℃下加入1.8 g 4-溴丁酸甲酯与2.8 g K2CO3,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到化合物6,
(f)舍曲林酸衍生物的合成:将2.6 g化合物6加入50 ml MeOH中溶解,然后加入0.25 g LiOH,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到舍曲林酸衍生物,
(g)舍曲林马来酰亚胺衍生物的合成:将0.8 g舍曲林酸衍生物加入5 ml二甲基甲酰胺中溶解,然后的加入1 ml 二异丙基乙胺,0.9 g HATU和0.3 g化合物7,将此反应混合物在室温下搅拌12小时,反应结束后在混合物中加入20 mL纯化水,然后进行过滤,滤渣采用制备级高效液相色谱进行纯化,得到舍曲林马来酰亚胺衍生物,
本发明还提供一种如上所述的舍曲林检测试剂的使用方法,包括以下步骤:
(一)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;
(二)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舍曲林的含量;
所述试剂R1与试剂R2按1:1~4:1的体积比使用,优选按4∶1的体积比使用。
本发明所述的舍曲林检测试剂的使用方法,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。
本发明的有益效果是:通过反复的试验获得了一种全新的舍曲林马来酰亚胺衍生物,并使用该舍曲林马来酰亚胺衍生物制备得到高免疫原性的舍曲林免疫原,进而免疫实验动物得到高效价的抗舍曲林特异性抗体;同时,使用该舍曲林马来酰亚胺衍生物制备得到舍曲林酶标偶联物。含有上述抗舍曲林特异性抗体与舍曲林酶标偶联物的舍曲林检测试剂可以实现在全自动生化分析仪上对舍曲林高通量、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,还能有效降低舍曲林检测成本,有利于临床推广使用。
附图说明
图1是实施例6舍曲林均相酶免疫检测方法的标准曲线;
图2是实施例8舍曲林临床标本均相酶免疫检测与高效液相色谱检测对比数据的散点图。
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.舍曲林马来酰亚胺衍生物的合成
舍曲林马来酰亚胺衍生物的化学结构式如式(Ⅲ)所示:
式(Ⅲ)。
上述舍曲林马来酰亚胺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2的合成:称取7.8 g化合物1溶解于100 ml三氟乙酸中,然后加入7ml三氟甲磺酸和2.3 g硝酸钾,制成反应混合液,室温下搅拌1.5 小时,在此反应混合液中加入100 ml纯化水,然后用100ml乙酸乙酯萃取2次,将萃取得到的有机物用200ml卤水冲洗,用MgSO4干燥后进行过滤,再通过减压的方法除去溶剂,最后将得到的粗产物经硅胶纯化得到3.8 g化合物2,产率44%,
(b)化合物3的合成:将15 g化合物2溶解于300 mL乙醇中,然后加入21 g铁粉混合,将以上混合物回流加热16小时,而后将反应物冷却并进行过滤,将收集到的滤液浓缩得到粗品,再用SiO2快速色谱分析柱对粗品进行纯化,得到9 g化合物3,产率67%,
(c)化合物4的合成:将13.7 g化合物3加入26 mL甲酸中溶解,然后在0℃下加入由25 mL甲酸和49 mL乙酸酐配制的溶液,将此混合物在室温下搅拌2小时,再加入冰水,并用二氯甲烷进行萃取,萃取得到的有机物用1mol/L的氢氧化钠水溶液冲洗,再用卤水洗涤,用Na2SO4进行干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0-95:5的二氯甲烷:甲醇溶处理,得到11g化合物4,产率74%,
(d)化合物5的合成:将10 g化合物4加入285 ml丙酮中溶解,然后加入57 ml浓硫酸和285 ml纯化水组成的混合物,并放入冰水浴中,再加入2.2 g亚硝酸钠,将此混合物在冰水浴中搅拌0.5小时,然后加热至室温,将上述混合物在75℃下加热反应2小时,在冰水浴中冷却,再用高浓度的氢氧化铵水溶液终止反应,将反应混合物用乙酸乙酯萃取3次,萃取得到的有机物用纯化水冲洗,用Na2SO4干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0的二氯甲烷:甲醇到95:5的二氯甲烷:甲醇处理,得到6.6 g化合物5,产率66%,
(e)化合物6的合成:将3.5 g化合物5加入50 ml二甲基甲酰胺中溶解,然后在0℃下加入1.8 g 4-溴丁酸甲酯与2.8 g K2CO3,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到2.9 g化合物6,产率64%,
(f)舍曲林酸衍生物的合成:将2.6 g化合物6加入50 ml MeOH中溶解,然后加入0.25 g LiOH,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到1.9 g舍曲林酸衍生物,产率80%,
(g)舍曲林马来酰亚胺衍生物的合成:将0.8 g舍曲林酸衍生物加入5 ml二甲基甲酰胺中溶解,然后的加入1 ml 二异丙基乙胺,0.9 g HATU和0.3 g化合物7,将此反应混合物在室温下搅拌12小时,反应结束后在混合物中加入20 mL纯化水,然后进行过滤,滤渣采用制备级高效液相色谱进行纯化,得到0.6 g舍曲林马来酰亚胺衍生物,为白色固体,产率55%,
对上述合成步骤得到的舍曲林马来酰亚胺衍生物利用VARIAN MERCURY plus400MHz进行1H核磁共振光谱扫描,采用TMS作为内标进行结构鉴定,并用LC-MS方法检测最终产物纯度,结果显示:纯度>95%。
实施例2.舍曲林免疫原的制备
本实施例中的舍曲林免疫原由式(Ⅲ)所示的舍曲林马来酰亚胺衍生物与甲基化牛血清白蛋白(MBSA)连接而成,其结构式如下述式(Ⅴ)所示:
式(Ⅴ)。
该舍曲林免疫原的合成方法具体步骤如下:
(1)将质量分数1.25%的MBSA溶解于0.3 mmol/L,pH=8.0的磷酸缓冲液中,得到MBSA溶液;
(2)将质量分数0.8%的舍曲林马来酰亚胺衍生物、7.5%的二甲基甲酰胺、7.5%的乙醇溶解于20 mmol/L,pH=5.2的磷酸钾缓冲液中,再加入质量分数0.8%的1-乙基-3-(-3-二甲氨丙基)碳二亚胺,将上述化学物质在0℃下搅拌反应120分钟;
(3)将步骤(2)中溶解好的溶液缓慢滴加至步骤(1)中的MBSA溶液中,并在-8℃下搅拌48小时,得到偶联物溶液,将反应后的偶联物溶液进行透析纯化,纯化后所得溶液即为舍曲林免疫原溶液,在舍曲林免疫原溶液中加入质量分数0.08%的NaN3,于-20℃下储存。
实施例3.抗舍曲林特异性抗体的制备
将实施例2制备得到的舍曲林免疫原采用常规方法接种实验动物山羊,加强免疫后取抗血清,具体步骤如下:
a.用PBS缓冲液将舍曲林免疫原稀释至2.5 mg/ml,得到免疫原溶液,然后用2.5ml所述免疫原溶液与等量弗氏完全佐剂混合,对实验动物山羊进行注射;
b. 2周后,再用2.5 ml相同的免疫原溶液与等量弗氏不完全佐剂混合,对上述实验动物山羊注射一次,之后每隔4周注射一次,共计注射5次;
c.对免疫后的实验动物山羊取血,分离纯化得到抗舍曲林特异性抗体,测定其效价为1∶12500。
实施例4.舍曲林酶标偶联物的制备
本实施例中的舍曲林酶标偶联物由式(Ⅲ)所示的舍曲林马来酰亚胺衍生物与葡萄糖-6-磷酸脱氢酶(G6PDH)连接而成,其结构式如下述式(Ⅱ)所示:
式(Ⅱ);
该舍曲林酶标偶联物的合成方法具体步骤如下:
①葡萄糖-6-磷酸脱氢酶溶液的制备:将质量分数5.0%的规格为300KU的葡萄糖-6-磷酸脱氢酶,室温下溶解于含有0.08 mol/L Tris、6.0 mmol/L MgCl2和3.3 mmol/LNaCl,pH=9.2的溶液中;在此溶液中加入质量分数12.5%的还原态的烟酰胺腺嘌呤二核苷酸、7.5%的葡萄糖-6-磷酸以及1.25%的卡必醇;加热到37℃,再缓慢加入质量分数0.75%的二甲基亚砜,摇匀后静置60秒;
②舍曲林马来酰亚胺衍生物的激活:在无水状态下将质量分数0.8%的舍曲林马来酰亚胺衍生物,溶解于7.5%的二甲基甲酰胺中;将此溶液温度降到-8℃;然后加入2.25%的三丁胺、2.75%的氯甲酸异丁酯、1.75%的碳二亚胺;0.75%的N-羟基硫代琥珀酰亚胺;0℃搅拌120分钟;
③葡萄糖-6-磷酸脱氢酶与舍曲林马来酰亚胺衍生物的连接:将步骤②中激活的温度为0℃的舍曲林马来酰亚胺衍生物溶液逐滴加入到步骤①中溶解的温度为37℃的葡萄糖-6-磷酸脱氢酶溶液中;-4℃搅拌48小时;
④纯化产物:将反应后的连接产物通过G-25葡聚糖凝胶层析柱进行纯化,纯化后所得溶液即为舍曲林酶标偶联物溶液,在舍曲林酶标偶联物溶液中加入质量分数0.8%的BSA和质量分数0.08%的NaN3,于-20℃下储存。
实施例5.舍曲林均相酶免疫检测试剂的制备
A.试剂R1的制备:将质量分数3.5%的氧化态的烟酰胺腺嘌呤二核苷酸、3.5%的葡萄糖-6-磷酸、0.12%的甲基化牛血清白蛋白、0.03%的 NaN3的用75 mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗舍曲林特异性抗体加入所述均相酶底物溶液中混匀,得到试剂R1,所述抗舍曲林特异性抗体与均相酶底物溶液的体积比为1: 900;
B.试剂R2的制备:将质量分数0.12%的甲基化牛血清白蛋白、0.03%的NaN3用150mmol/L、pH=8.5的Tris缓冲液溶解制成R2缓冲液,再将舍曲林酶标偶联物加入所述R2缓冲液中混匀,得到试剂R2,所述舍曲林酶标偶联物与R2缓冲液的体积比为1: 3600。
实施例6. 舍曲林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(表1)。
(2)操作步骤为:先加试剂R1,再加入标准品,最后加入试剂R2。加入试剂R2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R1和试剂R2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1 迈瑞BS480全自动生化分析仪反应参数
2. 样本检测:通过本发明的舍曲林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:将舍曲林标准品溶解于空白人造血浆中,至浓度分别为3.00,15.00,30.00 ng/ml。检测结果及数据分析见表2。
表2 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) | 3.00 | 15.00 | 30.00 |
1 | 3.14 | 15.47 | 31.19 |
2 | 3.26 | 15.03 | 30.31 |
3 | 2.98 | 15.54 | 30.06 |
4 | 3.15 | 15.28 | 29.87 |
5 | 3.09 | 14.67 | 29.98 |
6 | 3.15 | 15.19 | 30.99 |
7 | 2.97 | 15.32 | 29.45 |
8 | 3.02 | 14.75 | 30.61 |
9 | 3.08 | 15.77 | 30.53 |
10 | 3.21 | 15.50 | 30.24 |
平均值(ng/ml) | 3.11 | 15.25 | 30.32 |
标准差(SD) | 0.95 | 3.51 | 5.24 |
精密度(CV%) | 3.05 | 2.30 | 1.73 |
回收率(%) | 103.67 | 101.67 | 101.07 |
检测结果:本发明的舍曲林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7: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作为干扰物,进行干扰试验,调整浓度至100.0 ng/mL,采用实施例5的舍曲林均相酶免疫检测试剂进行测定:将待测干扰物与实施例5制备的试剂R1接触反应,再加入试剂R2;检测上述混合溶液的OD340吸光值,根据图1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详见表3。
表3常见干扰物测定结果
ID# | 化合物名称 | 等价于舍曲林的浓度(ng/mL) | ID# | 化合物名称 | 等价于舍曲林的浓度 (ng/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 |
23 | 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) | 0.00 | 64 | 醛固酮 | 0.00 |
65 | 雄烯二酮 | 0.00 | 66 | 雄甾酮 | 0.00 |
67 | 皮质脂酮 | 0.00 | 68 | 皮质酮 (可的松) | 0.00 |
69 | 去氧皮质酮 | 0.00 | 70 | 脱氢表雄酮 | 0.00 |
71 | 硫酸脱氢表雄酮 | 0.00 | 72 | 二氢睾酮 | 0.00 |
73 | 雌二醇 | 0.00 | 74 | 雌三醇 | 0.00 |
75 | 雌酮 | 0.00 | 76 | 本胆烷醇酮 | 0.00 |
77 | 17-羟孕烯醇酮 | 0.00 | 78 | 17-羟孕酮 | 0.00 |
79 | 孕烯醇酮 | 0.00 | 80 | 孕酮 | 0.00 |
81 | 睾酮 | 0.00 | 82 | 孕三醇 | 0.00 |
83 | 孕二醇 | 0.00 | 84 | 17α-羟基黄体酮 | 0.00 |
85 | 雄烯二酮 | 0.00 | 86 | 17-酮类固醇 | 0.00 |
87 | 17-羟皮质类固醇 | 0.00 | 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舍曲林的浓度均小于1.00 ng/mL。由此可见,本发明的抗体是抗舍曲林的特异性抗体,与常见干扰物无交叉反应。
实施例8:相关性分析
对100例临床标本分别使用高效液相色谱法和实施例5的舍曲林均相酶免疫检测试剂进行测定,并作相关性分析,测定的数据详见表4。
表4 临床标本测定结果对比数据
样本号 | 均相酶免疫检测试剂测定值(ng/mL) | 高效液相色谱法测定值(ng/mL) |
1 | 14.11 | 14.32 |
2 | 15.98 | 16.09 |
3 | 5.49 | 5.32 |
4 | 2.32 | 2.38 |
5 | 13.23 | 13.35 |
6 | 11.02 | 11.06 |
7 | 14.85 | 14.66 |
8 | 17.85 | 17.94 |
9 | 7.13 | 7.24 |
10 | 2.04 | 2.05 |
11 | 5.95 | 6.08 |
12 | 5.10 | 5.23 |
13 | 10.03 | 10.17 |
14 | 2.25 | 2.36 |
15 | 3.24 | 3.36 |
16 | 15.3 | 15.26 |
17 | 11.81 | 11.98 |
18 | 14.58 | 14.69 |
19 | 14.54 | 14.33 |
20 | 3.98 | 4.03 |
21 | 15.01 | 15.21 |
22 | 18.17 | 18.25 |
23 | 16.05 | 16.22 |
24 | 14.26 | 14.25 |
25 | 17.21 | 17.55 |
26 | 0.36 | 0.33 |
27 | 4.98 | 5.03 |
28 | 1.99 | 2.02 |
29 | 16.01 | 16.23 |
30 | 15.05 | 15.25 |
31 | 17.85 | 17.89 |
32 | 17.15 | 17.23 |
33 | 11.41 | 11.22 |
34 | 10.59 | 10.55 |
35 | 18.03 | 18.25 |
36 | 13.96 | 14.06 |
37 | 18.56 | 18.69 |
38 | 17.15 | 17.22 |
39 | 16.04 | 16.23 |
40 | 13.09 | 13.12 |
41 | 2.58 | 2.56 |
42 | 5.87 | 6.01 |
43 | 1.15 | 1.18 |
44 | 1.20 | 1.23 |
45 | 5.19 | 5.23 |
46 | 2.35 | 2.36 |
47 | 5.21 | 5.23 |
48 | 18.59 | 18.79 |
49 | 5.31 | 5.22 |
50 | 17.03 | 17.25 |
51 | 9.56 | 9.69 |
52 | 5.02 | 5.02 |
53 | 12.69 | 12.87 |
54 | 14.16 | 14.35 |
55 | 17.01 | 17.22 |
56 | 2.06 | 2.03 |
57 | 3.99 | 4.03 |
58 | 1.18 | 1.22 |
59 | 11.35 | 11.96 |
60 | 10.02 | 10.39 |
61 | 13.88 | 14.21 |
62 | 11.23 | 11.56 |
63 | 2.01 | 2.03 |
64 | 15.21 | 15.36 |
65 | 8.27 | 8.03 |
66 | 18.05 | 17.96 |
67 | 12.49 | 12.33 |
68 | 16.34 | 16.13 |
69 | 4.03 | 4.28 |
70 | 5.88 | 6.01 |
71 | 2.26 | 2.23 |
72 | 4.52 | 4.47 |
73 | 15.69 | 15.38 |
74 | 17.15 | 17.29 |
75 | 14.41 | 14.35 |
76 | 10.96 | 10.99 |
77 | 16.23 | 16.33 |
78 | 15.86 | 15.79 |
79 | 3.41 | 3.22 |
80 | 4.21 | 4.05 |
81 | 5.46 | 5.55 |
82 | 17.91 | 17.58 |
83 | 10.76 | 10.99 |
84 | 17.89 | 17.68 |
85 | 2.42 | 2.35 |
86 | 4.02 | 4.22 |
87 | 8.79 | 8.99 |
88 | 15.48 | 15.23 |
89 | 4.42 | 4.39 |
90 | 8.49 | 8.56 |
91 | 7.36 | 7.22 |
92 | 5.16 | 5.23 |
93 | 8.67 | 8.97 |
94 | 1.96 | 1.88 |
95 | 12.34 | 12.58 |
96 | 2.45 | 2.55 |
97 | 6.09 | 6.34 |
98 | 15.62 | 15.79 |
99 | 17.52 | 17.69 |
100 | 2.38 | 2.33 |
对上述数据作图,参见图2,得到的线性方程为:y = 1.0034x + 0.0271,相关系数R2 = 0.9993,表明本发明的舍曲林检测试剂测定舍曲林临床标本的准确度高,与高效液相色谱法的一致性较好。
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 (12)
1.一种舍曲林检测试剂,其特征在于,包括试剂R1和试剂R2,所述试剂R1中包含抗舍曲林特异性抗体和均相酶底物溶液;所述试剂R2包含舍曲林酶标偶联物和R2缓冲液;
所述的舍曲林检测试剂,制备方法包括以下步骤:
A.试剂R1的制备:将质量分数3.5%的氧化态的烟酰胺腺嘌呤二核苷酸、3.5%的葡萄糖-6-磷酸、0.12%的甲基化牛血清白蛋白、0.03%的NaN3用75mmol/L、pH=7.8的Tris缓冲液溶解制成均相酶底物溶液;再将抗舍曲林特异性抗体加入所述均相酶底物溶液中混匀,得到试剂R1,所述抗舍曲林特异性抗体与均相酶底物溶液的体积比为1:100~1:10000;
B.试剂R2的制备:将质量分数0.12%的甲基化牛血清白蛋白、0.03%的NaN3用150mmol/L、pH=8.5的Tris缓冲液溶解制成R2缓冲液,再将舍曲林酶标偶联物加入所述R2缓冲液中混匀,得到试剂R2,所述舍曲林酶标偶联物与R2缓冲液的体积比为1:100~1:10000;
所述的抗舍曲林特异性抗体,由舍曲林免疫原免疫实验动物后产生,所述抗体为完整抗体分子,或者为保留与舍曲林特异性结合能力的抗体片段或抗体衍生物,所述实验动物为山羊、绵羊、小鼠、豚鼠、兔或马中的一种;
所述抗舍曲林特异性抗体的制备方法,包括以下步骤:
a.用PBS缓冲液将舍曲林免疫原稀释至2.5mg/ml,得到免疫原溶液,然后用2.5ml所述免疫原溶液与等量弗氏完全佐剂混合,对实验动物进行注射;
b.2周后,再用2.5ml相同的免疫原溶液与等量弗氏不完全佐剂混合,对上述实验动物注射一次,之后每隔4周注射一次,共计注射5次;
c.对免疫后的实验动物取血,分离纯化得到抗舍曲林特异性抗体;
所述舍曲林免疫原由舍曲林马来酰亚胺衍生物与载体连接而成,其结构式如下述式(Ⅰ)所示:
所述载体为具有免疫原性的甲基化蛋白质或多肽,选自甲基化血清蛋白、甲基化卵清蛋白、甲基化血蓝蛋白或甲基化甲状腺球蛋白中的一种;
所述舍曲林免疫原的制备方法,包括以下步骤:
(1)将质量分数1.25%的载体蛋白溶解于0.3mmol/L,pH=8.0的磷酸缓冲液中,得到载体蛋白溶液;
(2)将质量分数0.8%的舍曲林马来酰亚胺衍生物、7.5%的二甲基甲酰胺、7.5%的乙醇溶解于20mmol/L,pH=5.2的磷酸钾缓冲液中,再加入质量分数0.8%的1-乙基-3-(3-二甲氨丙基)碳二亚胺,将上述化学物质在0℃下搅拌反应120分钟;
(3)将步骤(2)中溶解好的溶液缓慢滴加至步骤(1)中的载体蛋白溶液中,并在-8℃下搅拌48小时,得到偶联物溶液,将反应后的偶联物溶液进行透析纯化,纯化后所得溶液即为舍曲林免疫原溶液,在舍曲林免疫原溶液中加入质量分数0.08%的NaN3,于-20℃下储存;
所述的舍曲林酶标偶联物由舍曲林马来酰亚胺衍生物与葡萄糖-6-磷酸脱氢酶连接而成,其结构式如下述式(Ⅱ)所示:
所述舍曲林酶标偶联物的制备方法,包括以下步骤:
①葡萄糖-6-磷酸脱氢酶溶液的制备:将质量分数5.0%的规格为300KU的葡萄糖-6-磷酸脱氢酶,室温下溶解于含有0.08mol/L Tris、6.0mmol/L MgCl2和3.3mmol/L NaCl,pH=9.2的溶液中;在此溶液中加入质量分数12.5%的还原态的烟酰胺腺嘌呤二核苷酸、7.5%的葡萄糖-6-磷酸以及1.25%的卡必醇;加热到37℃,再缓慢加入质量分数0.75%的二甲基亚砜,摇匀后静置60秒;
②舍曲林马来酰亚胺衍生物的激活:在无水状态下将质量分数0.8%的舍曲林马来酰亚胺衍生物,溶解于7.5%的二甲基甲酰胺中;将此溶液温度降到-8℃;然后加入2.25%的三丁胺、2.75%的氯甲酸异丁酯、1.75%的碳二亚胺;0.75%的N-羟基硫代琥珀酰亚胺;0℃搅拌120分钟;
③葡萄糖-6-磷酸脱氢酶与舍曲林马来酰亚胺衍生物的连接:将步骤②中激活的温度为0℃的舍曲林马来酰亚胺衍生物溶液逐滴加入到步骤①中溶解的温度为37℃的葡萄糖-6-磷酸脱氢酶溶液中;-4℃搅拌48小时;
④纯化产物:将反应后的连接产物通过G-25葡聚糖凝胶层析柱进行纯化,纯化后所得溶液即为舍曲林酶标偶联物溶液,在舍曲林酶标偶联物溶液中加入质量分数0.8%的BSA和质量分数0.08%的NaN3,于-20℃下储存;
所述的舍曲林马来酰亚胺衍生物,其结构式如下述式(Ⅲ)所示:
2.根据权利要求1所述的舍曲林检测试剂,其特征在于,所述抗舍曲林特异性抗体与均相酶底物溶液的体积比为1:900;所述舍曲林酶标偶联物与R2缓冲液的体积比为1:3600。
3.根据权利要求1所述的舍曲林检测试剂,其特征在于,所述实验动物为山羊。
4.根据权利要求1所述的舍曲林检测试剂,其特征在于,所述载体为甲基化血清蛋白。
5.根据权利要求4所述的舍曲林检测试剂,其特征在于,所述甲基化血清蛋白为甲基化牛血清白蛋白。
7.根据权利要求6所述的舍曲林检测试剂,其特征在于,所述的舍曲林马来酰亚胺衍生物的合成路线,具体制备步骤如下:
(a)化合物2的合成:称取7.8g化合物1溶解于100ml三氟乙酸中,然后加入7ml三氟甲磺酸和2.3g硝酸钾,制成反应混合液,室温下搅拌1.5小时,在此反应混合液中加入100ml纯化水,然后用100ml乙酸乙酯萃取2次,将萃取得到的有机物用200ml卤水冲洗,用MgSO4干燥后进行过滤,再通过减压的方法除去溶剂,最后将得到的粗产物经硅胶纯化得到化合物2,
(b)化合物3的合成:将15g化合物2溶解于300mL乙醇中,然后加入21g铁粉混合,将以上混合物回流加热16小时,而后将反应物冷却并进行过滤,将收集到的滤液浓缩得到粗品,再用SiO2快速色谱分析柱对粗品进行纯化,得到化合物3,
(c)化合物4的合成:将13.7g化合物3加入26mL甲酸中溶解,然后在0℃下加入由25mL甲酸和49mL乙酸酐配制的溶液,将此混合物在室温下搅拌2小时,再加入冰水,并用二氯甲烷进行萃取,萃取得到的有机物用1mol/L的氢氧化钠水溶液冲洗,再用卤水洗涤,用Na2SO4进行干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0-95:5的二氯甲烷:甲醇溶处理,得到化合物4,
(d)化合物5的合成:将10g化合物4加入285ml丙酮中溶解,然后加入57ml浓硫酸和285ml纯化水组成的混合物,并放入冰水浴中,再加入2.2g亚硝酸钠,将此混合物在冰水浴中搅拌0.5小时,然后加热至室温,将上述混合物在75℃下加热反应2小时,在冰水浴中冷却,再用高浓度的氢氧化铵水溶液终止反应,将反应混合物用乙酸乙酯萃取3次,萃取得到的有机物用纯化水冲洗,用Na2SO4干燥,过滤后减压除去溶剂,粗产物用硅胶进行纯化,最后用溶剂浓度梯度为100:0的二氯甲烷:甲醇到95:5的二氯甲烷:甲醇处理,得到化合物5,
(e)化合物6的合成:将3.5g化合物5加入50ml二甲基甲酰胺中溶解,然后在0℃下加入1.8g 4-溴丁酸甲酯与2.8g K2CO3,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到化合物6,
(f)舍曲林酸衍生物的合成:将2.6g化合物6加入50mlMeOH中溶解,然后加入0.25gLiOH,再将此混合物在室温下搅拌12小时,然后通过减压蒸发溶剂,最后用SiO2快速色谱分析柱纯化得到舍曲林酸衍生物,
(g)舍曲林马来酰亚胺衍生物的合成:将0.8g舍曲林酸衍生物加入5ml二甲基甲酰胺中溶解,然后加入1ml二异丙基乙胺,0.9gHATU和0.3g化合物7,将此反应混合物在室温下搅拌12小时,反应结束后在混合物中加入20mL纯化水,然后进行过滤,滤渣采用制备级高效液相色谱进行纯化,得到舍曲林马来酰亚胺衍生物,
8.一种如权利要求1所述的舍曲林检测试剂的使用方法,其特征在于,包括以下步骤:
(一)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;
(二)加入R2试剂,混匀,37℃恒温5-10分钟后,340nm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舍曲林的含量;
所述试剂R1与试剂R2按1:1~4:1的体积比使用。
9.根据权利要求8所述的舍曲林检测试剂的使用方法,其特征在于,所述试剂R1与试剂R2按4∶1的体积比使用。
10.根据权利要求8所述的舍曲林检测试剂的使用方法,其特征在于,所述的待测样本为生理样本。
11.根据权利要求10所述的舍曲林检测试剂的使用方法,其特征在于,所述的生理样本为血清、血浆、尿液、唾液。
12.根据权利要求11所述的舍曲林检测试剂的使用方法,其特征在于,所述的生理样本为血清或血浆。
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