Production of Monoclonal Antibody for Okadaic Acid and Its Utilization in an Ultrasensitive Enzyme-Linked Immunosorbent Assay and One-Step Immunochromatographic Strip
Production of Monoclonal Antibody for Okadaic Acid and Its Utilization in an Ultrasensitive Enzyme-Linked Immunosorbent Assay and One-Step Immunochromatographic Strip
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DOI：
10.1021/jf404827s
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发表时间：
2014-02-12
影响因子：
6.1
通讯作者：
Yu, Feng-Yih
中科院分区：
文献类型：
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作者：
Liu, Biing-Hui;Hung, Chun-Tse;Yu, Feng-Yih
冈田酸 (OA) 是一种常见的海洋生物毒素，在双壳类动物体内积聚并导致腹泻性贝类中毒 (DSP)。本研究从杂交瘤细胞系 6BIA3 中产生了一种针对 OA 的单克隆抗体 (mAb)，该细胞系是通过将骨髓瘤细胞 (P3/NS1/1-AG4-1) 与从用 OA-γ-球蛋白免疫的 BALB/c 小鼠分离的脾细胞融合而获得的。 6BIA3 mAb 属于免疫球蛋白 GI（κ 链）同种型。建立了竞争性直接和间接酶联免疫吸附测定 (ELISA) 来表征抗体。在cdELISA中计算出OA对OA-辣根过氧化物酶与抗体的结合产生50%抑制的浓度为0.077ng/mL。还建立了快速、灵敏的基于单克隆抗体的金纳米粒子免疫层析试纸条。该试纸条对 OA 的检测限为 5 ng/mL，可在 10 分钟内完成。通过 ELISA 对 20 个海鲜样品进行广泛分析，发现其中 10 个样品受到 OA 轻微污染，平均浓度为 0.892 ng/g。对贝类样品中 OA 的分析表明，免疫层析条获得的数据与 ELISA 获得的数据非常吻合。本研究开发的基于 mAb 的 ELISA 和免疫层析试纸条检测对于快速筛查贝类样品中的 OA 具有足够的灵敏度和准确性。
Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6BIA3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-gamma-globulin. The 6BIA3 mAb belongs to the immunoglobulin GI (kappa chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.