直接竞争酶联免疫法测定食品中的丙烯酰胺含量
吴璟等
摘要丙烯酰胺由于分子量小、结构简单,制备其特异性抗体难以实现。本研究将丙烯酰胺与对巯基苯乙酸衍生合成半抗原,偶联载体蛋白并免疫动物制备针对丙烯酰胺衍生物的特异性抗体,并进行辣根过氧化物酶标记,进而建立通过检测丙烯酰胺衍生物实现对丙烯酰胺定量分析的直接竞争酶联免疫分析方法。本方法对丙烯酰胺的检出限为3.0 μg/L,线性范围为9.2~195 μg/L,对饼干、薯片及咖啡样品中丙烯酰胺的平均添加回收率为83.6%~112.7%,结果与标准检测方法HPLCMS/MS符合。本方法可用于食品样品中丙烯酰胺的快速检测。
1引言
丙烯酰胺(Acrylamide)广泛存在于油炸或焙烤的马铃薯和谷物类食品中\[1\],被国际癌症研究机构列为2A类致癌物质。目前,丙烯酰胺的检测方法主要有色谱法\[2~5\]、毛细管电泳\[6\]、质子传递反应质谱\[7\]等,这些方法需要昂贵的检测仪器、费时费力,难以适应量大面广的检测需求。
基于抗原抗体的免疫分析方法具有灵敏度好、特异性高、操作简便及检测成本低等特点\[8\],可以作为仪器检测方法的有效补充。但是,由于丙烯酰胺的结构简单,分子量小(71.08 Da),即使与载体蛋白偶联后也难以有效刺激动物免疫应答产生抗体\[1\]。尽管文献\[9,10\]以丙烯酰胺的结构类似物N丙烯酰氧基琥珀酰亚胺(NAS)作为半抗原,并制备了针对丙烯酰胺的多克隆抗体,但此法重复性差,本实验室通过多次重复均无法制备得到针对丙烯酰胺的抗体。Chappey等\[11\]也通过大量文献总结出,当化合物(半抗原)的分子量<300 Da时,其特异性的高亲和力抗体制备难度增大;而当半抗原分子量<150 Da时,其高特异性高亲和力的抗体几乎无法获得。
本研究针对丙烯酰胺的结构特点,利用其双键易于发生迈克尔加成反应\[12\]的特性,使其与对巯基苯乙酸发生快速、简便的衍生反应, 制备全新半抗原; 将半抗原与载体蛋白偶联, 制备针对半抗原的高特异性抗体,随后对抗体进行辣根过氧化物酶标记,进而建立通过检测丙烯酰胺衍生产物的丙烯酰胺直接竞争酶联免疫分析方法(dcELISA),经过条件优化后用于实际样品检测,且与HPLCMS/MS方法所得结果进行了对照。
2实验部分
2.1仪器与试剂
DRX400/600核磁共振仪(德国Burker公司),UV160A紫外可见扫描仪(日本Kyoto公司),Multiskan MK3多功能酶标仪(美国Thermo公司),AB SCIEX 5500 三重四级杆质谱仪(美国AB SCIEX公司)。
丙烯酰胺(99%)、对巯基苯乙酸(99.5%)购自北京百灵威试剂公司;辣根过氧化物酶(HRP)、匙孔血蓝蛋白(KLH)购自美国Sigma公司;包被液、洗涤液、封闭液均按文献方法配制;稀释液为磷酸盐缓冲液(0.2 g/L KH2PO4,2.9 g/L Na2HPO
2.2.2人工抗原的合成与鉴定采用活泼酯法\[14\]将半抗原MPA与载体蛋白KLH和OVA分别偶联,如图1所示。采用紫外光谱法对偶联产物进行鉴定\[15\]。
2.2.3酶标记多克隆抗体的制备与纯化将制备的抗原MPAKLH按照文献\[16\]的免疫方案,免疫两只1.8~2.2 kg雌性新西兰大白兔,得兔抗血清,用辛酸硫酸铵\[17\]法纯化抗血清,并按照文献\[16\]制备酶标记多克隆抗血清,
Symbolm@@ 20 ℃保存备用。
2.2.4dcELISA方法的建立根据文献\[18\]所述的dcELISA的操作步骤,通过棋盘滴定法确定最佳抗体稀释倍数及包被原浓度;通过单因素实验,考察吐温20浓度,离子浓度对dcELISA检测方法的影响。以Amax/IC50的比值作为评价各影响因素的标准,比值越大,则代表灵敏度越高\[18\]。选取最佳的反应条件,使用Origin8.5软件,按照四参数对数拟合绘制MPA标准曲线。
2.2.5方法的特异性以交叉反应率评价dcELISA方法的特异性,交叉反应率(CR)越高,特异性越差\[19\]。用公式(1)计算各竞争物与抗MPA抗体的交叉反应率。
2.3添加回收率的测定
对市售薯片、饼干、咖啡样品,按0, 50, 200和1000 μg/kg的比例,分别添加丙烯酰胺标准品,粉碎混匀。每份样品称取2 g,加入20 mL蒸馏水,漩涡振荡2 min,8000 r/min离心10 min,取10 mL上清液再次离心,加入20 mL正己烷漩涡振荡脱脂取水层。取2 mL提取液用于HPLCMS/MS分析;另取5 mL上述提取液, 加入2 mg对巯基苯乙酸标准品,调至pH 9.6, 50 ℃下暗处反应30 min,反应结束后调至pH 7.4, 用乙酸乙酯萃取衍生产物MPA,氮气吹干乙酸乙酯后, 加1 mL 5% 甲醇PBST溶液复溶衍生产物,用于dcELISA分析。每个添加量做3个平行实验,计算本方法的添加回收率。
3.2人工抗原的鉴定
采用活泼酯法制备得到人工抗原,并通过测定其紫外吸收图谱对其进行结构鉴定。从图3可知,人工抗原MPAKLH和MPAOVA的紫外吸收图谱同时具备了MPA和载体蛋白的紫外吸收特征,说明人工抗原偶联成功。
3.3血清效价及抑制率的测定
采用间接ELISA测得抗血清的效价为1∶64000;采用间接竞争ELISA测定抗血清的抑制率,结果表明,此抗血清可以识别半抗原MPA,但无法识别游离丙烯酰胺。当使用半抗原MPA作为抑制药物时,1 mg/L对抗体效价的抑制率为80.6%。
3.4MPA抗血清dcELISA方法的建立
对包被原浓度、抗体稀释倍数、稀释液体系、吐温20浓度、PO3
Symbolm@@ 4浓度、甲醇体积分数等工作条件进行优化,以Amax/IC50值为判断标准,其值越大,方法的检测效果越好\[18\]。通过对ELISA工作条件优化,确定的最佳工作条件参数如下:包被浓度为250 μg/L,抗体稀释倍数为16000倍,缓冲体系为含500 μL/L吐温、 PO3
Symbolm@@ 4浓度为400 mmol/L的PBST溶液。甲醇体积分数低于5%时,IC50值及Amax/IC50没有显著变化,对dcELISA工作效果没有太大影响。根据上述条件,绘制标准曲线(图4)。本方法的半抑制浓度(IC50)为45.5 μg/L,检出限(LOD)为3.0 μg/L,线性检测范围为9.2~195 μg/L。
3.5方法特异性分析
选取衍生反应物、丙烯酰胺类似物及其类似衍生物做交叉反应。由表1可知,抗体与其它药物的交叉反应率均小于1%,说明本方法所用的多克隆抗体对MPA具有很高的特异性。
[TS(16Y][
3.6添加回收实验
由于丙烯酰胺在食品内可通过麦拉德反应自发形成\[1\],存在于高温加工淀粉类食品中,不含丙烯酰胺的高温加工淀粉类阴性样品极少见。为此,本实验先用HPLCMS/MS法准确测定空白样品中丙烯酰胺的含量,在此基础上添加已知浓度的丙烯酰胺,采用dcELISA进行检测,扣除空白样品丙烯酰胺的含量后,计算回收率;两种方法对比结果见表2。dcELISA对样品的平均回收率在83.6%~112.7% 之间,HPLCMS/MS对样品的平均回收率在74.5%~96.5%之间,说明所建立的dcELISA方法准确度和精密度好,可用于实际样品检测。由于制备直接针对丙烯酰胺的特异性抗体难度大,本实验利用丙烯酰胺的双键易于发生迈克尔加成的特点,将其与对巯基苯乙酸衍生,制备获得特征结构显著的半抗原,将其与载体蛋白偶联并免疫动物,获得了可以特异性识别半抗原的高特异性抗体。检测丙烯酰胺前,通过进行衍生化,即可利用所建立的ELISA方法对衍生物含量进行检测,从而推算出样品中丙烯酰胺的含量。丙烯酰胺的衍生时间约为30 min,衍生率为87.1%,且所建立的ELISA方法检测时间少于1 h,检测成本低,特别适合大量样品的快速筛查。本研究对于今后开展食品样品中丙烯酰胺含量的监测与监管具有重要意义。
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18Liang C, Jin R, Gui W, Zhu G. Environ. Sci. Technol., 2007, 41(19): 6783-6788
19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization
15Shen Y D, Wang Y, Zhang S W, Xiao Z L, Sun Y M, Bu X Z, Gu L Q. Chinese Chem. Letter., 2007, 18: 1490-1492
16Meulenberg E P. J. Toxins, 2012, 4(4): 244-266
17CHEN Dan, SUN GuangRui, LIU ZengShan. J. Anhui Agric. Sci., 2007, 35(26): 8105-8108
陈 丹, 孙广瑞, 柳增善. 安徽农业科学, 2007, 35(26): 8105-8108
18Liang C, Jin R, Gui W, Zhu G. Environ. Sci. Technol., 2007, 41(19): 6783-6788
19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization
15Shen Y D, Wang Y, Zhang S W, Xiao Z L, Sun Y M, Bu X Z, Gu L Q. Chinese Chem. Letter., 2007, 18: 1490-1492
16Meulenberg E P. J. Toxins, 2012, 4(4): 244-266
17CHEN Dan, SUN GuangRui, LIU ZengShan. J. Anhui Agric. Sci., 2007, 35(26): 8105-8108
陈 丹, 孙广瑞, 柳增善. 安徽农业科学, 2007, 35(26): 8105-8108
18Liang C, Jin R, Gui W, Zhu G. Environ. Sci. Technol., 2007, 41(19): 6783-6788
19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization
摘要丙烯酰胺由于分子量小、结构简单,制备其特异性抗体难以实现。本研究将丙烯酰胺与对巯基苯乙酸衍生合成半抗原,偶联载体蛋白并免疫动物制备针对丙烯酰胺衍生物的特异性抗体,并进行辣根过氧化物酶标记,进而建立通过检测丙烯酰胺衍生物实现对丙烯酰胺定量分析的直接竞争酶联免疫分析方法。本方法对丙烯酰胺的检出限为3.0 μg/L,线性范围为9.2~195 μg/L,对饼干、薯片及咖啡样品中丙烯酰胺的平均添加回收率为83.6%~112.7%,结果与标准检测方法HPLCMS/MS符合。本方法可用于食品样品中丙烯酰胺的快速检测。
1引言
丙烯酰胺(Acrylamide)广泛存在于油炸或焙烤的马铃薯和谷物类食品中\[1\],被国际癌症研究机构列为2A类致癌物质。目前,丙烯酰胺的检测方法主要有色谱法\[2~5\]、毛细管电泳\[6\]、质子传递反应质谱\[7\]等,这些方法需要昂贵的检测仪器、费时费力,难以适应量大面广的检测需求。
基于抗原抗体的免疫分析方法具有灵敏度好、特异性高、操作简便及检测成本低等特点\[8\],可以作为仪器检测方法的有效补充。但是,由于丙烯酰胺的结构简单,分子量小(71.08 Da),即使与载体蛋白偶联后也难以有效刺激动物免疫应答产生抗体\[1\]。尽管文献\[9,10\]以丙烯酰胺的结构类似物N丙烯酰氧基琥珀酰亚胺(NAS)作为半抗原,并制备了针对丙烯酰胺的多克隆抗体,但此法重复性差,本实验室通过多次重复均无法制备得到针对丙烯酰胺的抗体。Chappey等\[11\]也通过大量文献总结出,当化合物(半抗原)的分子量<300 Da时,其特异性的高亲和力抗体制备难度增大;而当半抗原分子量<150 Da时,其高特异性高亲和力的抗体几乎无法获得。
本研究针对丙烯酰胺的结构特点,利用其双键易于发生迈克尔加成反应\[12\]的特性,使其与对巯基苯乙酸发生快速、简便的衍生反应, 制备全新半抗原; 将半抗原与载体蛋白偶联, 制备针对半抗原的高特异性抗体,随后对抗体进行辣根过氧化物酶标记,进而建立通过检测丙烯酰胺衍生产物的丙烯酰胺直接竞争酶联免疫分析方法(dcELISA),经过条件优化后用于实际样品检测,且与HPLCMS/MS方法所得结果进行了对照。
2实验部分
2.1仪器与试剂
DRX400/600核磁共振仪(德国Burker公司),UV160A紫外可见扫描仪(日本Kyoto公司),Multiskan MK3多功能酶标仪(美国Thermo公司),AB SCIEX 5500 三重四级杆质谱仪(美国AB SCIEX公司)。
丙烯酰胺(99%)、对巯基苯乙酸(99.5%)购自北京百灵威试剂公司;辣根过氧化物酶(HRP)、匙孔血蓝蛋白(KLH)购自美国Sigma公司;包被液、洗涤液、封闭液均按文献方法配制;稀释液为磷酸盐缓冲液(0.2 g/L KH2PO4,2.9 g/L Na2HPO
2.2.2人工抗原的合成与鉴定采用活泼酯法\[14\]将半抗原MPA与载体蛋白KLH和OVA分别偶联,如图1所示。采用紫外光谱法对偶联产物进行鉴定\[15\]。
2.2.3酶标记多克隆抗体的制备与纯化将制备的抗原MPAKLH按照文献\[16\]的免疫方案,免疫两只1.8~2.2 kg雌性新西兰大白兔,得兔抗血清,用辛酸硫酸铵\[17\]法纯化抗血清,并按照文献\[16\]制备酶标记多克隆抗血清,
Symbolm@@ 20 ℃保存备用。
2.2.4dcELISA方法的建立根据文献\[18\]所述的dcELISA的操作步骤,通过棋盘滴定法确定最佳抗体稀释倍数及包被原浓度;通过单因素实验,考察吐温20浓度,离子浓度对dcELISA检测方法的影响。以Amax/IC50的比值作为评价各影响因素的标准,比值越大,则代表灵敏度越高\[18\]。选取最佳的反应条件,使用Origin8.5软件,按照四参数对数拟合绘制MPA标准曲线。
2.2.5方法的特异性以交叉反应率评价dcELISA方法的特异性,交叉反应率(CR)越高,特异性越差\[19\]。用公式(1)计算各竞争物与抗MPA抗体的交叉反应率。
2.3添加回收率的测定
对市售薯片、饼干、咖啡样品,按0, 50, 200和1000 μg/kg的比例,分别添加丙烯酰胺标准品,粉碎混匀。每份样品称取2 g,加入20 mL蒸馏水,漩涡振荡2 min,8000 r/min离心10 min,取10 mL上清液再次离心,加入20 mL正己烷漩涡振荡脱脂取水层。取2 mL提取液用于HPLCMS/MS分析;另取5 mL上述提取液, 加入2 mg对巯基苯乙酸标准品,调至pH 9.6, 50 ℃下暗处反应30 min,反应结束后调至pH 7.4, 用乙酸乙酯萃取衍生产物MPA,氮气吹干乙酸乙酯后, 加1 mL 5% 甲醇PBST溶液复溶衍生产物,用于dcELISA分析。每个添加量做3个平行实验,计算本方法的添加回收率。
3.2人工抗原的鉴定
采用活泼酯法制备得到人工抗原,并通过测定其紫外吸收图谱对其进行结构鉴定。从图3可知,人工抗原MPAKLH和MPAOVA的紫外吸收图谱同时具备了MPA和载体蛋白的紫外吸收特征,说明人工抗原偶联成功。
3.3血清效价及抑制率的测定
采用间接ELISA测得抗血清的效价为1∶64000;采用间接竞争ELISA测定抗血清的抑制率,结果表明,此抗血清可以识别半抗原MPA,但无法识别游离丙烯酰胺。当使用半抗原MPA作为抑制药物时,1 mg/L对抗体效价的抑制率为80.6%。
3.4MPA抗血清dcELISA方法的建立
对包被原浓度、抗体稀释倍数、稀释液体系、吐温20浓度、PO3
Symbolm@@ 4浓度、甲醇体积分数等工作条件进行优化,以Amax/IC50值为判断标准,其值越大,方法的检测效果越好\[18\]。通过对ELISA工作条件优化,确定的最佳工作条件参数如下:包被浓度为250 μg/L,抗体稀释倍数为16000倍,缓冲体系为含500 μL/L吐温、 PO3
Symbolm@@ 4浓度为400 mmol/L的PBST溶液。甲醇体积分数低于5%时,IC50值及Amax/IC50没有显著变化,对dcELISA工作效果没有太大影响。根据上述条件,绘制标准曲线(图4)。本方法的半抑制浓度(IC50)为45.5 μg/L,检出限(LOD)为3.0 μg/L,线性检测范围为9.2~195 μg/L。
3.5方法特异性分析
选取衍生反应物、丙烯酰胺类似物及其类似衍生物做交叉反应。由表1可知,抗体与其它药物的交叉反应率均小于1%,说明本方法所用的多克隆抗体对MPA具有很高的特异性。
[TS(16Y][
3.6添加回收实验
由于丙烯酰胺在食品内可通过麦拉德反应自发形成\[1\],存在于高温加工淀粉类食品中,不含丙烯酰胺的高温加工淀粉类阴性样品极少见。为此,本实验先用HPLCMS/MS法准确测定空白样品中丙烯酰胺的含量,在此基础上添加已知浓度的丙烯酰胺,采用dcELISA进行检测,扣除空白样品丙烯酰胺的含量后,计算回收率;两种方法对比结果见表2。dcELISA对样品的平均回收率在83.6%~112.7% 之间,HPLCMS/MS对样品的平均回收率在74.5%~96.5%之间,说明所建立的dcELISA方法准确度和精密度好,可用于实际样品检测。由于制备直接针对丙烯酰胺的特异性抗体难度大,本实验利用丙烯酰胺的双键易于发生迈克尔加成的特点,将其与对巯基苯乙酸衍生,制备获得特征结构显著的半抗原,将其与载体蛋白偶联并免疫动物,获得了可以特异性识别半抗原的高特异性抗体。检测丙烯酰胺前,通过进行衍生化,即可利用所建立的ELISA方法对衍生物含量进行检测,从而推算出样品中丙烯酰胺的含量。丙烯酰胺的衍生时间约为30 min,衍生率为87.1%,且所建立的ELISA方法检测时间少于1 h,检测成本低,特别适合大量样品的快速筛查。本研究对于今后开展食品样品中丙烯酰胺含量的监测与监管具有重要意义。
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19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization
15Shen Y D, Wang Y, Zhang S W, Xiao Z L, Sun Y M, Bu X Z, Gu L Q. Chinese Chem. Letter., 2007, 18: 1490-1492
16Meulenberg E P. J. Toxins, 2012, 4(4): 244-266
17CHEN Dan, SUN GuangRui, LIU ZengShan. J. Anhui Agric. Sci., 2007, 35(26): 8105-8108
陈 丹, 孙广瑞, 柳增善. 安徽农业科学, 2007, 35(26): 8105-8108
18Liang C, Jin R, Gui W, Zhu G. Environ. Sci. Technol., 2007, 41(19): 6783-6788
19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization
15Shen Y D, Wang Y, Zhang S W, Xiao Z L, Sun Y M, Bu X Z, Gu L Q. Chinese Chem. Letter., 2007, 18: 1490-1492
16Meulenberg E P. J. Toxins, 2012, 4(4): 244-266
17CHEN Dan, SUN GuangRui, LIU ZengShan. J. Anhui Agric. Sci., 2007, 35(26): 8105-8108
陈 丹, 孙广瑞, 柳增善. 安徽农业科学, 2007, 35(26): 8105-8108
18Liang C, Jin R, Gui W, Zhu G. Environ. Sci. Technol., 2007, 41(19): 6783-6788
19DENG Hao, KONG DeBin, Yang JinYi, XU ZhenLin, SHEN YuDong, YANG XingXing, SUN YuanMing. Chinese J. Anal. Chem., 2013, 41(2): 247-252
邓 浩, 孔德彬, 杨金易, 徐振林, 沈玉栋, 杨星星, 孙远明. 分析化学, 2013, 41(2): 247-252
AbstractDue to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative (hapten) was obtained. The antibody was labeled with horseradish peroxidase (HRP) and used to develop a direct competitive enzymelinked immunosorbent assay (dcELISA). The dcELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45.49 μg/L, a limit of detection of 3.0 μg/L and the linear range of 9.2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83.6% to 112.7%. Good correlations between the results of dcELISA and standard HPLCMS/MS were obtained. The proposed dcELISA is suitable for the determination of acrylamide in food samples.
KeywordsAcrylamide; Polyclonal antibody; Direct competitive enzymelinked immunosorbent assay; Derivatization