我国贝类中赤潮毒素腹泻性贝毒免疫检测技术研究
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摘要
腹泻性贝毒(Diarrhetic Shellfish Poisoning,DSP)是由有毒赤潮藻类鳍藻属和原甲藻属的一些种类产生的一类脂溶性多环醚类生物活性物质,主要成分是软海绵酸(Okadaic Acid,OA)及其衍生物。腹泻性贝毒可在贝等滤食性动物体内富集,危害食用者健康。腹泻性贝毒是世界范围内具有最严重威胁的赤潮藻毒素之一。我国近年来每年均有发现腹泻性贝毒的报道。腹泻性贝毒的主要分析方法有液相色谱法、免疫学法以及生物毒性法。本文采用碳二亚胺缩合方法合成了软海绵酸与不同蛋白载体偶联物,分别用作免疫抗原和包被抗原,免疫Balb/C鼠,采用细胞融合技术,制备了抗腹泻性贝毒主要成份软海绵酸的1种多克隆抗体、2种单克隆抗体,研究了应用这些抗体发展建立软海绵酸的间接竞争酶联免疫检测和胶体金标记免疫层析检测技术。结果表明:用抗OA-BSA单克隆抗体建立的间接竞争酶联免疫检测OA方法,最低检出限为31.2ng/ml,平均回收率99.8%,平均变异系数8.7%。用抗OA-BSA单克隆抗体建立的胶体金标记免疫层析快速检测OA方法,检测时间15min,检出限为500ng/ml。用抗OA-KLH多克隆抗体建立的间接竞争酶联免疫检测OA方法,海水样品加标回收率在62～114%之间,批内变异系数在22～24%之间;贝类样品加标回收率在51～78%之间,批内变异系数在1～10%之间;用3#多克隆抗体建立的方法分析OA的检出限为0.1ng/ml,对于海水相当于10 ng/ml,对于贝类相当于50 ng/g。用抗OA-KLH单克隆抗体建立的间接竞争酶联免疫检测OA方法,海水的加标平均回收率96.6%,扇贝的加标平均回收率86.2%;用1# OA-KLH单克隆抗体建立的分析OA的方法检出限小于0.8ng/ml,对于海水31.2ng/ml,对于贝156.2ng/g贝肉;用抗OA-KLH单克隆抗体建立的胶体金标记免疫层析快速检测OA方法,检测时间15min,检出限12ng/ml。本研究建立的酶联免疫吸附分析和胶体金标记免疫层析检测贝类中软海绵酸的方法可以满足规定的20μg/100g贝肉的安全阈值,应用于贝类实际样品的分析,为我国腹泻性贝毒的监测、保障食品安全提供技术基础和快速检测产品。
Diarrhetic shellfish poisoning (DSP) is a kind of lipophylic natural biotoxin produced by the Dinophysis and Prorocentrum.Okadaic acid (OA) and its derivatives have been reported to be the principal components of DSP toxin which may cause eaters to suffer from diarrhea, nausea, vomit, and gastrointestinal cramping pain and are a strong tumor promoter . At present, DSP are mainly analyzed by mouse bioassay, high-performance liquid chromatography(HPLC) and some immunological methods.OA were conjugated to KLH, BSA and OVA using carbodiimide reaction. The Balb/C mice were immunized by ip injection with OA-KLH and OA-BSA. Monoclonal antibody were prepared throughout fusing the spleen cells of immunized mice with Sp2/O cells. The polyclonal antibodies and monoclonal antibodies against OA were producted.
An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using 3# polyclonal antibody against OA. Under optimal condition, the detection limit of OA was 0.1ng/ml. The quantitative limit for OA was 10ng/ml for sea water, 50ng/g for shellfish, respectively. The recovery of OA added to sea water was 62%～114%, with a coefficient of variation of 22%～24%;the recovery of OA added to shellfish was 51%～78%, with a coefficient of variation of 1%～10%.
The Monoclonal antibody prepared using OA-KLH as the immunogen were applied in development the idc-ELISA(indirect competitive enzyme-linked immunosorbent assay) for detection OA. The idc-ELISA method with detection limit of 0.8ng/ ml was developed using 1# monoclonal antibody with the highest specificity against OA. An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using monoclonal antibody against OA prepared using OA-BSA conjugate as immunogen, the detection limit of OA was 31.2ng/ml, the mean recovery was 99.8%, with a mean coefficient of variation of 8.7%. An immunochromatographic strip test was developed for detection of okadaic acid by using colloidal gold-labeled monoclonal antibody(OA-BSA) of the okadaic acid molecule as the detection antibody.The lower detection limit for OA was 50ng/strip, 500ng/ ml.A Gold Immunochromatography Assay(GICA) was developed for detection of okadaic acid by using an colloidal gold-labeled monoclonal antibody(OA-KLH) of the okadaic acid molecule as the detection antibody. The test works on the principle of lateral flow immunochromatography using a strip format,providing a qualitative (yes/no) indication of the presence of OA within 15 min. At 12ng/ ml OA levels, the test is applicable only as a qualitative assay. The major advantages of the step strip test are that results can be obtained within 15 min and that all reagents are included in the test device.
Diarrhetic shellfish poisoning (DSP) is a kind of lipophylic natural biotoxin produced by the Dinophysis and Prorocentrum.Okadaic acid (OA) and its derivatives have been reported to be the principal components of DSP toxin which may cause eaters to suffer from diarrhea, nausea, vomit, and gastrointestinal cramping pain and are a strong tumor promoter . At present, DSP are mainly analyzed by mouse bioassay, high-performance liquid chromatography(HPLC) and some immunological methods.OA were conjugated to KLH, BSA and OVA using carbodiimide reaction. The Balb/C mice were immunized by ip injection with OA-KLH and OA-BSA. Monoclonal antibody were prepared throughout fusing the spleen cells of immunized mice with Sp2/O cells. The polyclonal antibodies and monoclonal antibodies against OA were producted.
An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using 3# polyclonal antibody against OA. Under optimal condition, the detection limit of OA was 0.1ng/ml. The quantitative limit for OA was 10ng/ml for sea water, 50ng/g for shellfish, respectively. The recovery of OA added to sea water was 62%～114%, with a coefficient of variation of 22%～24%;the recovery of OA added to shellfish was 51%～78%, with a coefficient of variation of 1%～10%.
The Monoclonal antibody prepared using OA-KLH as the immunogen were applied in development the idc-ELISA(indirect competitive enzyme-linked immunosorbent assay) for detection OA. The idc-ELISA method with detection limit of 0.8ng/ ml was developed using 1# monoclonal antibody with the highest specificity against OA. An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using monoclonal antibody against OA prepared using OA-BSA conjugate as immunogen, the detection limit of OA was 31.2ng/ml, the mean recovery was 99.8%, with a mean coefficient of variation of 8.7%. An immunochromatographic strip test was developed for detection of okadaic acid by using colloidal gold-labeled monoclonal antibody(OA-BSA) of the okadaic acid molecule as the detection antibody.The lower detection limit for OA was 50ng/strip, 500ng/ ml.A Gold Immunochromatography Assay(GICA) was developed for detection of okadaic acid by using an colloidal gold-labeled monoclonal antibody(OA-KLH) of the okadaic acid molecule as the detection antibody. The test works on the principle of lateral flow immunochromatography using a strip format,providing a qualitative (yes/no) indication of the presence of OA within 15 min. At 12ng/ ml OA levels, the test is applicable only as a qualitative assay. The major advantages of the step strip test are that results can be obtained within 15 min and that all reagents are included in the test device.
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