氟甲磺氯黴素之抗體製備與其應用於酵素連結免疫分析法和奈米金粒子免疫層析試紙分析法之開發

Production of Antibodies and Their Application to ELISA and Gold Nanoparticle Immunochromatographic Strip for Florfenicol

趙子惟(Tzu-Wei Chao)

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(2019年)

https://doi.org/10.6834/CSMU201900070

摘要

氟甲磺氯黴素 (Florfenicol, FF)，為一種廣效型抗生素。由於 FF 對多種革蘭氏陽性與陰性菌的抑菌效果顯著，因此被廣泛應用於畜禽、水產養殖業及養蜂業中多種傳染病的治療，因為被廣泛的應用，動物體內常有殘留。然而許多研究指出，高劑量的 FF 會導致男性生殖毒性，如睾丸萎縮，精子細胞的減少，得到間質細胞瘤的風險增加，因此本實驗欲利用抗體與抗原之間的專一性，建立了一套可以快速檢測食品中 FF 殘留量之分析法。本實驗先使用 Succinic anhydride (SH) 將 FF 衍生為 FF-SH，再利用薄層層析法 (TLC) 進行確認與純化，衍生後產物再與牛的甲狀腺球蛋白 (Bovine thyroglobulin, BTG) 進行接合，接著使用 FF-SH-BTG 作為抗原注射入紐西蘭大白兔體內，進而產生對 FF 有專一性之多株抗體；由 ELISA 結果顯示其抑制 50 % FF 多株抗體結合至 FF-SH-HRP 所需的 FF 濃度 (IC50) 為 4.08 ng/mL，本實驗進一步使用此多株抗體開發出免疫層析試紙，而此免疫試紙之目視檢測極限值為 5 ng/mL；此外本實驗為了製備出對 FF 高度專一性之單株抗體，也以 FF-SH-BTG 作為抗原來免疫小鼠，由 ELISA 結果顯示三隻小鼠血清中抗體的 IC50 分別為 1.61、2.3 和 0.094 ng/mL，且三隻皆已犧牲進行融合瘤實驗，但並未篩選出可分泌對 FF 專一性抗體的細胞株。本實驗利用 FF 多株抗體開發之兩種檢測分析法，皆比目前使用之high-performance liquid chromatography (HPLC), liquid chromatography–tandem mass spectrometry (LC–MS/MS) 等分析法簡單並且容易操作，希望可以提供給大眾使用並為食品安全盡一份心力。

關鍵字

抗體 ； 免疫層析試紙 ； 氟甲磺氯黴素 ； 酵素連結免疫分析法

並列摘要

Florfenicol (FF) is a synthetic and broad-spectrum antibiotic belonging to the fenicol drug family. It is widely used in poultry, livestock and aquaculture. Due to the residue concerns of FF in animals and the potential drug resistance of bacteria, FF is strictly regulated in many countries. FF was also reported with male reproductive toxicity and might cause testicular atrophy, decrease sperm cells production and increase the risk of Leydig cell tumors. Therefore, developing a rapid and sensitive detection method for screening the level of FF is urgent. First, FF was derivatized with succinic anhydride (SH) and the result was confirmed by thin layer chromatography (TLC). Then, the FF-SH was coupled with bovine thyroglobulin (BTG) and New Zealand rabbit was immunized with FF-SH-BTG to produce the anti-FF antibody. The results of cdELISA from rabbit antiserum showed that the concentration causing 50% inhibition (IC50) of binding of FF-horseradish peroxidase (FF-HRP) to the antibody by FF was calculated to be 4.08 ng/mL. The gold nanoparticle immunochromatographic strips (immunostrip) were developed based on the antibody described above. The visual detection limit of immunostrip was 5 ng/mL. To produce the monoclonal antibody for FF, the Balb/c mice were immunized and the IC50 values of FF-antibody in three mice serum were calculated to be 1.61, 2.3 and 0.094 ng/mL, respectively. The spleens of these mice were used to fuse with the myeloma cells for screening the hybridoma. However, we didn’t screen the hybridoma cell that can secret FF antibody. The ELISA and immunostrip that we established are more convenient and rapid than HPLC and LC–MS/MS to detect the FF contamination in foods.

並列關鍵字

antibody ； immunostrip ； florfenicol ； ELISA

參考文獻

Alechaga, É., Moyano, E., & Galceran, M. T. (2012). Ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of phenicol drugs and florfenicol-amine in foods. Analyst, 137(10), 2486-2494.

Bailes, J., Mayoss, S., Teale, P., & Soloviev, M. (2012). Gold nanoparticle antibody conjugates for use in competitive lateral flow assays. Methods in Molecular Biology, 906, 45-55.

Blickwede, M., Valentin‐Weigand, P., Rohde, M., & Schwarz, S. (2004). Effects of subinhibitory concentrations of florfenicol on morphology, growth, and viability of Staphylococcus aureus. Journal of Veterinary Medicine, Series B banner, 51(6), 293-296.

Booker, C. W., Jim, G. K., Guichon, P. T., Schunicht, O. C., Thorlakson, B. E., & Lockwood, P. W. (1997). Evaluation of florfenicol for the treatment of undifferentiated fever in feedlot calves in western Canada. The Canadian Veterinary Journal, 38(9), 555.

Cannon, M., Harford, S., & Davies, J. (1990). A comparative study on the inhibitory actions of chloramphenicol, thiamphenicol and some fluorinated derivatives. Journal of Antimicrobial Chemotherapy, 26(3), 307-317.