| Abstract: | 克倫特羅 (Clenbuterol, CLE) 及沙丁胺醇 (Salbutamol, Sal) 為一種腎上腺素受體的乙型促進劑 (β- adrenergic agonist)，與萊克多巴胺 (Ractopamine, Rac) 具有相似功能，通常作為支氣管擴張劑，近年來畜牧業者常將 CLE、Sal、Rac 加入飼料中，增加動物的瘦肉比例獲取較高的利潤，因此CLE、Sal 及 Rac 又統稱為瘦肉精。民眾若誤食殘留瘦肉精的肉品，可能會造成心跳過速、心律不整等中毒症狀，因此本研究希望利用抗體-抗原之間具有專一性的特性，製備抗體來建立一套快速且靈敏的檢測方式，並應用於檢測肉品中的 CLE 及 Sal 含量。 在 CLE 部分，首先將 CLE 接合至載體蛋白質 γ- globulin 使其具有免疫原性，並以此接合物免疫紐西蘭大白兔，製備出可辨識 CLE 並可與 Sal 有交叉反應的多株抗體，並用此抗體開發檢測 CLE 及 Sal 的直接競爭型酵素連結免疫吸附法 (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) 分別為 0.3 ng/mL 及 1.49 ng/mL；而 CLE 的非直接競爭型酵素連結免疫吸附法 (Competitive indirect enzyme-linked immunosorbent assay, ciELISA)，其抑制 50 % 抗原-載體蛋白與抗體結合所需的抗原濃度 (IC50) 為 0.77 ng/mL；在 Sal 部分，首先將 Sal 接合至載體蛋白質 γ- globulin 使其具有免疫原性，並以此接合物免疫紐西蘭大白兔，製備出可辨識 Sal 並可與 CLE 有交叉反應的多株抗體，並用此抗體開發檢測 CLE 及 Sal 的直接競爭型酵素連結免疫吸附法 (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) 皆為 20 ng/mL；而 Sal 的非直接競爭型酵素連結免疫吸附法 (Competitive indirect enzyme-linked immunosorbent assay, ciELISA)，其抑制 50 % 抗原-載體蛋白質與抗體結合所需的抗原濃度 (IC50) 為 35 ng/mL。 由於 ciELISA 及 cdELISA 需要在實驗室中才能操作，為了開發一套簡便且適合一般大眾的檢測方式，本研究成功利用 CLE 多株抗體開發快速免疫層析試紙 (immunochromatographic strip, immunostrip) 用於檢測 CLE 及 Sal，其檢測限制量 (detection limit) 分別為 10 ng/mL、20 ng/mL；本研究也成功利用 Sal 多株抗體開發快速免疫層析試紙 (immunochromatographic strip, immunostrip) 用於檢測 CLE 及 Sal，其檢測限制量 (detection limit) 皆為 100 ng/mL。利用 cdELISA 及 immunostrip 檢測樣品時，兩種檢測方式都具有良好的一致性。因此本研究開發的 cdELISA 及 immunostrip 能快速且有效的檢測樣品中瘦肉精的含量，以避免大眾受到這些瘦肉精的危害。
Clenbuterol (CLE) and Salbutamol (Sal), β2 agonist similar to Ractopamine (Rac), have been widely used in livestock due to theirs remarkable effectiveness of muscle growth and protein-to-fat ratio improvement. CLE and Sal may cause acute toxic responses, such as cardiac palpitation, tachycardia, nervousness, muscle tremors and confusion. In addition, the toxicity of Sal was less than the CLE. For detecting the level of CLE and Sal in pork or beef, rabbit polyclonal antibodies for CLE and Sal were produced in our studies. Using the antibodies from the rabbit immunized with CLE-γ-globulin or Sal-γ-globulin, both sensitive indirect and direct competitive enzyme-linked immunosorbent assays (ci and cdELISA) were developed. In the ciELISA of CLE, the concentration causing 50% inhibition (IC50) of binding of antibodies to the solid-phase Sal-OVA by free CLE was 0.77 ng/mL. In the ciELISA of Sal, the concentration causing 50% inhibition (IC50) of binding of antibodies to the solid-phase Sal-OVA by free Sal was 35 ng/mL. In the cdELISA of CLE, the concentration causing 50% inhibition (IC50) of binding of Salbutamol-horseradish peroxidase (Sal-HRP) to the antibody by CLE was calculated to be 0.3 ng/mL. In the cdELISA of Sal, the concentration causing 50% inhibition (IC50) of binding of Salbutamol-horseradish peroxidase (Sal-HRP) to the antibody by Sal was calculated to be 35 ng/mL. The antibody-gold nanoparticle immunochromatographic strip (immunostrip) for CLE was established for detecting the level of CLE and Sal in pork or beef. The detection limit of strip were 10 ng/mL and 20 ng/mL for CLE and Sal , respectively. In addition, Immunostrip of Sal was established for detecting the level of Sal and CLE in pork or beef. The detection limit of strip were both 100 ng/mL for CLE and Sal. Therefore, we have been successfully generated polyclonal antibodies, developed sensitive ELISAs for detection of CLE or Sal and established an immunostrip for rapid on-site detection of CLE and Sal in beef or pork samples. |
