制备岗田酸单克隆抗体及其在超灵敏酶联免疫吸附测定和一步免疫层析中的应用。

Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

机构信息

Graduate Institute of Toxicology, College of Medicine, National Taiwan University , Taipei, Taiwan.

出版信息

J Agric Food Chem. 2014 Feb 12;62(6):1254-60. doi: 10.1021/jf404827s. Epub 2014 Jan 30.

Abstract

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, 6B1A3, 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-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ 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.

摘要

岗田酸（OA）是一种常见的海洋生物毒素，在双壳贝类中积累并导致腹泻性贝类中毒（DSP）。本研究从杂交瘤细胞系 6B1A3 中产生了一种针对 OA 的单克隆抗体（mAb），该细胞系通过融合骨髓瘤细胞（P3/NS1/1-AG4-1）和用 OA-γ球蛋白免疫的 BALB/c 小鼠的脾细胞获得。6B1A3 mAb 属于免疫球蛋白 G1（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）来表征该抗体。在 cdELISA 中，通过 OA 竞争抑制 OA-辣根过氧化物酶与抗体结合的浓度，计算出 OA 抑制 50%结合的浓度为 0.077 ng/mL。还建立了一种快速灵敏的基于 mAb 的金纳米粒子免疫层析条。该条带对 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表明，其中 10 个样品被 OA 轻微污染，平均浓度为 0.892 ng/g。贝类样品中 OA 的分析表明，免疫层析条带获得的数据与 ELISA 获得的数据吻合良好。本研究开发的基于 mAb 的 ELISA 和免疫层析条带检测法具有足够的灵敏度和准确性，可用于贝类样品中 OA 的快速筛选。