Developing a kit to detect catechin EGCG via its antibodies generated by artificial oil bodies

由於動物的免疫系統無法辨識分子量小於1 kDa 的小分子抗原，因此無法產生相對應的小分子抗體，但利用載體蛋白與小分子連接則可以成功引發免疫反應，進而產生相對應的小分子抗體。於本實驗室先前研究發現，基因改造後富含離胺酸的油體鈣蛋白可用於連接小分子，如生物素，其形成的人造油體可用於引發動物的免疫反應並產生生物素的抗體。因此富含離胺酸的油體鈣蛋白被認為適合作為小分子載體。富含離胺酸的油體鈣蛋白連接上兒茶素EGCG後，可成功製備成人造油體並能將其放入動物體內引發免疫反應而產生抗體，並應用於ELISA和免疫試紙的檢測。本研究將EGCG連接上OVA和BSA製備成人造抗原，應用於ELISA和免疫試紙之檢測分析；使用ELISA進行EGCG檢測時，EGCG本身的化學特性可能會使抗體沉澱，影響檢測結果；另一方面，免疫試紙上的BSA-EGCG人造抗原可和樣品內的EGCG競爭接上奈米金的EGCG抗體，檢測範圍約落在115-230 ppm，不過呈色效果仍有待改善以供實際的應用。Molecules of molecular masses less than 1 kDa are usually non-immunogenic and fail to elicit immune response in animal. However, antibodies against a small molecule can be generated when it is conjugated with a carry protein to form an artificial antigen prior to animal immunization. In our previous study, polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with biotinylated Lys-rich caleosins. It appears that engineered Lys-rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. In this study, a lys-rich caleosin was used to render catechin EGCG for the production of its polyclonal antibodies. The raised antibodies were used to develop an ELISA-based immunochromatographic strip in company with BSA or ovalbumin conjugated with EGCG. In my detection, EGCG seemed to resulted in antibody precipitation. However, the strip could be used to detect EGCG at concentrations of 0.25~0.5 mM. Nevertheless, the color intensity of strip should be improved prior to commercial applications.摘要........................................ I Abstract.................................. II 目次...................................... III 1. 緒論.................................... 1 1.1. 茶................................... 1 1.2. 兒茶素(Catechins)..................... 1 1.3. 分子檢測－免疫層析法.................... 2 1.4. 小分子抗體............................ 5 1.5. 油體................................. 7 1.6. 油體蛋白.............................. 7 1.7. 油體技術應用－小分子抗體製備.............. 8 1.8. 研究目的.............................. 9 2. 材料與方法............................... 10 2.1. 藥品及材料............................ 10 2.2. 溶液配置 ............................. 11 2.3. 儀器 ................................ 12 2.4. EGCG抗體來源 ......................... 12 2.5. EGCG蛋白載體製備 ...................... 13 2.6. 蛋白質電泳............................ 14 2.7. ELISA檢測............................ 15 2.8. 奈米金抗體製備.......................... 16 2.9. 免疫試紙檢測 ........................... 17 3. 結果 .................................... 18 3.1. EGCG蛋白載體製備 ....................... 18 3.2. ELISA於EGCG濃度的檢測 .................. 18 3.3. 免疫試紙於EGCG濃度的檢測 ................. 19 4. 討論 ..................................... 20 4.1. EGCG蛋白載體製備 ........................ 20 4.2. ELISA於EGCG濃度的檢測 ................... 21 4.3. 免疫試紙於EGCG濃度的檢測 .................. 22 5. 結論 ...................................... 24 6. 參考文獻.................................... 25 圖表目次 圖表 ......................................... 28 圖一. OVA-EGCG及BSA-EGCG蛋白質電泳分析........... 28 圖二. ELSIA對EGCG濃度檢測結果.................... 29 圖三. ELSIA檢測EGCG之模擬卡通示意圖............... 30 圖四. 免疫試紙檢測結果-1 ........................ 31 圖五. 免疫試紙檢測結果-2 ........................ 32 附錄.......................................... 33 附錄一. 茶裡主要兒茶素、黃酮類及黃酮醇之結構........ 33 附錄二. 免疫層析試紙主要的分析形式................ 34 附錄三. 常見的免疫試紙呈色方法.................... 35 附錄四. 免疫層析原理：比較競爭法和三明治法.......... 36 附錄五. 油體胞器模型............................ 37 附錄六. 模型：油體鈣蛋白和油體膜蛋白............... 38 附錄七. 修改後的油體鈣蛋白示意圖.................. 39 附錄八. 利用油體產生多株抗體模型.................. 40 附錄九. 生物素的多株抗體的西方墨點分析............. 41 附錄十. BSA和OVA的個別胺基酸(K,Y,S,C)數量........ 42 附錄十一. EGCG人造抗原合成方法示意圖.............. 43 附錄十二. EGCG多株抗體之西方墨點分析.............. 4

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies