頭孢氨苄抗體之製備及酵素免疫吸附分析法與奈米金粒子免疫層析試紙分析法之開發

頭孢氨苄 (CEX) 是第一代頭孢菌素，頭孢菌素屬於乙內醯胺類抗生素，與青黴素結構類似。頭孢菌素藉由破壞細菌細胞壁的方式，殺死葛蘭氏陽性菌以及部分葛蘭氏陰性菌，而 CEX 常用於治療細菌感染。為維護消費者的健康，根據我國衛生福利部之動物用藥殘留標準規定，CEX 在家禽、家畜的肌肉、肝臟及脂肪相關製品中，不得檢出超過 200 ng/g；而腎臟相關製品中，不得檢出 1000 ng/g。因此，為了從家禽家畜相關製品中檢測出 CEX，本實驗欲利用抗原抗體間的專一性，開發了高靈敏性且方便的檢測方法。由於 CEX 分子量約 347 Dalton，屬於小分子化合物的範圍，需要與載體蛋白質接合進而對實驗動物進行免疫。本實驗利用碳二亞胺 (carbodiimide) 方法將頭孢氨苄與牛甲狀腺球蛋白質 (Bovine thyroglobulin, BTG) 接合，以 CEX-BTG 作為抗原免疫紐西蘭大白兔，進而刺激實驗動物產生對頭孢氨苄具有專一性的抗體。本實驗以免疫紐西蘭大白兔所產生之多株抗體成功建立了直接競爭型酵素連結免疫吸附法 (Competitive direct enzyme-linked immunosorbent assay, cdELISA) 及非直接競爭型酵素連結免疫吸附法 (Competitive indirect enzyme-linked immunosorbent assay, ciELISA)，其 IC50 分別為 0.146 與 0.32 ng/mL，並以此抗體更進一步建立免疫層析試紙，此免疫層析試紙之目視極限值為 50 ng/mL。為了製備出對 CEX 專一性的單株抗體，同樣以 CEX-BTG 作為抗原免疫小鼠，ELISA 結果顯示兩隻小鼠抑制 50% 頭孢氨苄多株抗體結合至 CEX-HRP 所需之頭孢氨苄的濃度 (IC50) 分別為 2.79 及 37.3 ng/mL。本研究已成功利用 CEX 多株抗體建立 ELISA 分析法以及免疫試紙分析法，以期快速且大量的篩檢方法來檢驗CEX，以避免大眾受此抗生素過量的危害。

Cephalexin (CEX) is the first generation cephalosporins, that are a family of bactericidal antibiotics structurally related to penicillin. It kills gram-positive and some gram-negative bacteria by disrupting the growth of the bacterial cell wall. The presence of antibiotic residues can increase the proliferation of antibiotic-resistant pathogens and threats to public health. The symptoms of an overdose include nausea, vomiting, difficulty urinating, abdominal pain, and diarrhea. In order to protect consumer health, the maximum residue limits (MRLs) of CEX was established by the standards for veterinary drug residue limits in foods at 200 ng/g in livestock and poultry for muscle, fat, and liver products, and at 1000 ng/g for kidney products. Thus, to detect the presence of CEX in the products, developing a high sensitive and convenient detection method for CEX is required. CEX was conjugated with thyroglobulin from bovine thyroid (BTG) by carbodiimide method. Then, the polyclonal antibody (pAb) against CEX was produced using CEX-BTG conjugates as the immunogen to immune the New Zealand rabbit. Based on the antiserum describe above, the direct and indirect competitive enzyme-linked immunosorbent assay were successful developed with the IC50 of 0.146 and 0.32 ng/mL, respectively. The gold nanoparticle immunochromatographic strips (immunostrip) were developed based on the antibody described above. The visual detection limit of immunostrip was 50 ng/mL. To produce the monoclonal antibody of CEX, the Balb/c mice were immunized by CEX-BTG conjugates as the immunogen. The concentration causing 50% inhibition (IC50) of binding of CEX-horseradish peroxidase (CEX-HRP) by the antibodies produced by mice were calculated to be 2.79, and 37.3 ng/mL, respectively. The spleen of one of the mice was used to fuse with the myeloma cells for screening the hybridoma. In this study, both of ELISA and immunochromatographic strip have been based on polyclonal antibody developed for sample analysis.