CN106496296A - 一种雌二醇人工抗原及其制备方法与应用 - Google Patents
一种雌二醇人工抗原及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种雌二醇半抗原和相应的人工抗原,同时本发明也公开了所述雌二醇半抗原和相应的人工抗原的制备方法及其应用。本发明提供的雌二醇半抗原是式1所示产物,用式1所示产物与载体蛋白连接可以得到雌二醇抗原。所述雌二醇抗原可应用于制备雌二醇特异性抗体。本发明制备方法简便可行、成本较低,半抗原产率较高。本发明的雌二醇人工抗原,通过免疫动物可产生了针对雌二醇的特异性抗体,可用于制备检测雌二醇残留的酶联免疫检测试剂盒,具有简单、快速、处理样品量大、灵敏度高、特异性强等诸多优点。
Description
技术领域
本发明属于食品安全检测技术领域,具体涉及一种雌二醇半抗原、抗原制备方法及其应用。
背景技术
雌激素是国家明令禁止在食品中添加的违禁药物,雌二醇是雌激素的主要成分之一,在畜禽养殖业中有着广泛的药理作用,可促进雌性未成年动物性器官的形成和第二性征的发育,促进母畜发情,具有促进动物机体生长,显著提高生产性能和畜禽瘦肉比等功效。近年来“速成鸡”、“早熟儿童”等食品安全事件的发生,食品中雌激素残留问题已经成为了当前食品安全的热点问题之一。由于这些雌激素类药物不仅会引起动物本身生理特征的异变,而且残留于动物源食品中,儿童食用后长期积累,造成性早熟和第二特征异常,还促进各种癌症和肿瘤疾病的并发,进而影响人类健康。因此,防控雌激素非法添加,保障人民的食品安全和身体健康十分重要。
目前食品中雌二醇药物检测方法多为仪器方法,如生物测定法、以气相色谱法和高效液相色谱法等。仪器法检测结果准确,但价格昂贵、操作复杂,不适合大规模推广使用。酶联免疫检测方法具有结果准确、操作简便、不需要专业人士操作等优点。本发明公开的雌二醇半抗原、抗原为进一步研制雌二醇抗体及雌二醇酶联免疫试剂盒提供了原料。雌二醇酶联免疫反应试剂盒利用竞争性酶联免疫反应原理,对组织(牛肉、鸡肉和猪肉)和其他样品中雌二醇的残留进行定量检测。
发明内容
本发明的目的是提供一种雌二醇半抗原、抗原制备方法及其应用。
本发明提供的雌二醇半抗原,是式1所示化合物:
本发明还公开了式1所示产物的制备方法,包括如下步骤:
包括如下步骤:50ml圆底烧瓶中加入雌二醇原料药540mg,5ml DMF搅拌15min后20-24℃加入70%的NaH 83.3mg;20-24℃滴加4-溴丁酸乙酯468mg,将反应液滴加到45ml冰水中析出类白色固体,过滤,收集滤饼鼓风干燥得雌二醇丁酸乙酯570mg中间体;
50ml圆底烧瓶中加入雌二醇丁酸乙酯中间体500mg,THF 10ml,20-24℃磁力搅拌15-30min后滴加1M氢氧化锂水溶液3.27ml,20-24℃磁力搅拌3-4小时后35-40℃减压浓缩,残留物中加入20ml水,1M盐酸调PH到5.0-6.5,析出类白色固体,过滤,收集滤饼鼓风干燥得到式1所示的雌二醇丁酸半抗原。
本发明提供的雌二醇抗原,是将式1所示产物和载体蛋白偶联得到的偶联物。
本发明还保护所述雌二醇抗原的制备方法,包括如下步骤:
将15.96mg雌二醇半抗原用1.5ml DMF溶解,加入EDC 21.5mg,加入NHS 13mg,室温搅拌,形成反应液;称取BSA 50mg溶于3.5ml 0.1M碳酸氢钠溶液中,形成BSA溶液,将所述反应液在冰浴条件下边搅拌边逐滴加入到BSA溶液中,搅拌转速为1000rpm;将反应产物装入蒸馏水冲洗干净透析袋,1L 0.01M PBS 4℃搅拌,透析,制备得到雌二醇抗原。同法制备包被原。
常用载体蛋白均可采用,如牛血清白蛋白(BSA),卵清蛋白(OVA),人血清白蛋白(HSA),鼠血清白蛋白(MSA),甲状腺蛋白(TG)或血蓝蛋白(KLH)等。
所述雌二醇抗原可以作为免疫原免疫动物制备雌二醇特异性抗体,也可以作为包被原制备酶标板。
所述抗体具体可为单克隆抗体。
式1所示产物、所述雌二醇抗原、所述抗体均可应用于检测雌二醇。
本发明还公布了应用雌二醇抗原和雌二醇单克隆抗体制备得到的酶联免疫试剂盒。
所述酶联免疫检测试剂盒,是由包被有雌二醇抗原的酶标板、酶标抗体工作液、雌二醇系列标准品、底物显色液、终止液、浓缩复溶液、浓缩洗涤液。
本发明依靠免疫学、免疫化学基本原理和残留分析技术手段,设计、合成小分子目标分析物半抗原,并与载体蛋白偶联,制备有效人工抗原。本发明制备方法简便可行、成本较低,半抗原产率较高。本发明的雌二醇人工抗原,通过免疫动物可产生了针对雌二醇的特异性抗体,用于快速检测食品中的雌二醇残留。
附图说明
图1为雌二醇半抗原的质谱检测结果。
图2为雌二醇抗原“雌二醇半抗原+BSA”的MALDI-TOF-MAS图。
图3为雌二醇酶联免疫检测试剂盒标准曲线。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1、雌二醇半抗原的制备
一、雌二醇半抗原的制备
50ml圆底烧瓶中加入雌二醇原料药540mg,5ml的DMF搅拌15min后20-24℃加入70%的氢化钠83.3mg,20-24℃磁力搅拌反应1小时。20-24℃滴加4-溴丁酸乙酯468mg20-24℃磁力搅拌反应1小时,将反应液滴加到45ml冰水中析出类白色固体,过滤,滤饼用10ml水洗,收集滤饼50℃鼓风干燥5-6小时得雌二醇丁酸乙酯570mg中间体。
50ml圆底烧瓶中加入雌二醇丁酸乙酯中间体500mg,THF10ml,20-24℃磁力搅拌15-30min后滴加1M氢氧化锂水溶液3.27ml,20-24℃磁力搅拌3-4小时后35-40℃减压浓缩,残留物中加入20ml水,1M盐酸调PH到5.0-6.5,析出类白色固体,过滤,滤饼用10ml水洗,收集滤饼50℃鼓风干燥5-6小时得到410mg雌二醇丁酸半抗原。
二、雌二醇半抗原的鉴定
对所得产品进行质谱鉴定,-MS:M-1=248.7(图1)。
结果显示其化学结构式如式I所示,即为雌二醇半抗原。
实施例2、雌二醇人工抗原的制备和鉴定
一、雌二醇免疫抗原的合成
将15.96mg雌二醇半抗原用1.5ml DMF溶解,200rpm搅拌10min,加入EDC 21.5mg溶解后再加入NHS 13mg,室温搅拌(500rpm)活化2-3h。
称取BSA 50mg溶于3.5ml 0.1M碳酸氢钠溶液中,200rpm搅拌10min,使其充分溶解,将步骤1反应液在反应盒中冰浴(<4℃)搅拌(1000rpm)中逐滴加入(1ml/min),搅拌(500rpm)反应24h.
将反应产物装入蒸馏水冲洗干净透析袋(10cm),1L0.01M PBS(1L,pH7.2)4℃搅拌(100rpm)透析3d,每天换液3次(早中晚各一次),共计换液9次,将透析产物5000rpm离心6min,1.5ml/管分装,将抗原编号,-20℃保存备用。
同法制备包被原
二、雌二醇人工抗原的鉴定
BSA的分子量为66288.367,雌二醇半抗原分子量为358.48,E2-BSA的分子量为70852.951,E2-BSA偶联比=(70852.951-66288.367)/358.48=12.73。即免疫原中,所述雌二醇半抗原与牛血清白蛋白(BSA)偶联的摩尔比为12.73∶1。
实施例3、酶标单抗的制备和特异性鉴定
一、雌二醇单抗的制备
1、用上述制备出的免疫原按100μg/只,以生理盐水溶解免疫原与弗氏完全佐剂等体积混匀,颈背部皮下注射免疫6~8周龄Balb/c雌鼠,初次免疫后第7、14、28天以免疫原与弗氏不完全佐剂等体积混匀,各追加免疫一次,融合前3天以免疫复合物100μg/只,不加弗氏佐剂再追加免疫一次。
2、按常规方法进行,取免疫小鼠的脾细胞与处于对数生长期的小鼠骨髓瘤细胞(SP2/0)混合,然后在45s内缓慢加入预热的融合剂(PEG4000)进行融合,用HAT培养基悬浮均匀,再加入适量的饲养细胞,培养于96孔培养板,于37℃,5%CO2培养箱中培养,5天后用HT培养基半换液,9天时候进行全换液。
3、细胞融合后,待细胞长到培养孔面积的1/4时,采用分步筛选法筛选杂交瘤细胞。初选采用间接ELISA方法,以包被抗原(预先用方阵法常规滴定其最佳包被浓度和阳性血清稀释度)包被酶标板,加入被测孔培养上清,孵育,清洗后加入羊抗鼠IgG-HRP和IgM-HRP,OPD进行显色反应。筛选出的阳性孔再用间接竞争ELISA方法筛选,先将细胞上清与100μg/mL的雌二醇等体积混合,37℃水浴作用30min,再加入到包被好的酶标板中。同时用PBS取代雌二醇作对照,其余步骤同上。若经雌二醇阻断后的OD450nm值下降到对照孔的50%以下,则判为阳性,经2~3次检测都为阳性的孔,立即用有限稀释法进行亚克隆化。
4、将2~3次亚克隆建株后的杂交瘤细胞扩大培养,收集上清液用间接ELISA测定效价,冻存;并取8~10周龄Balb/c小鼠腹腔注射液体石蜡0.5mL/只,7~10日后腹腔注射杂交瘤细胞1~2×106/只,7~10日后抽取小鼠腹水,离心取上清,测定效价,并冻存备用。
二、酶标抗体的制备
(1)称取辣根过氧化物酶(HRP)2mg溶解于0.5mL水中,加入0.5mL 0.06mol/LNaIO4溶液,4℃避光作用30min;
(2)加入160mmol/L的乙二醇0.5mL,室温作用30min;
(3)加入步骤一制备的雌二醇单抗2mg,混匀后装入处理过的透析袋中,置1000mL的0.05mmol/L碳酸钠缓冲液中透析,4℃过夜;
(4)透析液吸至10mL的离心管中,加0.25mL 5g/L的NaBH4溶液,混匀后置4℃2h;
(5)加入等体积的饱和硫酸铵溶液,4℃作用30min后4℃下3000r/min离心25min,弃上清;
(6)将沉淀溶于1.5mL0.02mol/L pH 7.4的PBS中,吸入透析袋内,在0.02mol/L pH7.4PBS透析,4℃过夜(中途更换PBS 3次);
(7)将透析袋中液体吸至微量离心管中,4℃下10000r/min离心30min,将上清液吸出,加等量甘油,混匀,-20℃保存备用。
三、酶标雌二醇抗体效价的测定
雌二醇标准品购自Sigma公司。
用方阵滴定法确定雌二醇包被抗原和步骤一制备的单抗的工作浓度,雌二醇包被抗原的工作浓度为1∶60000μg/mL,单克隆抗体的工作浓度为1∶7000。
用不同浓度的雌二醇标准品溶液做实验溶液,其浓度如下:0、0.5、1.5、4.5、13.5、40.5μg/L。采用8组平行试验(n=8)。竞争性ELISA方法:
(1)用上述工作浓度的雌二醇抗原包被酶标板,将雌二醇标准品实验溶液与酶标抗体溶液同时加入酶标板微孔中,同时设置空白孔(将添加的抗体溶液换成高纯水,其它一致)和阴性对照孔(将标准品实验溶液用PBS溶液代替,其它一致),25℃避光环境中反应30min;
(2)倒出孔内液体,用洗涤液洗涤3~5次,将酶标板倒置在吸水纸上拍干;
(3)加入底物显色溶液到酶标板微孔中,25℃避光环境中反应15min;
(4)加入终止液,轻轻振荡混匀,用酶标仪在波长450nm处测定OD值。
以OD值为纵坐标,以雌二醇实验溶液浓度的log10值为横坐标,绘制半对数标准曲线图。标准曲线具有完整的反S形状,并具有上平台和下平台,标准曲线的平行测定次数8次,实验重复性良好,相对标准偏差(变异系数)均在10%以内。
根据标准曲线得出半数抑制量(IC50),确定检测灵敏度。
抑制率用以下式计算:
式中:ODmax:为不加标准品时的吸光值,ODx为标准品x时的吸光值,ODmin为空白对照孔的吸光值。
由上述公式计算得雌二醇抗体在缓冲液中的半数抑制量(IC50)为0.369ppb。
实施例4、检测雌二醇的酶联免疫试剂盒及其制备
一、酶联免疫试剂盒由下述物质组成:
(1)包被雌二醇半抗原的酶标板;
(2)酶标雌二醇抗体工作液:实施例3中所述酶标抗体溶液;
(3)雌二醇标准品:雌二醇标准品溶液浓度分别为0、0.1、0.3、0.9、2.7、8.1ppb;
(4)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(TMB)的水溶液;
(5)终止液:0.2M硫酸水溶液;
(6)浓缩洗涤液:每1升所述洗涤液是按照如下方法配制得到的:将10mL吐温-20、5g叠氮化钠和990mL磷酸盐缓冲液混合,得到所述洗涤液;所述磷酸盐缓冲液的浓度为0.01M pH值为7.4;
二、包被有E2-OVA的酶标板及其制备
包被E2-OVA的聚苯乙烯酶标板:用0.05M的碳酸盐溶液将抗原稀释至2.5μg/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10s,拍干,然后在每孔中加入150μL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。
包被缓冲液:pH9.6,0.05mol/L的碳酸钠缓冲液;
封闭液:每1升封闭液按照如下方法配制:将5mL马血清、1g叠氮化钠、30g酪蛋白混合,用磷酸盐缓冲液溶解并定容至1000mL,得到封闭液;其中,磷酸盐缓冲液的浓度为0.02M,pH值为7.2。
三、试剂盒检测方法
(一)样品前处理
原奶、还原乳、鸡肉、鸭肉、猪肉、牛肉、羊肉、饲料(稀释系数:8)
a)准确量取1±0.01g新鲜样品于50mL离心管中;
b)加入5mL乙腈和3mL丙酮,充分涡动1min;
c)4000g以上,离心5min;
d)取2mL上清液于干净离心管中;
e)50-60℃水浴中,氮气吹干;
f)加入0.5mL三氯甲烷,充分涡动30s;
g)加入2mL 2M氢氧化钠溶液,充分涡动30s;
h)4000g以上,离心5min;
i)取1mL上清液于干净离心管中;
j)加入150μL 6M磷酸溶液;
k)依次加入3mL乙腈、2mL丙酮,充分涡动30s;
l)4000g以上,离心5min;
m)取3mL上层有机相于干净离心管中;
n)50-60℃水浴中,氮气吹干;
o)加入0.5mL样品稀释液,充分涡动30s;
p)取50μL进行检测。
(二)用试剂盒检测
1、标准曲线的制作
向包被有E2-OVA的酶标板微孔中加入雌二醇标准品溶液50μL,然后加入酶标抗体工作液50μL/孔,轻轻振荡混合均匀,用盖板膜盖板后置25℃避光环境中反应30min。小心揭开盖板膜,将孔内液体甩干,加入洗涤工作液250μL/孔,充分洗涤4~5次,每次间隔10s,泼掉板孔内洗涤液,用吸水纸拍干。加入底物A液50μL/孔、底物B液50μL/孔,轻轻振荡混匀,25℃恒温箱避光显色15min,每孔加入终止液50μL,轻轻振荡混匀,用酶标仪,测定每孔吸光度值。
用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)的吸光度值(B0)再乘以100%,得到百分吸光度值。以雌二醇标准品浓度(μg/L)的半对数值为X轴,百分吸光度值为Y轴,绘制标准曲线图。得到的标准曲线如附图3所示。
百分吸光度值(%)=(B/B0)×100%
2、样品中雌二醇浓度的测定
用每个检测样本溶液的吸光度平均值(B)除以第一个标准品溶液(0标准)的吸光度值(B0)再乘以100%,得到百分吸光度值。相对应每一个检测样本溶液的百分吸光度值,则可从标准曲线上读出检测样本溶液的吸光度值,再根据标准品溶液的浓度值换算出样本溶液中雌二醇的残留量,最后再乘以各样品前处理过程的稀释倍数,即可计算出样品中雌二醇的浓度。
Claims (10)
1.一种雌二醇半抗原,为式1所示产物:
2.根据权利要求1所述的一种雌二醇半抗原,式1所示产物的制备方法,包括如下步骤:50ml圆底烧瓶中加入雌二醇原料药540mg,5ml DMF搅拌15min后20-24℃加入70%的NaH83.3mg;20-24℃滴加4-溴丁酸乙酯468mg,将反应液滴加到45ml冰水中析出类白色固体,过滤,收集滤饼鼓风干燥得雌二醇丁酸乙酯570mg中间体;
50ml圆底烧瓶中加入雌二醇丁酸乙酯中间体500mg,THF 10ml,20-24℃磁力搅拌15-30min后滴加1M氢氧化锂水溶液3.27ml,20-24℃磁力搅拌3-4小时后35-40℃减压浓缩,残留物中加入20ml水,1M盐酸调PH到5.0-6.5,析出类白色固体,过滤,收集滤饼鼓风干燥得到式1所示的雌二醇丁酸半抗原。
3.一种雌二醇抗原,是将式1所示产物和载体蛋白偶联得到的偶联物。
4.根据权利要求3所述的雌二醇抗原,其特征在于,所述载体蛋白包括牛血清白蛋白,卵清蛋白,人血清白蛋白,鼠血清白蛋白,甲状腺蛋白或血蓝蛋白。
5.权利要求3所述雌二醇抗原的制备方法,包括如下步骤:将15.96mg雌二醇半抗原用1.5ml DMF溶解,加入EDC 21.5mg,加入NHS 13mg,室温搅拌,形成反应液;称取BSA 50mg溶于3.5ml 0.1M碳酸氢钠溶液中,形成BSA溶液,将所述反应液在冰浴条件下边搅拌边逐滴加入到BSA溶液中,搅拌转速为1000rpm;将反应产物装入蒸馏水冲洗干净透析袋,1L 0.01MPBS 4℃搅拌,透析,制备得到雌二醇抗原。
6.权利要求3所述雌二醇抗原在制备雌二醇特异性抗体中的应用。
7.应用权利要求3所述雌二醇抗原制备得到的特异性抗体。
8.权利要求1所述产物、权利要求3所述雌二醇抗原、权利要求7所述抗体在检测雌二醇中的应用。
9.应用权利要求1所述产物、权利要求3所述雌二醇抗原、权利要求7所述特异性抗体制备得到的酶联免疫检测试剂盒。
10.权利要求9所述酶联免疫检测试剂盒,其特征在于,它包括:包被有雌二醇抗原的酶标板、酶标抗体工作液、雌二醇系列标准品、底物显色液、终止液、浓缩复溶液、浓缩洗涤液。
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