Absorbance at 405
Figure 24：可溶性單株抗體片段的表現。利用 ELISA 測試表現之單株蛋白片 段與anti-cMyc 單株抗體間的親和性。
具有抗原高專一性之噬菌體株(Fig. 14-A 之 LAA5)感染 E.Coli /HB2151 表 現可以得到single epitope antibody fragment，以 mouse monoclonal antibody against c-Myc 最為 1o Ab 及 peroxidase conjugated goat anti-mouse IgG 為 2o Ab 進行ELISA 實驗，圖中得表現之單株蛋白片段與 anti-cMyc 單株抗體間的親 和性dose-respousive 曲線。
研究成果
2001~2003 年承蒙國科會補助從事檢測牛乳品質的相關性研究，本計畫研究目的共 7 項，其中包含乳蛋白之物理化學分析、單株抗體的製作、抗體晶片之研發及建立乳蛋 白變性指標，研究成果遠超出所提之研究項目，研究成果如下：
(1). 2001 年利用 native-gel、SDS-gel、Western blot 及 circular dichroic spectrum 分析，發 現牛乳中生乳及奶粉中有一主要蛋白(lactoglobulin)，經熱處理後在結構上有相當大 的改變，更發現在台灣市售的鮮乳中乳蛋結構嚴重改變，更進一步分析生乳及美國 購得的鮮乳，其乳中蛋白大致相同並未有改變，台灣市售的鮮乳乳中蛋白甚至比還 原乳(由奶粉沖泡的乳)結構改變更嚴重，此結果已在台灣生物醫學年會及美國生物 醫學年會中發表，並準備投稿至Journal of Dairy Science
進一步分析乳中lactoglobulin 經熱處理後結構的改變，研究發現結構隨著加熱 溫度及加熱時間的增加改變越嚴重，結果大約在加熱70℃, 15 秒鐘以上結構開始嚴 重改變。
(2). 2002 年利用 2001 年的研究結果推測還原乳乳蛋白結構上必定與生乳中乳蛋白不 同，因此想利用單株抗體來區分還原乳及生乳，研究結果成功的製作出四株單株抗 體可區分生乳及還原乳，更成功的解決台灣四十年無法處理得奶粉摻雜問題。更有 趣的是此四株單株抗體所辨認的乳蛋白皆為lactoglobulin，此結果與 2001 年用不同 方式分析的結果相符。結果已在台灣生物醫學年會及美國生物醫學年會中發表，並 已被Journal of Dairy Science 所接受於 2004 年八月刊出。
在同年上半年(2002)利用噬菌體表現的重組抗體(phage display)，成功的篩選出 會辨認乳中蛋白lactoglobulin 及 lactalbumin 的重組抗體，此結果已在台灣生物醫學 年會中發表。
(3). 2003 年深入研究 lactoglobulin 的功能，結果發現 lactoglobulin 具有抗氧化的效果，
並且已研究出抗氧化的機制是利用蛋白質本身被氧化，保護其他蛋白質不會因氧化 壓力而氧化，此結果已在生化工程年中發表，且預計於 2004 年在生物醫學年會中 發表，並投稿至Journal of Dairy Science。
同時深入研究科區分生乳及還原乳單株抗體所辨認的區段，結果發現利用 trypsin、CNBr 分解及化學修飾 lactoglobulin，再用 Western blot 分析，抗體所辨認 的區段應含有Lys 胺基酸，且在靠近 N 端部位，此結果或許可說明 lactoglobulin 加 熱變性後所呈現新的抗體辨認區(eptope)應靠近 N 端部位。此結果將在 2004 年生物 醫學年會中發表。並投稿至Journal of biological chemistry
2001~2003 年執行牛乳相關計畫所發表之論文期刊 國際及國內會議(international conference)
1. Chen W. L., M. T. Hwang, H. C. Liu, C. W. Li, and S. J. T. Mao, Distinction between dry and raw milk using monoclonal antibodies prepared against dry milk proteins. J Dairy Sci. 2004, 87:2720-2729.
2. Song C. Y, W. L. Chen, M. C. Yang, J. P. Huang, S. J. T. Mao. Epitope mapping of a monoclonal antibody specific to bovine dry milk. Involvement of residues 66-76 of strand D in thermal denatured beta -lactoglobulin. J Biol Chem. 2004 Nov 9; [Epub ahead of print]
3. Chen W.L., C.Y. Liau, and S. J. T. Mao. Analyses of β-Lactoglobulin as a thermal marker in raw and commercially processed milk using native gel electrophoresis, Westernblot, and circular dichroic spectrum. (Accepted by J Dairy Sci. 2005)
4. Liu H. C. and S. J. T. Mao. Effect of disulfide bond for the antioxidant activity of β-Lactoglobulin (2004) (Submitted to J Dairy Sci.)
國際及國內會議(international conference)
1. Chen W. L., S. J. T. Mao. Analyses of β-lactoglobulin as a thermal marker in raw and commercially processed milk using a native gel electrophoresis and circular dichroic spectrum. Experimental Biology 2002 New Orleans, LA, USA. FASEBJ.16: A1187 2. Chen W. L., S. J. T. Mao. Distinguish between dry and raw milk using monoclonal
antibodies prepared against dry milk proteins: LG is a sensitive thermal marker in dry milk. Experimental Biology 2003 San Diego, USA. FASEB. 17:A1127
3. Chen W. L., M. T. Huang, C. W. Li, H. C. Liu, S. J. T. Mao. (2004) Thermal denaturation of β-lactoglobulin as probed by an monoclonal antibody. Experimental Biology 2004 Washington, DC, USA. FASEB.18 :A144.
4. Chen W. L., Ho J. C., Hoang K. C., and S. J. T. Mao. Differentiation between fresh and powdered milk using chemical, physical, and immunochemical analyses. The sixteenth joint annual conference of biomedical sciences. (2001) Programs & abstracts.
5. Liau C. Y., Chen W. L., Wu Y. J., Lin C. C., and S. J. T. Mao. Differentiation of Milk Proteins Using Phage Displayed Antibodies. The Seventeenth Joint Annual Conference of Biomedical Sciences. (2002) oral presentation.
6. Chen W. L., Huang M. T., Liu H. C., Li C. W., S. J. T. Mao. Production of conformationally dependent monoclonal antibodies prepared against thermal-denatured milk. The Eighteenth Joint Annual Conference of Biomedical Sciences. (2003) oral
presentation.
7. Song C. Y., W. L. Chen, S. S. Wang, S. J. T. Mao. (2004) Thermal denaturation of milk β-lactoglobulin. The nineteenth joint annual conference of biomedical sciences.
Programs & abstracts.
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