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題名 | 乙內醯胺類抗生素酵素連結免疫吸附法殘留檢驗試劑之開發與應用=Development of Enzyme-linked Immunosorbent Assay Residual Detection Kits for β-lactam Antibiotics |
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作者 | 林育興; 王渭賢; 林毓芬; 張鎮璿; 涂青宇; 許添桓; 洪紹文; | 書刊名 | 中國畜牧學會會誌 |
卷期 | 45:3 2016.09[民105.09] |
頁次 | 頁197-222 |
分類號 | 412.37 |
關鍵詞 | 抗體; 乙內醯胺類抗生素; 酵素連結免疫吸附分析法; 高效液相層析法; 殘留; Antibody; β-lactam antibiotics; ELISA; High performance liquid chromatography; Residues; |
語文 | 中文(Chinese) |
中文摘要 | 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、快速與精確的檢測試劑檢測禽畜水產品中殘留的乙內醯胺類(β-lactams)抗生素:安默西林(amoxicillin, AM)、安比西林(ampicillin, AM)與盤尼西林(penicillin G, PG)。結果顯示,利用 carbodiimide-mediated coupling method(CD)、glutaraldehyde-mediated coupling method(GA)與 physiological coupling method(PH)三種不同螯合法均可與人類血清白蛋白(human serum albumin, HSA)和牛血清白蛋白(bovine serum albumin, BSA)成功地製備藥物-蛋白螯合物。利用高效液相層析儀(high performance liuid chromatography, HPLC)搭配紫外光檢測器可以用來檢測藥物-HSA和藥物-BSA螯合物。螯合物經皮下免疫紐西蘭白兔 5 次後,血清中抗體力價可達 65,536 倍以上。利用 PH 螯合法製備藥物-BSA螯合物是最適合用於產生乙內醯胺類抗生素之多株抗體。之後將 AM、AM 及 PG 以 PH 螯合法結合 BSA 所得的螯合物,用以生產單株抗體。結果顯示經腹腔免疫 BALB/c 小 鼠7 次後,血清中抗體力價可達 32,768 倍以上,並發現抗 AM、AM 及 PG 融合瘤的陽性率分別為 1.96%、1.88% 及 1.67%。以 AMELISA 檢測盤在磷酸緩衝液(phosphate buffered saline, PBS)、牛乳、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;在 AM-ELISA 檢測盤的檢測極限分別為 0.5、0.5、0.5、0.5、10 及 10 /mL;在 PG-ELISA 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 AMELISA 檢測盤之精確度最高, PG-ELISA 檢測盤次之,AM-ELISA 檢測盤最低。在 AMELISA、AM-ELISA 及 PG-ELISA 檢測盤中,其分析內與分析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,AMELISA、AM-ELISA 及 PG-ELISA 檢測盤對乙內醯胺類抗生素有較高的交叉反應率(4.7-97.9%)外,對於非乙內醯胺類抗生素的交叉反應率皆小於 0.01%。綜合上述結果顯示,本研究所開發之乙內醯胺類抗生素檢測試劑具有簡單、快速與精確之特性,所需時間為 2 小時,此檢測法一次能針對至少 3 種乙內醯胺類抗生素進行檢測。未來可應用於現場,針對禽畜水產品中殘留之乙內醯胺類抗生素進行初步篩檢。 |
英文摘要 | The aim of this study was to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay ELISA method for detecting the residues of β-lactams antibiotics such as amoxicillin AM, ampicillin AM, and penicillin G PG in the animal biomatrix. Carbodiimide-medicated coupling CD, glutaraldehyde-medicated coupling GA, and physiological coupling PH methods were used to conjugate β-lactam antibiotics AM AM, and PG, respectively and carrier proteins human serum albumin and bovine serum albumin, respectively. The different absorption characteristics of β-lactam antibiotics, BSA, HSA, and β-lactam antibiotic-protein conjugates were identified by the high performance liuid chromatography HPLC with ultraviolet detector. All conjugates were individually used for producing polyclonal antibodies in New ealand rabbits. The results showed that the titers of the serum polyclonal antibodies were 65,536-fold after five boosts. PH method was the most suitable conjugation method to form the conjugates for producing polyclonal antibody. At the same time, bovine serum albumin was better than human serum albumin as the carrier protein for β-lactam antibiotics-carrier protein conjugation. By using the PH method it was possible to form the conjugates to produce the monoclonal antibody mAb in BALB/c mice. Test samples were detected by indirect competitive ELISA method. The results showed that the titers of the serum mAb were 32,768-fold after seven boosts. The positive hybridoma ratios against AM AM, and PG were 1.96%, 1.88%, and 1.67%, respectively. The lowest detection limit LDL of the ELISA kits we developed for AM 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 /mL, and 50, 100, 50, 50, 100 and 100 ng/mL, respectively. The coefficient variation value of the intra-assay and inter-assay of AM, AM-, and PG-ELISA kits were 3.72% and 6.4%, 8.83% and 11.29%, 10.27% and 9.0%, respectively. The 50% cross-reactivities of all the AM, AM-, and PG-ELISA plates for non-β-lactam antibiotics were below 0.01%. From the above assay results, two hours were reuired for assay time of ELISA kits we developed. At least three kinds of β-lactam antibiotics could be detected each run by using the kits. Thus, the ELISA kits we developed seemed to be a simple, rapid, and reproducible tool for clinical detection of the residues of β-lactam antibiotics in the animal biomatrix in the future. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。