Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13478
標題: β-lactams藥物酵素連結免疫吸附法殘留檢驗試劑之開發
Development of Enzyme-Linked Immunosorbent Assay Residual Detection Kit for β-lactams
作者: 林毓芬
Lin, Yuh-Fen
關鍵字: residues
殘留
ELISA
monoclonal antibody
酵素連結免疫吸附分析法
單株抗體
出版社: 獸醫學系暨研究所
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摘要: 本研究之目的在於嘗試以間接競爭型酵素連結免疫吸附法開發出一套簡單、快速、精確檢測禽畜產品中β-lactams類抗菌劑 amoxicillin (AMX), ampicillin (AM), and penicillin G (PG)的試劑。實驗結果顯示利用carbodiimide coupling (CD), glutaraldehyde coupling (GA), and physiological coupling (PH)等三種不同螯合技術均可以和human serum albumin (HSA)及bovine serum albumin (BSA)成功地製備藥物蛋白質衍生物之結合體。我們利用HPLC搭配紫外光檢測器可以偵測藥物-HSA和-BSA之結合體。經皮下免疫紐西蘭白兔五次後,抗體力價可達65,536倍以上,同時根據結果顯示,利用PH螯合技術製備藥物BSA蛋白衍生物之結合體是最適合用於產生β-lactams之多株抗體。將所得AMX、AM及PG以PH螯合技術結合BSA蛋白嘗試以生產單株抗體。由試驗結果顯示,經腹腔免疫小鼠七次後抗體力價可達32,768倍以上,並且發現抗AMX、AM及PG融合瘤的陽性率分別為: 1.96%、1.88% 及 1.67%。以AMX檢測盤在磷酸緩衝液PBS、牛奶、魚肉、雞肉、牛肉及豬肉的檢測極限分別為: 50、50、50、50、100 及100 ng/mL;而在AM檢測盤的檢測極限分別為: 0.5、0.5、0.5、0.5、10及10µg/m; 另外在PG檢測盤的檢測極限分別為: 50、100、50、50、100 及100 ng/mL。在精確度方面,以AMX檢測盤之精確度最高,PG檢測盤次之,以AM檢測盤最低。在AMX、AM及PG檢測盤中其組內與組間檢測變異值分別為3.72%,6.4%;8.83%,11.29%;及10.27%,9.0 %。在交叉反應試驗中,AMX、AM及PG檢測盤對β-lactams類藥物有較高的反應率外(4.7%~97.9%),而對於非β-lactams類藥物皆小於0.01。因此,綜合上述所有結果顯示,本研究所開發之檢測試劑具有簡單、快速及精確之特性,可應用於現場針對魚肉、雞肉β-lactams類抗菌劑之殘留檢驗。
The aim of this study is to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay (ELISA) method for detecting the residues of β-lactams and as amoxicillin (AMX), ampicillin (AM), and penicillin G (PG) in the varietal samples. Using the carbodiimide coupling (CD), glutaraldehyde coupling (GA), and physiological coupling (PH) methods, the AMX, AM, and PG was productively conjugated with human serum albumin (HSA) and bovine serum albumin(BSA), respectively. According to the different absorption characteristics of β-lactams, BSA, HSA, and β-lactam-conjugated proteins, the three drug-carrier protein conjugations made using three different conjugated techniques were able to be identified by the high performance liquid chromatography (HPLC) with ultraviolet detector. All conjugated proteins were individually used for producing polyclonal antibodies in rabbits. The results showed that the titers of the serum antibodies from rabbit were 65,536-fold after five boosts. According our results, we suggested that the PH method was the most suitable technique to form the conjugated protein for producing polyclonal antibody. At the same time, we found that BSA was better than HSA as the carrier protein for β-lactams. We boosted the β-lactam-conjugated proteins by using the PH methods to producing the monoclonal antibody. The results showed that the titers of the serum antibodies from mice were 32,768-fold after seven boosts. From the mAb tests, our results indicated that the positive hybridoma ratio for anti-AMX, anti-AM, and anti-PG was 1.96%, 1.88%, and 1.67%, respectively. The lowest detection limit (LDL) of our developed ELISA kit for the AMX, AM, and PG in PBS, milk, fish, chicken, beef, and pork meats were (50, 50, 50, 50, 100 and 100) ng/mL, (0.5, 0.5, 0.5, 0.5, 10 and 10) µg/mL, and (50, 100, 50, 50, 100 and 100) ng/mL, respectively. The coefficient variation value of the intra-assay and inter-assay of our AMX, AM, and PG-ELISA kits were (3.72% and 6.4%), (8.83% and 11.29%), (10.27% and 9.0%), respectively. We found the cross-reactive ratio of the AMX, AM, and PG-ELISA plates for tested quinolones were below 0.01%. From above assay results, our developed ELISA kits seemed to be a simple, rapid, and reproducible method to detect the residues of the tested β-lactams in the chicken and fish meats.
URI: http://hdl.handle.net/11455/13478
其他識別: U0005-3107200701371500
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