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软骨藻酸多克隆抗体的制备及间接竞争ELISA检测法的建立

李炳喜 刘靖 李扬 叶剑敏

李炳喜, 刘靖, 李扬, 叶剑敏. 软骨藻酸多克隆抗体的制备及间接竞争ELISA检测法的建立[J]. 华南师范大学学报(自然科学版), 2020, 52(2): 68-76. doi: 10.6054/j.jscnun.2020029
引用本文: 李炳喜, 刘靖, 李扬, 叶剑敏. 软骨藻酸多克隆抗体的制备及间接竞争ELISA检测法的建立[J]. 华南师范大学学报(自然科学版), 2020, 52(2): 68-76. doi: 10.6054/j.jscnun.2020029
LI Bingxi, LIU Jing, LI Yang, YE Jianmin. Preparation of Polyclonal Antibody Against Domoic Acid and Establishment of Indirect Competitive ELISA Assay[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(2): 68-76. doi: 10.6054/j.jscnun.2020029
Citation: LI Bingxi, LIU Jing, LI Yang, YE Jianmin. Preparation of Polyclonal Antibody Against Domoic Acid and Establishment of Indirect Competitive ELISA Assay[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(2): 68-76. doi: 10.6054/j.jscnun.2020029

软骨藻酸多克隆抗体的制备及间接竞争ELISA检测法的建立

doi: 10.6054/j.jscnun.2020029
基金项目: 

国家自然科学基金项目 31472302

广州市科技计划项目 201607010370

详细信息
    通讯作者:

    李扬,研究员,Email:liyang@scnu.edu.cn

    叶剑敏,教授,青年千人计划获得者,Email:yjmying@126.com

  • 中图分类号: S965.3

Preparation of Polyclonal Antibody Against Domoic Acid and Establishment of Indirect Competitive ELISA Assay

  • 摘要: 软骨藻酸(Domoic Acid, DA)是记忆缺失性贝毒(Amnesic Shellfish Poisoning,ASP)的主要成分,能在鱼类、贝类富集,通过食物链的传递作用对人产生毒性作用. DA是小分子半抗原,运用活泼酯法将DA与载体蛋白钥孔血蓝蛋白(KLH)和牛血清白蛋白(BSA)偶联,用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)提高偶联效率,通过紫外-可见分光光度法和聚丙烯凝胶电泳(SDS-PAGE)检测鉴定,成功制备偶联比为44 : 1的完全免疫抗原DA-KLH和偶联比为16 : 1的包被抗原DA-BSA; 用DA-KLH免疫小鼠后获得高达210 000 units/mL效价的抗血清,并通过不断优化间接竞争ELISA(ic-ELISA)检测DA的条件,确定各项最佳工作质量浓度及条件,最后成功绘制100 ng/mL~10 μg/mL范围内的DA检测标准曲线.通过优化ic-ELISA检测方法成功实现了快速定量检测DA,这对水产品藻毒素检测试剂盒的开发具有指导意义,也为建立赤潮毒素监督体系提供了良好的实验基础.
  • 图  1  免疫抗原DA-KLH的紫外光谱扫描图

    Figure  1.  The UV absorption of DA-KLH

    图  2  包被抗原DA-BSA的紫外光谱扫描图

    Figure  2.  The UV absorption of DA-BSA

    图  3  SDS-PAGE的凝胶电泳检测分析

    Figure  3.  The analysis of SDS-PAGE

    图  4  小鼠抗血清效价

    Figure  4.  The antibody titer of mice antisera

    图  5  间接竞争ELISA法测定DA质量浓度的标准曲线拟合曲线

    Figure  5.  The fitting DA curve with ic-ELISA

    图  6  间接竞争ELISA法测定DA质量浓度的标准曲线

    Figure  6.  The standard DA curve with ic-ELISA

    表  1  不同包被抗原质量浓度及多抗稀释比的OD

    Table  1.   The optimal concentration of coating antigen and the diluted multiple of polyclonal antibody

    ρ(DA-BSA)/
    (μg·mL-1)
    DA-KLH多抗稀释比
    1:100 1:200 1:400 1:800 1:1 600 1:3 200 1:6 400 1:12 800 1:25 600 1:51 200 1:102 400 空白
    8.000 1.142 1.910 2.511 3.062 3.136 2.572 2.298 1.593 0.991 0.581 0.348 0.108
    4.000 1.517 2.016 2.678 2.993 3.217 2.839 2.096 1.614 0.960 0.590 0.354 0.095
    2.000 1.440 1.542 2.380 2.599 2.635 2.349 1.759 1.194 0.697 0.410 0.239 0.076
    1.000 1.188 1.358 1.877 2.145 2.012 1.872 1.299 0.974 0.621 0.398 0.265 0.076
    0.500 1.056 1.002 1.282 1.512 1.399 1.115 0.749 0.521 0.326 0.199 0.145 0.070
    0.250 0.974 0.985 1.182 1.205 1.152 0.859 0.559 0.377 0.242 0.169 0.116 0.074
    0.125 0.723 0.676 0.726 0.735 0.624 0.492 0.322 0.236 0.182 0.123 0.100 0.077
    0 0.218 0.174 0.166 0.150 0.133 0.113 0.092 0.088 0.080 0.088 0.084 0.083
    下载: 导出CSV

    表  2  二抗稀释比对vmax和OD的影响

    Table  2.   The effect of different diluted multiple of the HRP-IgG

    二抗稀释比 vmax/(OD·min-1) OD
    平均vmax SD Cv 平均OD SD Cv P/N 阴性对照 空白对照
    1:1 000 12.833 2.374 18.502 1.577 0.190 12.030 26.746 0.059 0.087
    1:2 000 7.937 1.541 19.420 1.164 0.191 16.373 19.412 0.060 0.090
    1:4 000 4.475 1.223 27.343 0.702 0.154 21.999 12.660 0.055 0.035
    1:8 000 2.465 0.475 19.273 0.428 0.090 20.910 7.544 0.057 0.023
    注:Vmax:最大反应速率;SD:方差;OD:吸光值;Cv:孔间差异系数;P/N:及阳性孔与阴性孔OD的比值.
    下载: 导出CSV

    表  3  抗原包被温度和时间对vmax和OD的影响

    Table  3.   The effect of different temperatures and time lengths of the coating antigen

    温度/℃ 时间/h vmax/(OD·min-1) OD
    平均vmax SD Cv 平均OD SD Cv P/N 阴性对照 空白对照
    4 过夜 10.986 0.805 7.323 1.327 0.094 7.049 19.828 0.067 0.067
    37 0.5 1.780 0.370 20.786 0.233 0.036 15.224 4.150 0.056 0.053
    37 1.0 11.790 0.269 2.281 1.478 0.024 1.643 25.326 0.058 0.058
    37 2.0 12.546 0.448 3.573 1.507 0.092 6.076 20.130 0.075 0.069
    25 1.0 6.395 1.016 15.895 0.844 0.135 15.962 12.721 0.066 0.057
    25 2.0 12.654 1.295 10.237 1.461 0.032 2.176 25.660 0.057 0.382
    下载: 导出CSV

    表  4  封闭时间对vmax和OD的影响

    Table  4.   The effect of different blocking time lengths

    封闭时间/h vmax/(OD·min-1) OD
    平均vmax SD Cv 平均OD SD Cv P/N 阴性对照 空白对照
    0.5 11.350 0.340 2.998 1.691 0.037 2.167 15.471 0.109 0.112
    1.0 12.868 0.565 4.389 1.800 0.074 4.137 15.034 0.120 0.117
    2.0 11.977 6.035 50.386 1.534 0.752 49.056 15.036 0.102 0.106
    下载: 导出CSV

    表  5  抗原与多抗作用时间对vmax和OD的影响

    Table  5.   The effect of time of antigen and polyclonal antibody incubation

    作用时间/h vmax/(OD·min-1) OD
    平均vmax SD Cv 平均OD SD Cv P/N 阴性对照 空白对照
    0.5 8.425 0.811 9.624 2.398 0.100 4.160 19.734 0.122 0.137
    1.0 14.428 1.850 12.822 2.087 0.311 14.899 15.054 0.139 0.109
    2.0 5.318 4.897 92.088 0.677 0.641 94.588 6.261 0.108 0.100
    下载: 导出CSV

    表  6  二抗作用时间对vmax和OD的影响

    Table  6.   The effect of time of enzyme marked secondary antibody incubation

    作用时间/h vmax/(OD·min-1) OD
    平均vmax SD Cv 平均OD SD Cv P/N 阴性对照 空白对照
    0.5 11.018 0.448 4.066 1.384 0.103 7.471 7.353 0.188 0.129
    1.0 13.589 0.969 7.128 1.769 0.270 15.257 19.800 0.089 0.067
    2.0 18.430 2.392 12.976 2.286 0.092 4.031 19.992 0.114 0.113
    下载: 导出CSV

    表  7  间接ELISA法测定不同质量浓度DA的结果

    Table  7.   The analysis of ic-ELISA determination of different DA concentrations

    ρ(DA)/(ng·mL-1) OD B/B0/%
    0 0.351 100
    1.0 0.227 64.606
    2.5 0.223 63.495
    5.0 0.238 67.825
    10 0.232 66.030
    25 0.223 63.467
    50 0.197 56.146
    100 0.162 46.233
    250 0.099 28.315
    500 0.078 22.077
    1 000 0.065 18.402
    2 500 0.059 16.778
    5 000 0.060 17.092
    10 000 0.054 15.382
    下载: 导出CSV

    表  8  贝类样品中DA质量分数的检测

    Table  8.   The concentration of DA in shellfish

    样品 组织 平均OD 抑制率/% w(DA)/
    (ng·g-1)
    紫贻贝(青岛) 消化腺 0.172 49.110 57.009
    肌肉 0.087 24.688 675.943
    扇贝(獐子岛) 消化腺 0.125 35.589 224.156
    肌肉 0.144 41.048 128.960
    北极贝(日本) 消化腺 0.118 33.613 273.781
    肌肉 0.181 51.702 43.848
    美国贝(朝鲜) 消化腺 0.141 40.032 142.933
    肌肉 0.127 28.249 213.430
    下载: 导出CSV
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  • 收稿日期:  2019-01-21
  • 刊出日期:  2020-04-25

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