CN105175531A - 羟脯氨酸免疫原、特异性抗体、检测试剂及其制备方法 - Google Patents
羟脯氨酸免疫原、特异性抗体、检测试剂及其制备方法 Download PDFInfo
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- CN105175531A CN105175531A CN201510496894.2A CN201510496894A CN105175531A CN 105175531 A CN105175531 A CN 105175531A CN 201510496894 A CN201510496894 A CN 201510496894A CN 105175531 A CN105175531 A CN 105175531A
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- oxyproline
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Abstract
本发明公开了一种羟脯氨酸免疫原及其合成方法,以及由该免疫原获得的抗羟脯氨酸抗体、检测试剂及其制备方法。本发明制备的羟脯氨酸免疫原,免疫原性高,可以诱导得到高效价的抗羟脯氨酸特异性抗体,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羟脯氨酸检测试剂,可以精确快速地确定尿液等生物样品中的羟脯氨酸含量。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,还能有效降低羟脯氨酸检测成本,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,涉及羟脯氨酸免疫原、抗羟脯氨酸特异性抗体和羟脯氨酸检测试剂。
背景技术
羟脯氨酸(HydroProline),其结构式如式(Ⅲ)所示:
式(Ⅲ)
羟脯氨酸是胶原蛋白所特有的组成成分,占其氨基酸总量的13%,与胶原的螺旋结构形成并能承受巨大压力有关。羟脯氨酸经脯氨酸羟化酶和羟脯氨酸脱氢酶在肝脏中降解,未降解的羟脯氨酸由肾脏清除。羟脯氨酸大约有一半由尿排出体外。羟脯氨酸具有多种重要的生理功能及独特的生物活性,尿液中羟脯氨酸的浓度可代表骨吸收水平,严重骨折、烧伤、重症肺结核和肝硬化、Hodgkin’s病、甲状腺功能亢进、羟脯氨酸血症,氟骨症都会导致尿液中羟脯氨酸排泄量的增加,因而测定尿液中羟脯氨酸含量可反应胶原蛋白的代谢情况及某些疾病对结缔组织尤其是骨组织的损害程度,具有重要的临床参考价值。此外,羟脯氨酸可作为肺纤维化的特异性指标以及人体衰老检测的参考指标等。
目前,羟脯氨酸测定方法主要有:比色法、高效液相色谱法、氨基酸分析仪法、电泳法等。但这些方法检测灵敏度相对较低,步骤繁琐,成本较高,不适合临床大量尿液样本的检测。目前市场上缺乏稳定性好、灵敏度高、特异性强的羟脯氨酸检测试剂,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羟脯氨酸检测试剂势在必行。
发明内容
本发明为了克服现有技术存在的缺陷,采用全新的羟脯氨酸衍生物制备免疫原性强的羟脯氨酸免疫原及其抗体,用该抗体制备的羟脯氨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羟脯氨酸高通量、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,还能有效降低羟脯氨酸检测成本,有利于临床推广使用。
本发明的一个目的在于提供一种羟脯氨酸衍生物。
本发明的另一个目的在于提供一种免疫原性强的羟脯氨酸免疫原。
本发明的另一个目的在于提供一种羟脯氨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羟脯氨酸免疫原制备得到的特异性强的抗羟脯氨酸特异性抗体。
本发明的再一个目的在于提供一种羟脯氨酸检测试剂。
免疫原性与所合成的羟脯氨酸衍生物分子结构及所选载体种类有关,现有技术中羟脯氨酸免疫原的免疫原性较弱,所得到抗体的特异性、与羟脯氨酸的结合力,敏感度都不如本发明。本发明的羟脯氨酸免疫原,免疫原性高,可以诱导得到高效价的抗羟脯氨酸特异性抗体。该抗体特异性高,与羟脯氨酸的结合力强。由该抗体制备得到的羟脯氨酸检测试剂,可以快速、准确地确定样品中的羟脯氨酸含量。本发明是通过以下技术方案实现的:
一种羟脯氨酸免疫原,其结构式如式(Ⅰ)所示:
式(Ⅰ)
式中,R为连接基团-(CH2)n-COO-,n是1至20之间的整数,优选R为-(CH2)5-COO-。
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。
当R为-(CH2)n-COO-时,该羟脯氨酸免疫原的合成途径和方法如下:
1.羟脯氨酸衍生物的制备方法:
一种羟脯氨酸衍生物,其结构式如式(Ⅱ)所示:
式(Ⅱ)
式中,R为连接基团-(CH2)n-COO-,n是1至20之间的整数。
取n=5时,该羟脯氨酸衍生物的具体合成步骤如下:
本发明中,羟脯氨酸衍生物的制备过程中选用了6-溴己酸甲酯为合成原料,故所得的最终产物羟脯氨酸衍生物的连接基团R为-(CH2)5-COO-。当n取其他数值时,选用其他6-溴己酸甲酯的类似物,即直链的、通式为Br-(CH2)n-COOCH3的化合物参加反应时,合成方法完全一致。
2.羟脯氨酸免疫原的制备步骤:
(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本发明合成的羟脯氨酸衍生物、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,得到羟脯氨酸免疫原。
本发明中当n取1~20范围内的其他整数时,用上述方法可以制备出如式(Ⅰ)所示的羟脯氨酸免疫原。载体仍为具有免疫原性的蛋白质,可以是血清蛋白,血蓝蛋白和甲状腺球蛋白。优选的,载体为血清蛋白。更优选的,载体为牛血清白蛋白。
由于连接基团主要起小分子衍生物与载体的连接作用,免疫原性强弱与所合成的羟脯氨酸衍生物分子结构及所选载体种类有关,因此理论上n取1至20之间的任意整数时,羟脯氨酸衍生物制备的羟脯氨酸免疫原无显著差异,均具备强免疫原性,都能制备高效价的特异性抗体。
一种抗羟脯氨酸特异性抗体,由上述的羟脯氨酸免疫原免疫动物后生产得到。
所述的抗羟脯氨酸特异性抗体由上述制得的羟脯氨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-羟脯氨酸免疫原稀释至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的抗羟脯氨酸特异性抗体。
本发明的抗羟脯氨酸特异性抗体为完整的抗体分子,也包括保留与羟脯氨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羟脯氨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。
本发明提供一种羟脯氨酸检测试剂,含有上述抗羟脯氨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,由羟脯氨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。
羟脯氨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羟脯氨酸特异性抗体混合在一起。因此,羟脯氨酸均相酶免疫检测试剂包括两类试剂:
(1)试剂A由抗羟脯氨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)将制备的抗羟脯氨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1:1500;
(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)羟脯氨酸衍生物的激活:
a.在无水状态下称取5~15mg羟脯氨酸衍生物,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与羟脯氨酸衍生物的连接:
a.将上述激活的羟脯氨酸衍生物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1:1000。
本发明的羟脯氨酸免疫原特异性强、免疫原性高,制备出的抗羟脯氨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;含有上述抗羟脯氨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羟脯氨酸含量,并且可以在全自动生化分析仪上同时测定多个样品,实现羟脯氨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是羟脯氨酸的ELISA检测反应曲线;
图2是羟脯氨酸的均相酶免疫反应曲线;
图3是羟脯氨酸均相酶免疫相关性分析图。
具体实施方式
实施例一羟脯氨酸衍生物的合成及其分析鉴定
羟脯氨酸衍生物的化学结构如式(Ⅳ)所示:
式(Ⅳ)
上述羟脯氨酸衍生物的合成路线及制备步骤如下:
具体的合成步骤如下:
化合物2的合成
1)称取10.0g(76.3mmol)化合物1溶解在100mL甲醇中,然后加入6M的HCl/甲醇溶液100mL,50℃下搅拌过夜。LCMS检测显示反应已完成,将合成后的溶液在真空中干燥浓缩得到9.9g白色固体化合物2,产率69.2%。
2)利用BrukerAvanceIIIplus400MHz和VARIANMERCURYplus300M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:1HNMR(D2O,400MHz):δ2.15-2.22(m,1H),2.36-2.41(m,1H),3.28-3.16(d,1H),3.91-3.43(dd,1H),4.57-4.70(m,2H)。表征为上式所示的化合物2。
化合物3的合成
1)称取9g(49.71mmol)化合物2、12.41g(59.65mmol)6-溴己酸甲酯和20.58g(149.12mmol)K2CO3共同溶解于200mL乙腈中,80℃下搅拌过夜。TLC检测显示反应已完成。将合成后所得溶液过滤,滤液在真空中干燥浓缩得到粗制品,再将此粗制品通过硅胶柱纯化,得到5.2g黄色油状化合物3,产率36.8%。
2)利用BrukerAvanceIIIplus400MHz和VARIANMERCURYplus300M对上述黄色油状化合物进行核磁共振光谱扫描,采用TMS作为内标。结果如下:1HNMR(CDCl3,400MHz):δ1.34-1.37(m,2H),1.42-1.52(m,2H),1.61-1.67(m,2H),2.03-2.05(m,1H),2.06-2.09(m,2H),2.17-2.24(m,2H),2.29-2.32(m,2H),2.41-2.49(m,2H),2.64-2.70(m,1H),3.40-3.53(m,2H),4.46(s,3H),4.47(s,3H),4.48-4.49(m,1H)。表征为上式所示的化合物3。
羟脯氨酸衍生物的合成
1)称取5g(18.3mmol)化合物3和1.3g(54.9mmol)LiOH共同溶解于50mL甲醇中,50℃下搅拌3小时。在真空中去除溶剂,再将残留物溶解于水中。在上述水溶液中加入离子交换树脂,然后在室温下搅拌过夜。将此反应体系过滤,过滤后得到的滤液经浓缩干燥最终得到1.2g棕色固体化合物,即羟脯氨酸衍生物,产率26.7%。
2)利用BrukerAvanceIIIplus400MHz和VARIANMERCURYplus300M对上述白色油状化合物进行核磁共振光谱扫描,采用TMS作为内标。结果如下:1HNMR(CDCl3,400MHz):δ1.27-1.33(m,2H),1.48-1.54(m,2H),1.55-1.65(m,2H),2.07-2.12(m,1H),2.25-2.29(t,2H),2.33-2.38(m,1H),3.10-3.28(m,3H),3.79-3.83(m,1H),4.09-4.13(m,1H),4.50-4.51(m,1H)。表征为上式所示的羟脯氨酸衍生物。
3)利用色谱/质谱技术(LC/MS)对得到的衍生物进行分析鉴定:MS246(M+1),确定该最终所得化合物为式(Ⅳ)所示的羟脯氨酸衍生物,纯度>95%。
本实施例中,羟脯氨酸衍生物的制备过程中选用了6-溴己酸甲酯为合成原料,故所得的最终产物羟脯氨酸衍生物的连接基团R为-(CH2)5-COO-。当n取其他数值时,选用其他6-溴己酸甲酯的类似物,即直链的、通式为Br-(CH2)n-COOCH3的化合物参加反应时,合成方法完全一致。
实施例二羟脯氨酸免疫原的合成
羟脯氨酸免疫原由牛血清白蛋白(BovineSerumAlbumin,BSA)与式(Ⅱ)所示的羟脯氨酸衍生物的-(CH2)n-COO-基团连接而成,在本实施例中,以n=5为例详细说明该免疫原的合成方法,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合成的羟脯氨酸衍生物、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,得到羟脯氨酸免疫原。
实施例三:抗羟脯氨酸特异性抗体的制备
将实施例二制备得到的羟脯氨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将上述合成的羟脯氨酸免疫原稀释至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的抗羟脯氨酸特异性抗体。
实施例四:羟脯氨酸的ELISA检验
1.羟脯氨酸的ELISA检测标准曲线的建立
(1)标准品的制备
将羟脯氨酸粉末(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200.00mg/L、100.00mg/L、50.00mg/L、25.00mg/L、12.50mg/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)利用羟脯氨酸的ELISA检验方法制备标准曲线
用PBS将实施例三中所制备的抗羟脯氨酸抗体稀释成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将上述包被有抗羟脯氨酸抗体的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-羟脯氨酸偶联物;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.待测样品中羟脯氨酸含量的检测
(1)制作待测样品
制备方法:将羟脯氨酸粉末(购于Sigma公司)溶解于甲醇溶液制成1mg/mL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0.00,10.00,100.00,200.00mg/L,形成空白、低、中、高浓度的尿液样本。该空白尿液为不含羟脯氨酸的健康人尿液。
(2)测试方法
利用上述羟脯氨酸的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中所示的羟脯氨酸的ELISA检验的标准曲线,计算每个样本中羟脯氨酸含量,并对每个样本进行3个复孔测定,根据上述样本中羟脯氨酸的实际含量计算回收率,结果如表1所示。
表1羟脯氨酸的ELISA检测回收实验
尿液样品 | 空白 | 低 | 中 | 高 |
样品浓度(mg/L) | 0.00 | 10.00 | 100.00 | 200.00 |
测试1 | 0.02 | 11.23 | 96.45 | 198.57 |
测试2 | 0.03 | 10.17 | 102.98 | 202.54 |
测试3 | 0.01 | 9.25 | 98.78 | 203.62 |
平均值(mg/L) | 0.02 | 10.22 | 99.40 | 201.58 |
回收率(%) | - | 102.20 | 99.40 | 100.80 |
由表1中结果可知:采用本发明羟脯氨酸的ELISA检测试剂测定不同浓度样品中的羟脯氨酸回收率都较高,均>90%,说明本发明所述的抗羟脯氨酸特异性抗体可以用于样本中羟脯氨酸的检测,并且结果准确度高。
实施例五: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)。
羟脯氨酸衍生物的激活:
(1)在无水状态下称取10mg上述羟脯氨酸衍生物,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。
与羟脯氨酸衍生物的连接:
(1)将上述激活的羟脯氨酸衍生物溶液逐滴加入到上述溶解的G6PDH溶液中。
(2)2-8℃搅拌过夜。
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。
实施例六:羟脯氨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;将上述制备的抗羟脯氨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1:1500。
2.试剂B的制备:将实施例五制备的葡萄糖-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1:1000。
实施例七:羟脯氨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:将羟脯氨酸标准品溶解于人尿液中,至浓度分别为10.00,100.00,200.00mg/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) | 10.00 | 100.00 | 200.00 |
1 | 10.26 | 99.82 | 200.78 |
2 | 11.05 | 102.35 | 199.69 |
3 | 9.87 | 104.29 | 198.72 |
4 | 10.09 | 101.96 | 203.55 |
5 | 9.89 | 98.63 | 205.32 |
6 | 9.43 | 99.95 | 199.74 |
7 | 10.29 | 103.56 | 197.96 |
8 | 9.87 | 101.37 | 200.23 |
9 | 10.33 | 99.87 | 199.98 |
10 | 10.12 | 100.28 | 201.09 |
平均值(mg/L) | 10.12 | 101.21 | 200.71 |
标准差(SD) | 0.40 | 1.72 | 2.09 |
精密度(CV%) | 3.97% | 1.70% | 1.04% |
回收率 % | 101.20 | 101.21 | 100.35 |
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%。
实施例八: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,采用实施例七的均相酶免疫方法进行测定:
1.将待测干扰药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。
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 | 等价于羟脯氨酸的浓度 (mg/L) | ID# | 化合物名称 | 等价于羟脯氨酸的浓度 (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种常见药物等价于羟脯氨酸的浓度均小于0.01mg/L。由此可见,本发明的抗体是抗羟脯氨酸的特异性抗体,与其它药物无交叉反应。
实施例九:相关性分析
对100例临床标本分别使用青岛博新生物技术有限公司的比色法试剂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) | 青岛博新比色法测定值(mg/L) |
1 | 56.34 | 55.73 |
2 | 98.72 | 95.35 |
3 | 136.94 | 135.96 |
4 | 8.26 | 8.30 |
5 | 29.33 | 27.54 |
6 | 112.35 | 110.38 |
7 | 40.73 | 39.50 |
8 | 19.40 | 20.75 |
9 | 60.52 | 59.67 |
10 | 183.64 | 179.32 |
11 | 122.27 | 125.00 |
12 | 77.35 | 76.03 |
13 | 33.62 | 34.10 |
14 | 56.47 | 55.55 |
15 | 121.62 | 118.49 |
16 | 192.39 | 189.93 |
17 | 42.50 | 41.00 |
18 | 23.03 | 24.32 |
19 | 87.99 | 88.20 |
20 | 36.66 | 35.27 |
21 | 53.00 | 55.25 |
22 | 10.85 | 9.80 |
23 | 78.26 | 75.01 |
24 | 70.33 | 75.67 |
25 | 78.44 | 79.35 |
26 | 58.55 | 56.99 |
27 | 98.29 | 97.32 |
28 | 89.45 | 86.36 |
29 | 95.61 | 92.03 |
30 | 95.82 | 91.33 |
31 | 105.38 | 100.38 |
32 | 189.45 | 178.00 |
33 | 47.80 | 48.22 |
34 | 27.53 | 29.35 |
35 | 16.72 | 17.93 |
36 | 19.77 | 20.50 |
37 | 77.98 | 77.37 |
38 | 38.35 | 39.73 |
39 | 87.43 | 89.50 |
40 | 27.11 | 27.92 |
41 | 42.32 | 45.66 |
42 | 15.24 | 14.98 |
43 | 58.45 | 57.65 |
44 | 83.99 | 85.44 |
45 | 67.33 | 66.89 |
46 | 60.10 | 59.31 |
47 | 112.50 | 115.02 |
48 | 69.05 | 68.30 |
49 | 153.21 | 150.17 |
50 | 85.63 | 86.63 |
51 | 99.22 | 96.35 |
52 | 114.57 | 112.11 |
53 | 156.34 | 154.43 |
54 | 19.98 | 20.90 |
55 | 136.42 | 132.68 |
56 | 48.37 | 49.04 |
57 | 116.52 | 119.80 |
58 | 178.39 | 174.32 |
59 | 29.26 | 33.64 |
60 | 88.19 | 85.39 |
61 | 25.41 | 26.97 |
62 | 62.52 | 63.15 |
63 | 45.58 | 46.11 |
64 | 123.88 | 125.36 |
65 | 132.00 | 136.48 |
66 | 45.66 | 48.29 |
67 | 78.99 | 80.83 |
68 | 98.34 | 100.30 |
69 | 59.45 | 62.55 |
70 | 78.97 | 81.36 |
71 | 45.62 | 48.28 |
72 | 25.33 | 26.54 |
73 | 36.87 | 36.92 |
74 | 56.15 | 60.20 |
75 | 25.72 | 29.35 |
76 | 45.83 | 46.40 |
77 | 58.95 | 60.03 |
78 | 46.82 | 48.37 |
79 | 15.90 | 16.29 |
80 | 48.54 | 47.91 |
81 | 75.66 | 79.32 |
82 | 23.98 | 25.89 |
83 | 56.80 | 58.53 |
84 | 15.45 | 16.43 |
85 | 75.00 | 76.76 |
86 | 23.67 | 25.00 |
87 | 58.30 | 60.26 |
88 | 23.06 | 26.55 |
89 | 36.04 | 39.17 |
90 | 56.15 | 59.38 |
91 | 26.96 | 27.14 |
92 | 55.68 | 54.04 |
93 | 78.50 | 76.50 |
94 | 45.09 | 46.27 |
95 | 25.83 | 24.30 |
96 | 58.45 | 57.18 |
97 | 58.87 | 55.13 |
98 | 66.99 | 65.39 |
99 | 36.43 | 34.85 |
100 | 56.83 | 55.32 |
对上述数据作图,参见图3,得到的线性方程为:y=0.9718x+1.9094,相关系数R2=0.9969,表明本发明的检测试剂测定羟脯氨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 (10)
1.一种羟脯氨酸免疫原,其结构式如式(Ⅰ)所示:
式(Ⅰ)
式中,R为连接基团-(CH2)n-COO-,n是1至20之间的整数,优选R为-(CH2)5-COO-;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所述的羟脯氨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羟脯氨酸衍生物、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,得到羟脯氨酸免疫原。
3.根据权利要求2所述的羟脯氨酸免疫原制备方法,其特征在于所述的羟脯氨酸衍生物结构式如式(Ⅱ)所示:
式(Ⅱ)
上述R为连接基团-(CH2)n-COO-,n为1至20之间的整数,优选n=5。
4.根据权利要求2-3任一项所述的羟脯氨酸衍生物,其特征在于,n=5时,具体合成步骤如下:
或者当n为5以外的其余整数时,羟脯氨酸衍生物的合成步骤与上述合成步骤的区别仅在于:由化合物2合成化合物3的步骤中,采用的原料6-溴己酸甲酯替换为其类似物。
5.一种抗羟脯氨酸特异性抗体,由权利要求1-2任意一项所述的羟脯氨酸免疫原免疫实验动物后产生的完整抗体分子,或者为保留与羟脯氨酸特异性结合能力的抗体片段或抗体衍生物。
6.根据权利要求5所述的一种抗羟脯氨酸特异性抗体,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,为采用单一的羟脯氨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。
7.一种如权利要求5-6中任一项所述的抗羟脯氨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将牛血清白蛋白-羟脯氨酸免疫原稀释至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的抗羟脯氨酸特异性抗体。
8.一种羟脯氨酸检测试剂,含有权利要求5-7所述的抗羟脯氨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;所述的酶试剂由羟脯氨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。
9.一种如权利要求8所述的羟脯氨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5-7中任一项所述的抗羟脯氨酸特异性抗体加到上述均相酶底物中,抗羟脯氨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:将羟脯氨酸酶标偶联物加到120mM、pH=8.2的Tris缓冲液中,羟脯氨酸酶标偶联物与Tris缓冲液的体积比为1:100~1:10000;
所述的抗羟脯氨酸特异性抗体与均相酶底物的体积比优选为1:1500;
所述的羟脯氨酸酶标偶联物与Tris缓冲液的体积比优选为1:1000。
10.根据权利要求8-9任一项所述的羟脯氨酸检测试剂,其特征在于所述的羟脯氨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)羟脯氨酸衍生物的激活:在无水状态下称取5~15mg羟脯氨酸衍生物,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-磷酸脱氢酶与羟脯氨酸衍生物的连接:将步骤(2)激活的羟脯氨酸衍生物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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