氟甲磺氯黴素單株抗體和萊克多巴胺多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

Production of Monoclonal Antibodies and Polyclonal Antibodies and Development of ELISA and Gold Nanoparticle Immunochromatographic Strip Assay for Florfenicol and Ractopamine

劉韋廷(Wei-Ting Liu)

指導教授 ： 余豐益

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2016年)

摘要

氟甲磺氯黴素 (Florfenicol, FF) 是一種人工合成 amphenicol 類之廣效型抗生素，藉由干擾胜肽轉換酶 (peptidyl transferase) 進行蛋白質轉譯，以達到抑制細菌滋生的目的，常應用於水產、畜牧動物細菌性疾病的預防與治療。民眾若誤食含有 FF 殘留的肉品或蛋類，研究指出 FF 具雄性生殖毒性，可能導致精細胞減少或增加間質細胞癌的風險；萊克多巴胺 (Ractopamine, Rac)，為一種人工合成酚乙醇胺（phenethanolamine）類的乙型感受體 (β-receptor) 興奮劑，能舒緩平滑肌，原本是想研發治療人類的氣喘疾病，由於心臟、脂肪組織及肌肉上也分布有乙型感受體，動物實驗中意外發現可增加老鼠的體重，並增加瘦肉的比例；後來添加在飼料餵食豬隻，促進豬隻的蛋白質合成且加速脂肪轉化分解，所以 Rac 俗稱為瘦肉精的一種。研究指出 Rac 具有心臟毒性，若民眾誤食含有瘦肉精殘留的肉品，可能造成心悸或心律不整等中毒症狀，嚴重更可能導致心肌受損。因此本實驗利用抗體–抗原之間專一性特性，製備抗體以建立一套快速且敏感的檢測方式，並應用於檢測食品中 FF 或是 Rac 含量。本研究利用 FF 及 Rac 和 Succinic anhydride (SH) 進行衍生，並利用 Thin layer chromatography (TLC) 結果確認衍生後產物，衍生產物經 TLC 純化後再接合載體蛋白使其具有免疫原性，之後用於免疫小鼠及紐西蘭大白兔，以產生具專一性抗體。FF 部分，本研究使用 FF-SH(L3)-DCC/NHS-BSA 作為抗原打入小鼠體內，使小鼠產生具 FF 專一性的多株抗體，並藉由此抗體來開發出檢測 FF 的直接競爭型酵素連結免疫分析法 (Competitive direct enzyme-linked immunosorbent assay, cdELISA)，其抑制 50％ 抗體結合至抗原-酵素接合物所需的 FF 濃度 (IC50) 為 3.14 ng/mL，之後取其小鼠脾臟細胞與骨髓瘤細胞融合，最後篩選出融合瘤單株細胞株 (7G81A3)，分泌對 FF 具有專一性的單株抗體，其單株抗體也可和類似物氯黴素 (Chloramphenicol, CAP) 交叉反應，抑制 50％ 抗體結合至抗原-酵素接合物所需 FF 及 CAP 濃度 (IC50) 分別為 3.7 ng/mL 和 32 ng/mL。在 Rac 部分，利用接合不同的載體蛋白質，以多種抗原來免疫紐西蘭大白兔及小鼠，製備出可辨識 Rac 的多株抗體，並用其開發 cdELISA，檢測兔子及小鼠血清的結果 (IC50) 分別為，40.2 ng/mL 和 20.4 ng/mL。其多株抗體也與其他兩種常見瘦肉精，克倫特羅 (Clenbuterol, CLE) 和沙丁胺醇 (Salbutamol, Sal) 有交叉反應，其抑制 50％ 抗體結合至抗原-酵素接合物所需的 CLE 及 Sal 濃度 (IC50) 分別為 187 ng/mL 和 32 ng/mL。由於 ELISA 仍需要在實驗室操作，為了希望能有適合一般大眾及更簡便的檢測方式，因此利用製備出的抗體與奈米金粒子相接合，開發 FF 及 Rac 的快速免疫層析試紙用於檢測 FF 或 Rac，其試紙的最低偵測量分別為 100 ng/mL、1000 ng/mL。本研究已成功利用 FF 單株抗體開發出靈敏並具有高度專一性的 ELISA 以及建立檢測試紙，此外本研究也成功產生對 Rac 具有專一性的多株抗體，並以此抗體開發 ELISA，但目前建立的免疫試紙仍需要改善，使其專一性與靈敏度更加提升，為食安問題更添一份心力，避免民眾受這些抗生素或瘦肉精影響健康。

關鍵字

氟甲磺氯黴素 ； 萊克多巴胺 ； 單株抗體 ； 多株抗體 ； 酵素連結免疫分析法 ； 免疫層析試紙

並列摘要

Florfenicol (FF) is a broad-spectrum, as a class of amphenicol synthetic antibiotics, has been widely used in poultry, livestock and aquaculture. FF has reported to have male reproductive toxicity and a long-term consumption may cause testicular atrophy, decreasing sperm cells production and increasing the risk of leydig cell tumors. Ractopamine (Rac) is a β-adrenoceptor agonist drug that used in asthma medication, which can increase protein synthesis, thereby making the animal more muscular. Rac is widely used as a feed additive for livestock, reducing the fat content of the meat and increasing the profit per animal. Rac may cause some toxic symptom, such as tachycardia, vasodilation, skeletal muscle tremor, nervousness and metabolic disturbances. For these reasons, it is quite urgent to develop a way to detect the level of FF or Rac in our daily food. In this study, we have developed the sensitive and rapid methods for both these small compounds. Firstly, we derivatived the haptens by coupling FF or Rac with SH, and then confirmed the results of derivative by TLC. Polyclonal antibodies specific for FF are generated from the mice after being immunized with FF-SH-BSA. Using the antibody from the mice, sensitive cdELISA were developed. In the cdELISA of polyclonal antibody of FF, the concentration causing 50％ inhibition (IC50) of binding of Florfenicol-horseradish peroxidase (FF-HRP) to the antibody by FF is calculated to be 3.14 ng/mL. Monoclonal antibody specific for FF was also produced from a stable hybridoma cell line, 7G81A3, which was generated by the fusion of P3/NS1/1-AG4-1 myeloma cell with spleen cells isolated from Balb/c mouse. In the cdELISA of monoclonal antibody of FF, the IC50 of binding of FF-HRP to the antibody by FF is calculated to be 3.7 ng/mL. We also produced polyclonal antibody of Rac from the mice and the rabbits, respectively, by being immunized different Rac conjugate antigens. According to the result of cdELISA of Rac, the IC50 of binding of Rac-HRP to the mice or rabbit antibody by Rac are calculated to be 40.2 ng/mL and 20.4 ng/mL respectively. Following, the immunostrip for detecting FF and Rac were established, respectively. The detection limit of strip were 100 ng/mL and 1000 ng/mL for FF and Rac, respectively. Therefore, we have been successfully generated polyclonal antibody for Rac and monoclonal antibody for FF, and apply in ELISAs. However, due to the regulatory limit level of Rac set by Taiwan-FDA is 10 ng/mL. The immunostrip established in the present study is not sensitive enough for Rac analysis.

並列關鍵字

Florfenicol ； Ractopamine ； ELISA ； monoclonal antibody ； polyclonal antibody ； immunostrip

參考文獻

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