Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13873
標題: Amoxicillin免疫酵素連結反應檢驗試劑開發及應用
Development of Amoxicillin Enzyme-Linked Immunosorbent Assay
作者: 葉俐君
Yeh, Li-Chun
關鍵字: ELISA;免疫酵素連結反應;Amoxicillin;Microdialysis;β-lactam;drug residues;藥物殘留檢測;微透析;乙內醯胺類抗生素;藥物組織動力學
出版社: 獸醫學系
摘要: 
畜產品及飼料中經常可發現乙內醯胺類(β-lactam)抗生素的殘留,造成動物過敏性反應或產生細菌的抗藥性等問題。抗生素殘留之檢測多以高效液相層析等各式層析法實施,但其耗時費資且可能製造有機廢液,故一般常利用微生物法(如抑菌圈)或免疫酵素連結反應(enzyme linked immunosorbent assay, ELISA)進行預先快速篩選大量樣品,以縮短確檢時間。Amoxicillin在國內為最常被使用的乙內醯胺類抗生素之一,但快速檢測篩選方法卻不多見。本實驗擬以所生產之amoxicillin特異性多株抗體(polyclonal antibody)製作快速篩選試劑檢驗amoxicillin之殘留。首先利用carbodiimide(CD)法及glutaraldehyde(GA)法螯合amoxicillin與牛血清白蛋白(bovine serum albumin)或聚苯乙烯聚合微粒為載體(polystyrene particle, 0.85μm)免疫紐西蘭大白兔。待產生抗體後,以間接免疫酵素連結反應(indirect ELISA)測定抗體力價。結果顯示以GA法螯合所引發之抗體力價優於CD法或使用聚苯乙烯聚合微粒為載體組。額外添加聚合微粒為佐劑無法進一步促進傳統呋喃氏佐劑(Freund’s adjuvant)之免疫效果。以棋盤方格法(checkerboard)決定之最佳抗體稀釋倍數可達1,600倍。以8種不同濃度(0.1 ng/mL ~ 1 mg/mL)amoxicillin標準品進行競爭性結合試驗顯示本反應試劑之amoxicillin檢測極限約為1 ng/mL。在交叉反應結果方面,所測試的九種乙內醯胺類抗生素中,以penicillin G與本抗體之交叉反應率最高(77.39%),接著依序為ampicillin(56.87%)、oxacillin(51.41%)、cloxacillin(48.79%)、6-aminopenillinic acid (6-APA)(37.71%)、carbenicillin(36.00%)、dicloxacillin(7.40%);對於同為乙內醯胺類之cefadroxil、cefazolin交叉反應率極低(<1%)。微透析活體實驗結果顯示,鴿子肌肉注射amoxicillin(25 mg ∕kg)後20分鐘,血中濃度急速上升至最高點(4.9 ug ∕mL),並於6小時內下降至約1 ug ∕mL。Amoxicillin濃度在胸部及腿部肌肉則上升幅度較緩,最高濃度分別為0.7 ug ∕mL及1.1 ug ∕mL,之後呈現緩慢下降趨勢,在8小時時濃度仍維持在約0.5 ug ∕mL。Amoxicillin之鴿血清蛋白結合率為28 ± 6%。
此結果顯示amoxicillin在肌肉組織內濃度遠低於血中濃度,未來可依此訂定合理給藥劑量,以達期望之療效。綜合所論,本實驗所開發之ELISA檢驗試劑除可有效的快速篩選amoxicillin及相似結構抗生素在血液、尿液及乳品中之殘留外,還可應用於amoxicillin相關組織動力學之研究。

The residues of β-lactam antibiotics in consumable meat and milk products pose potential risks for public health because of their propensity to induce allergic reactions in individuals and the increasing incidence of microbial resistance against these compounds. Amoxicillin is a β-lactam antibiotics widely used in human and veterinary clinical medicine. The aim of this study was to develop an enzyme -linked immunosorbent assay (ELISA) suitable for detection of amoxicillin in biological fluids.
Amoxicillin polyclonal antibody was raised from rabbits immunized with amoxicillin conjugated to bovine serum albumin (BSA) or carboxyl polystyrene particles by either carbodiimide or glutaraldehyde (GA) method. Polystyrene particle was further tested for their efficacy as adjuvant. The results indicated that the highest anti-amoxicillin titer (>102,400 fold dilution) was obtained from rabbits immunized with amoxicillin-BSA conjugates by GA method. Polystyrene particle did not significantly improve the immune response elicit with Freund's adjuvant. The optimal dilution of amoxicillin antibody for the developed assay was 1,600 fold. The assay has a limit of detection of 1 ng/mL and limit of quantitation of 5 ng/mL in bovine serum, urine and milk. Cross reactivity with penicillin G, ampicillin, oxacillin, cloxacillin, 6-aminopenillinic acid (6-APA), carbenicillin, cefadroxil, cefazolin and dicloxacillin were 77.39%, 56.87%, 51.41%, 48.79%, 37.71%, 36.00%, 7.40%,<1% and <1%, respectively.
The ELISA assay was combined with microdialysis sampling to measure venous and muscle tissue concentrations of amoxicillin in pigeons. After a single intramuscular injection of 25 mg ∕kg amoxicillin, the blood concentration peaked at 4.9 g ∕mL in 20 minutes and declined to about 1 g ∕mL after 6 hours. Amoxicillin concentrations in femoral and breast muscle tissues exhibited a delayed and minor increase compared to those in the blood. The peak concentration in femoral and breast reached 1.1 g ∕mL and 0.7 g ∕mL, respectively, at 60 minutes after drug administration. The protein binding of amoxicillin to pigeon serum was 28 ± 6%.
In conclusion, we have developed a sensitive amoxicillin ELISA that could efficiently detect amoxicillin and structurally related β-lactam antibiotics in various biological matrices. The application of this assay in combination with microdialysis has successfully demonstrated its use in tissue pharmacokinetic studies.
URI: http://hdl.handle.net/11455/13873
Appears in Collections:獸醫學系所

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