軟海綿酸單株抗體之製備並將其應用於酵素連結免疫分析法及奈米金粒子免疫層析試紙分析法之開發

小分子毒素常見於日常生活中，誤食這些小分子毒素容易對我們的健康造成危害，本研究以軟海綿酸 ( Okadaic acid, OA) 為主題。軟海綿酸是一常見的藻類毒素，經藻類釋放後會由貝類的濾食而累積於它們的消化道而被動物和人們誤食，因此希望建立一套有效的檢測方式來檢測食品中的 OA。我們以抗體抗原之間具有專一性的特性，利用製備抗體來建立其檢測方法。首先，將 OA 毒素接合至載體蛋白質 γ-globulin 使其具有免疫性，並且以此接合物免疫小鼠，取其脾臟細胞與骨髓瘤細胞 (P3/NS1/1-AG4-1 myeloma cells) 融合篩選出對 OA 具有專一性的細胞株，成功的製備了針對 OA 的單株抗體，6B1A3，並且開發出檢測 OA 的直接競爭型酵素免疫吸附分析法 (Competitive direct enzyme-linked immunosorbent assay，cdELISA)，其抑制 50 % 抗原-酵素接合物與抗體結合所需的抗原濃度 (IC50) 為 0.070 ng/mL。由於 cdELISA 需要在實驗室中才能運作，為了開發一套簡便且適合一般大眾的檢測方式，我們也成功的開發出檢測 OA 的快速免疫層析試紙 (immunochromatographic strip, immunostrip) 用於檢測 OA，其偵測限制量 (detection limit) 為 5 ng/mL。利用 cdELISA 與 immunostrip 檢測樣品時，兩種檢測方式的結果具有良好的一致性。我們開發的 cdELISA 及 immunostrip 能快速且有效的檢測樣品中的小分子毒素，以避免大眾受到這些小分子毒素的危害。

關鍵字

軟海綿酸；單株抗體；奈米金粒子；免疫層析試紙

並列摘要

Okadaic acid (OA) is a common algae toxin that accumulate in shellfish, clam and oyster. It has been identified clearly as a protein phosphatase inhibitor that generally was accumuated in gums causing diarrhea called diarrhea shellfish poisoning. This study focused on OA and developed an efficient method to detect the toxin. For producing the monoclonal antibody, OA was conjugated to carrier proteins (γ-globulin) as an immunogen to immunize the mice. A monoclonal antibody (mAb) specific to OA was produced from a stable hybridoma cell line, 6B1A3, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with OA-γ-globulin. To generate handy and sensitive detection methods, the direct competetive ELISA and gold nanoparticle were established for antibody characterization. The concentration causing 50% inhibition (IC50) of binding of OA-horseradish peroxidase to the antibody by okadaic acid was found to be 0.07 0 ng/ml. The immunochromatographic strip assay, which was generated by conjugating mAb with gold nanoparticle, has a detection limit of 5 ng/ml for OA. Results of samples analysis obtained from gold-nanoparticle based rapid immunochromatographic strips were in a good agreement with those obtained from cdELISA. The cdELISA and the rapid immunochromatographic strip can be rapid and efficient to detect the toxin in the samples. The result indicates that the specific and sensitive detection methods, which could rapidly screen the content of okadaic acid in shellfish samples, were established in this study.

並列關鍵字

Okadaic acid ； monoclonal antibody ； gold nanoparticle ； immunochromatographic strip

參考文獻

Aase, B., Rogstad, A., 1997. Optimization of sample cleanup procedure for determination of diarrhoeic shellfish poisoning toxins by use of experimental design. J Chromatogr A 764, 223-231.

Bialojan, C., Takai, A., 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256, 283-290.

Carmichael, W.W., 1992. Cyanobacteria secondary metabolites-the cyanotoxins. J Appl Bacteriol 72, 445-459.

Codd, G.A., Ward, C.J., Bell, S.G., 1997. Cyanobacterial toxins: occurrence, modes of action, health effects and exposure routes. Arch Toxicol Suppl 19, 399-410.

FAO, 2004. Marine biotoxins. FAO food and nutrition paper 80, 53.

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軟海綿酸單株抗體之製備並將其應用於酵素連結免疫分析法及奈米金粒子免疫層析試紙分析法之開發

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