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氯噻啉噬菌體展示酶免疫分析方法的研究

時間:2019-04-16 21:15來源:畢業論文
通過用特異性抗氯噻啉的單克隆抗體淘選噬菌體展示肽庫獲得六種噬菌體展示肽。選擇克隆L4-29建立噬菌體酶免疫吸附分析(P-ELISA)和噬菌體化學發光酶免疫分析(P-CLEIA)。 P-ELISA和P

摘要:通過用特異性抗氯噻啉的單克隆抗體淘選噬菌體展示肽庫獲得六種噬菌體展示肽。選擇克隆L4-29建立噬菌體酶免疫吸附分析(P-ELISA)和噬菌體化學發光酶免疫分析(P-CLEIA)。 P-ELISA和P-CLEIA的最佳緩沖液為pH 8,0.14 M NaCl和2.5%甲醇的PBS緩沖液。P-ELISA的半數最大抑制濃度(IC50)值,檢測限(LOD,IC10值)和線性范圍(IC10-IC90)分別為1.45,0.55和0.55至3.82 ng mL -1,P-CLEIA 分別為0.86,0.13和0.13-5.84 ng mL -1。與傳統ELISA相比,P-ELISA和P-CLEIA的靈敏度分別提高了40倍和68倍。P-CLEIA比P-ELISA更敏感,具有更寬的線性范圍。兩種分析方法與氯噻啉的類似物(除吡蟲啉)沒有顯著的交叉反應性。P-ELISA和P-CLEIA兩種方法在稻田水,土壤,甘藍,水稻,蘋果,梨和番茄樣品中對氯噻啉的添加回收率分別為72.3-101.3%和73.9-102.6%。P-ELISA和P-CLEIA與高效液相色譜(HPLC)對樣品中氯噻啉檢出結果的相關性良好,可用于實際樣品中的氯噻啉的檢測。34621
畢業論文關鍵詞:氯噻啉;酶免疫吸附分析;化學發光酶免疫分析;噬菌體展示肽;模擬表位
Development of phage enzyme immunoassays for the determination of imidaclothiz
Abstract:Six phage-displayed peptides were obtained by biopanning the phage-displayed peptide libraries with the specific anti-imidaclothiz monoclonal antibody. A phage enzyme-linked immunosorbent assay (P-ELISA) and a phage chemiluminescent enzyme immunoassay (P-CLEIA) for imidaclothiz were developed by using a clone L4-29. The optimal buffers of P-ELISA and P-CLEIA were pH 8, PBS buffer containing 0.14 M NaCl and 2.5% methanol. The half maximal inhibition concentration (IC50) values, limit of detection (LOD, IC10 values) and linear range (IC10 to IC90) of the P-ELISA were 1.45, 0.55 and 0.55 to 3.82 ng mL-1, while the P-CLEIA were 0.86, 0.13 and 0.13 to 5.84 ng mL-1, respectively. Compared with conventional ELISAs, the sensitivities of P-ELISA and P-CLEIA were increased by more than 40-fold and 68-fold, respectively. The P-CLEIA was more sensitive and showed wider linear range than the P-ELISA. Two analyses had no significant cross-reactivity with the analogues of imidaclothiz except for imidacloprid. Recoveries of the P-ELISA and P-CLEIA for imidaclothiz in paddy water, soil, cabbage, rice, apple, pakchoi, pear and tomato samples were 72.3-101.3% and 73.9-102.6%, respectively. The P-ELISA and P-CLEIA were feasible to detect the amounts of imidaclothiz in the authentic samples, which showed good correlation with that obtained from high-performance liquid chromatography (HPLC).
源¥自%六:維;論-文'網=www.aftnzs.live

Keywords:Imidaclothiz, Enzyme-linked immunosorbent assay, Chemiluminescent enzyme immunoassay, Phage-displayed peptide, Peptidomimetics
目錄
摘要    2
關鍵詞    2
Abstract    2
Keywords    2
引言    2
1.材料與方法     4
1.1材料     4
1.2主要儀器    4
1.3方法     4
1.3.1氯噻啉單克隆抗體的制備     4
1.3.2生物淘選     4
1.3.3噬菌體酶免疫吸附分析方法     5
1.3.4噬菌體化學發光酶免疫分析方法     5
1.3.5P-ELISA和P-CLEIA的優化     5
1.3.6標準曲線的建立     5
1.3.7交叉反應性(CR)     5
1.3.8 加標樣品的分析     5
1.3.9 HPLC分析驗證    6
2.結果與分析     6
2.1噬菌體展示肽的淘選     6
2.2噬菌體展示肽的選擇     6
2.3 P-ELISA和P-CLEIA的優化    7
2.4 P-ELISA和P-CLEIA的靈敏度     7
2.5 P-ELISA和P-CLEIA的特異性     8
2.6加標樣品的分析     9 氯噻啉噬菌體展示酶免疫分析方法的研究:http://www.aftnzs.live/yixue/20190416/32187.html
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