CN109134292B - 阿替洛尔半抗原、人工抗原和抗体及其制备方法和用途 - Google Patents
阿替洛尔半抗原、人工抗原和抗体及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及选择一种具有—COOH的、又最大可能包含阿替洛尔原有结构的化合物作为阿替洛尔半抗原,以此半抗原制成的人工抗原、特异性抗体;以及此类半抗原、人工抗原、特异性抗体的用途,和半抗原、人工抗原、抗体的制备方法。
背景技术
近年来,作为新型环境污染物,药物及个人护理品(PPCPs)由于对人类和水生生物的潜在危害受到极大的关注。这些药物在废水处理厂中不能被完全去除,因此废水处理厂的出水被认为是PPCPs进入水环境的主要来源。由于药物设计上的特点,很多药物对人类和动物具有生理作用,而且难以被生物降解,因此,很多药物在地表水、地下水和近海水中被频繁地检出。阿替洛尔(ATL)是一种常见的β-阻滞剂,主要用于治疗心血管疾病,如高血压、心绞痛和心律不齐。阿替洛尔在欧洲和北美有相当长的使用历史,环境检出浓度相对较高。此外,由于阿替洛尔具有潜在的生态毒性,比如它可以影响心率,使鱼类产生不正常精子或降低精子活动能力,其环境行为和生态风险也受到很大的关注,因此建立一系列速度快、成本低、准确性高的检测方法显得极为迫切。
目前对阿替洛尔的检测主要为仪器方法,如液相色谱法、液相色谱-质谱法等。仪器检测法存在样品前处理繁琐、检测时间长、仪器贵重等缺点,所以在我国无法得到广泛应用,并且不符合现场检测“在短时间内低成本对大量样品进行准确检测和筛选”的要求。酶联免疫吸附测定技术Enzyme-Linked Immunosorbent Assay(ELISA),是将免疫技术与现代测试手段相结合而建立的一种超微量的测定方法,具有成本低、速度快、灵敏度高、仪器设备简单的特点,适合大批量样品的快速分析。而免疫学检测方法的基础是相应的高特异性、高亲和力的抗体的制备,而阿替洛尔作为小分子化合物,本身不具备免疫原性,因此,其半抗原的结构改造和全抗原合成是建立免疫速测技术的关键和难点之一。
发明内容
针对现有技术中存在的不足之处,本发明提供一种能最大程度保留阿替洛尔的化学结构,又具有一定长度连接臂的半抗原以及这种半抗原的制备方法;以此半抗原制备的人工抗原、检测灵敏度高和特异性强的抗体;以及此半抗原、抗体的用途。
本发明为达到以上目的,是通过这样的技术方案来实现的:提供一种阿替洛尔半抗原,它的分子结构式为:
本发明的半抗原以4-(环氧乙基甲氧基)苯乙酰胺为起始原料,与L-β-高丙氨酸盐酸盐反应生成羧基阿替洛尔,即为阿替洛尔半抗原。反应式如下:
本发明的阿替洛尔半抗原的制备方法进一步描述如下:
取4-(环氧乙基甲氧基)苯乙酰胺0.5g,加60mL乙醇溶解,加1mol/L盐酸0.5mL,搅拌混匀,加L-β-高丙氨酸盐酸盐0.29g,油浴加热搅拌,80℃反应4h;停止反应,旋蒸,除去乙醇,加水80mL,加三氯甲烷100mL×3,萃取三次,合并有机相,蒸干,上硅胶柱层析纯化,体积比为10:1的二氯甲烷-甲醇洗脱,即可得到阿替洛尔半抗原。
上述方法制得的阿替洛尔半抗原,具有阿替洛尔特征结构的同时又具有可以与蛋白质偶联的—COOH结构。
本发明还提供了一种依靠上述阿替洛尔半抗原制得的阿替洛尔人工抗原,它的分子结构式为:
所述载体蛋白为牛血清白蛋白。
本发明的阿替洛尔人工抗原的制备方法描述如下:
取阿替洛尔半抗原7~11mg,加1.2~2mL N,N-二甲基甲酰胺溶解,加碳二亚胺6~8mg,N-羟基琥珀酰亚胺5~7mg,室温搅拌2h,得到半抗原活化液A液;取载体蛋白50mg,加0.1mol/L磷酸盐缓冲液3mL溶解,得到B液;将A液滴加到B液中,搅拌4h,0.02mol/L PB缓冲液透析纯化3天,每天换液3次,得到阿替洛尔人工抗原,分装,-20℃保存。
本发明同时提供了上述阿替洛尔半抗原的用途,是用作动物免疫的抗原体系的原料。
本发明同时提供了上述阿替洛尔人工抗原免疫白鼠所得到的、能与阿替洛尔发生特异性免疫反应的阿替洛尔单克隆抗体,及其在检测阿替洛尔残留中的应用。
基于阿替洛尔—NH2连接臂可能是其活性基团,对维系半抗原的免疫特性和特征结构十分重要,或若—NH2直接与载体连接后半抗原的特征结构易受载体的局部微化学环境或空间位阻的干扰,影响机体免疫系统的识别,因此,以4-(环氧乙基甲氧基)苯乙酰胺为原料,经过与L-β-高丙氨酸盐酸盐反应,合成了带有羧基的半抗原,最大可能保留了原来阿替洛尔的分子结构,使阿替洛尔分子的化学结构与电子分布几乎不受影响,这为获得对阿替洛尔有高度特异性的抗体提供了保证。
本发明中合成的阿替洛尔半抗原,既最大程度地保留了阿替洛尔的化学结构,又具有一定长度的连接臂,用这一半抗原制备的免疫抗原去免疫动物,所得的抗体的效价、特异性、亲和力都比较好,所得的抗体用于ELISA方法检测阿替洛尔,灵敏度可达0.2μg/L,与其他β-阻滞剂的交叉反应率低。
附图说明
图1阿替洛尔半抗原合成路线图
图2阿替洛尔ELISA竞争标准曲线图
具体实施方式
下面结合附图和具体实施例进一步详细说明本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1:半抗原合成
一种阿替洛尔半抗原,分子结构式为:
是用作动物免疫的抗原体系的原料。
上述阿替洛尔半抗原的制备方法如下(合成路线见附图1):
取4-(环氧乙基甲氧基)苯乙酰胺0.5g,加60mL乙醇溶解,加1mol/L盐酸0.5mL,搅拌混匀,加L-β-高丙氨酸盐酸盐0.29g,油浴加热搅拌,80℃反应4h;停止反应,旋蒸,除去乙醇,加水80mL,加三氯甲烷100mL×3,萃取三次,合并有机相,蒸干,上硅胶柱层析纯化,体积比为10:1的二氯甲烷-甲醇洗脱,即可得到阿替洛尔半抗原。
实施例2:人工抗原合成和鉴定
由实施例1所述阿替洛尔半抗原制成的一种阿替洛尔人工抗原,分子结构式为:
1、免疫原的合成
免疫原的合成方法如下:
取阿替洛尔半抗原11mg,加2mL N,N-二甲基甲酰胺(DMF)溶解,加碳二亚胺(EDC)8mg,N-羟基琥珀酰亚胺(NHS)7mg,室温搅拌2h,得到半抗原活化液A液;取牛血清白蛋白(BSA)50mg,加0.1mol/L磷酸盐缓冲液3mL溶解,得到B液;将A液滴加到B液中,搅拌4h,0.02mol/L PB缓冲液透析纯化3天,每天换液3次,得到阿替洛尔免疫原,分装,-20℃保存。
人工抗原的鉴定:
按合成阿替洛尔免疫原反应所用半抗原、载体蛋白与偶联产物的比例,进行紫外(200~400nm)扫描测定,通过比较三者分别在260nm和280nm的吸光值计算其结合比。偶联物阿替洛尔半抗原-BSA的最大吸收峰与阿替洛尔半抗原、BSA的最大吸收峰相比发生了明显的变化,表明人工抗原阿替洛尔半抗原-BSA的合成是成功的。经计算,半抗原与BSA的结合比为14:1。
2、包被原的合成
取阿替洛尔半抗原7mg,加1.2mL DMF溶解,加EDC 6mg,NHS 5mg,室温搅拌2h,得到半抗原活化液A液;取卵清白蛋白(OVA)50mg,加0.1mol/L磷酸盐缓冲液3mL溶解,得到B液;将A液滴加到B液中,搅拌4h,0.02mol/L PB缓冲液透析纯化3天,每天换液3次,得到阿替洛尔包被原,分装,-20℃保存。
人工抗原的鉴定:
按合成阿替洛尔包被原反应所用半抗原、载体蛋白与偶联产物的比例,进行紫外(200~400nm)扫描测定,通过比较三者分别在260nm和280nm的吸光值计算其结合比。偶联物阿替洛尔半抗原-OVA的最大吸收峰与阿替洛尔半抗原、OVA的最大吸收峰相比发生了明显的变化,表明人工抗原阿替洛尔半抗原-OVA的合成是成功的。经计算,半抗原与OVA的结合比为11:1。
实施例3:单克隆抗体制备
一种阿替洛尔特异性抗体,是将实施例2所述阿替洛尔免疫原免疫白鼠所得到的、能与阿替洛尔发生特异性免疫反应的单克隆免疫球蛋白G,用于检测阿替洛尔残留。
上述阿替洛尔单克隆抗体的制备方法如下:
1)动物免疫:将免疫原注入到Balb/c小鼠体内,免疫剂量为150μg/只,使其产生抗血清。
2)细胞融合和克隆化:取产生特异性抗体的Balb/c小鼠脾细胞与骨髓瘤细胞SP20融合,采用间接竞争酶联免疫分析方法测定细胞上清液,筛选阳性孔。利用有限稀释法对阳性孔进行克隆化,得到并建立产单克隆抗体的杂交瘤细胞株。
3)细胞冻存和复苏:取处于对数生长期的杂交瘤细胞用冻存液制成细胞悬液,分装于冻存管,在液氮中长期保存。复苏时取出冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养。
4)单克隆抗体的制备与纯化:采用体内诱生法,将Balb/c小鼠(8周龄)腹腔注入灭菌石蜡油,7~14天后腹腔注射杂交瘤细胞,7~10天后采集腹水。经辛酸-饱和硫酸铵法进行腹水纯化,纯度经SDS-PAGE电泳鉴定,小瓶分装,-20℃保存。
抗体的特异性测定:
用具有多种抗原决定簇的免疫原(蛋白质或多肽)制备的抗体,其中含的抗体分子往往是混合体。当有甲、乙两种抗原,其分子结构中具有相同或部分相同的抗原决定簇时,甲抗原可与乙抗原的抗体反应,而乙抗原也可与甲抗原的抗体反应,称为交叉反应。抗体的特异性就是指它同特异性抗原结合的能力与同该抗原类似物结合能力的比较。常用交叉反应活性作为评价的重要标准。交叉反应越小,抗体的特异性则越好。
将特异性抗原及其类似物做系列稀释,分别与同一种阿替洛尔半抗原-BSA抗体,按制作标准曲线同样方法制作标准曲线,并在曲线上找出抑制率50%的剂量和类似物抑制率50%时的用量。然后计算出各类似物的交叉反应率。
抗阿替洛尔半抗原-BSA抗体对阿替洛尔及各类似物的交叉反应率:阿替洛尔为100%,吲哚洛尔为11.5%、比索洛尔3.9%、美托洛尔6.3%、普萘洛尔5.7%。由此可见,所制备的抗体特异性较好。
实施例4:阿替洛尔酶联免疫吸附测定方法的建立与应用
一、阿替洛尔ELISA测定方法的基本原理
采用间接竞争酶联免疫分析方法,即将阿替洛尔分子与大分子载体(如蛋白质)偶联制得的复合物作为包被原吸附于固相载体(96孔酶标板)上,制备成固相抗原,然后加入待测样品和相应抗体。固相抗原、待测样品中的阿替洛尔与抗体进行竞争结合反应,待测样品中阿替洛尔含量多,则被结合在固相抗原上的抗体少,反之结合在固相抗原上的抗体多,然后加入酶标二抗,最后用底物进行显色加以测定。当抗体量一定时,加入的待测样本中阿替洛尔量越多,与固相抗原结合的抗体就越少,与抗体结合的酶标二抗也越少,发色反应就减弱,反之,则发色反应增强,因而可根据已知阿替洛尔量的标准曲线和待测样品的吸光度值,推算出待测样品中阿替洛尔的浓度。
二、阿替洛尔ELISA测定方法中涉及的组分
(1)包被有阿替洛尔人工抗原(阿替洛尔半抗原-卵清白蛋白偶联物)的酶标板;
(2)阿替洛尔系列标准品溶液:浓度分别为0μg/L、0.2μg/L、0.6μg/L、1.8μg/L、5.4μg/L;
(3)抗体工作液:实施例3中所述的阿替洛尔单克隆抗体工作液;
(4)酶标二抗:用辣根过氧化物酶标记的羊抗鼠抗抗体;
(5)底物显色液:由A液和B液组成,A液为2%过氧化脲的水溶液,B液为1%四甲基联苯胺的水溶液;
(6)终止液:2mol/L硫酸水溶液;
(7)洗涤液:含有1.0%吐温-20、0.02‰叠氮化钠,pH值为7.4、0.05mol/L的磷酸盐缓冲液;
(8)复溶液:含有5%牛血清白蛋白,pH值为7.4、0.02mol/L的磷酸盐缓冲液。
三、阿替洛尔ELISA测定方法中各组分的制备
1、包被有阿替洛尔人工抗原(阿替洛尔半抗原-卵清白蛋白偶联物)的酶标板的制备
用包被缓冲液将实施例2中所述阿替洛尔包被原稀释至0.2μg/mL,包被96孔聚苯乙烯酶标板,每孔100μL,37℃温育2h,倾去包被液,用洗涤液洗涤3次,每次30s,拍干,然后在每孔加入200μL封闭液,37℃温育2h,倾去孔内液体,干燥后用铝膜真空密封保存。
所用的包被缓冲液和封闭液如下:
包被缓冲液:pH9.6、0.05mol/L的碳酸盐缓冲液;
封闭液:含有0.5%马血清、0.1%叠氮化钠、3%酪蛋白,pH值为7.2、0.02mol/L的磷酸盐缓冲液。
2、阿替洛尔系列标准品溶液的制备
用标准品稀释液将阿替洛尔标准品稀释至浓度分别为0μg/L、0.2μg/L、0.6μg/L、1.8μg/L、5.4μg/L;标准品稀释液为含有5%牛血清白蛋白,pH值为7.4、0.02mol/L的磷酸盐缓冲液。
3、抗体工作液的制备
用抗体稀释液将实施例3中所述的阿替洛尔单克隆抗体稀释1000倍,得到抗体工作液;抗体稀释液为含有2.5%酪蛋白和0.03‰叠氮化钠的磷酸盐缓冲液。
4、酶标二抗的制备
(1)羊抗鼠抗抗体的制备
以羊作为免疫动物,以鼠源抗体为免疫原对无病原体羊进行免疫,得到羊抗鼠抗抗体。
(2)酶标二抗的制备
将羊抗鼠抗抗体与辣根过氧化物酶采用过碘酸钠法进行偶联,然后用酶标二抗稀释液将其稀释500倍;所述酶标二抗稀释液为含0.5%牛血清白蛋白,pH值为7.4、0.02mol/L的磷酸盐缓冲液。
四、用上述阿替洛尔ELISA测定方法检测样品中残留的阿替洛尔
(一)样品前处理
用复溶液将水样进行2倍稀释。
(二)检测
向包被有阿替洛尔半抗原-卵清白蛋白偶联物的酶标板微孔中加入系列标准品溶液或样品溶液50μL,随即加入酶标二抗50μL,再加入单克隆抗体工作液50μL,轻轻振荡混匀,用盖板膜盖板后置25℃避光环境中反应30min;倒出孔内液体,每孔加入洗涤液250μL,10s后倒出孔内液体,如此重复操作共洗板5次,用吸水纸拍干;每孔加入底物显色液A液和B液各50μL,轻轻振荡混匀,用盖板膜盖板后置25℃避光环境中反应15min;每孔加入终止液50μL,轻轻振荡混匀,设定酶标仪于450nm处,测定每孔的吸光度值。
(三)检测结果分析
用所获得的每个浓度的标准品溶液的吸光度平均值(B)除以第一个标准品溶液(0标准)的吸光度平均值(B0)再乘以100%,得到百分吸光度值。
以阿替洛尔标准品浓度的对数值为X轴,百分吸光度值为Y轴,绘制标准曲线图(图2)。用同样的方法计算样品溶液的百分吸光度值,相对应每一个样品的浓度,则可从标准曲线上读出样品中阿替洛尔的残留量。本发明中检测结果的分析也可以采用回归方程法,计算出样品溶液浓度。本发明中检测结果的分析还可以利用计算机专业软件,此法更便于大量样品的快速分析,整个检测过程只需1.0h可以完成。
五、上述阿替洛尔ELISA测定方法检测效果评价
(一)最低检测限
取不含阿替洛尔的阴性水样20份,将样品进行前处理后用上述阿替洛尔ELISA测定方法对其进行检测,以20份样品检测浓度的平均值加上3倍标准差表示检测限。
结果表明,该方法对水样的最低检测限为0.4μg/L。
(二)准确度和精密度
以回收率作为准确度评价指标,重复测定某一浓度样品的检测结果相对标准偏差(RSD%)作为精密度评价指标。计算公式为:回收率(%)=实际测定值/理论值×100%,其中理论值为样品的添加浓度;相对标准偏差RSD%=SD/X×100%,其中SD为标准偏差,X为测定数据的平均值。
向不含阿替洛尔的水样中分别添加阿替洛尔,使其终浓度为0.4μg/L、0.8μg/L、1.6μg/L,将添加后的样品进行前处理后用上述阿替洛尔ELISA测定方法对其进行检测,选用3批试剂,每个浓度做5个平行,分别计算回收率和批内、批间RSD,结果如表1所示。结果表明,水样的平均回收率在80%~100%,批内、批间RSD均在10%以内。
表1准确度和精密度实验结果
(三)保存期
上述阿替洛尔ELISA测定方法中所涉及到的主要试剂最终以工作液的形式提供,组装成试剂盒,大大降低了移液和操作的误差,同时体积小,易于携带和运送至终端客户处,更适合应用于现场操作检测和大批量样本检测,也节约了快递运输成本费用。
试剂盒保存条件为2-8℃,经过12个月的测定,试剂盒的最大吸光度值(零标准)、50%抑制浓度、阿替洛尔添加回收率均在正常范围内。考虑到运输和使用过程中会有非正常保存条件出现,将试剂盒在37℃下放置8天进行加速老化实验,结果该试剂盒的各项指标完全符合要求;考虑到试剂盒冷冻情况发生,将试剂盒在-20℃冰箱中放置8天,结果该试剂盒的各项指标也完全正常。从以上结果可得出,试剂盒可以在2-8℃至少保存12个月以上。
Claims (7)
2.一种权利要求1所述阿替洛尔半抗原的制备方法,其特征在于包括下列步骤:
取4-(环氧乙基甲氧基)苯乙酰胺0.5g,加60mL乙醇溶解,加1mol/L盐酸0.5mL,搅拌混匀,加L-β-高丙氨酸盐酸盐0.29g,油浴加热搅拌,80℃反应4h;停止反应,旋蒸,除去乙醇,加水80mL,加三氯甲烷100mL×3,萃取三次,合并有机相,蒸干,上硅胶柱层析纯化,体积比为10:1的二氯甲烷-甲醇洗脱,即可得到阿替洛尔半抗原。
4.一种权利要求3所述阿替洛尔人工抗原的制备方法,其特征在于包括下列步骤:
取阿替洛尔半抗原7~11mg,加1.2~2mL N,N-二甲基甲酰胺溶解,加碳二亚胺6~8mg,N-羟基琥珀酰亚胺5~7mg,室温搅拌2h,得到半抗原活化液A液;取载体蛋白50mg,加0.1mol/L磷酸盐缓冲液3mL溶解,得到B液;将A液滴加到B液中,搅拌4h,0.02mol/L PB缓冲液透析纯化3天,每天换液3次,得到阿替洛尔人工抗原,分装,-20℃保存。
5.一种权利要求1所述阿替洛尔半抗原在制备动物免疫的抗原体系方面的应用,其特征在于是用作动物免疫的抗原体系的原料。
6.一种采用权利要求3所述阿替洛尔人工抗原免疫白鼠所得到的、能与阿替洛尔发生特异性免疫反应的阿替洛尔单克隆抗体。
7.一种权利要求6所述阿替洛尔单克隆抗体在检测阿替洛尔残留中的应用。
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