Investigation of promiscuity in heavy chain and light chain on anti–alpha enolase antibodies

• Main Authors: Jing–Ling Hsu, 徐菁伶
• Other Authors: 呂思潔
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/39935067145937343156

碩士 === 臺北醫學大學 === 醫學科學研究所 === 98 === Abstract Since A. D. 1982, cancer has been the first leading causes of death in Taiwan. The morbidity and mortality rates have been rising in the recent years. According to the recent report in A. D. 2009 by Department Of Health, R. O. C., lung cancer is the third most common cancer for male population in Taiwan. It is in urgent need to develop a rapid test with high specificity and sensitivity for diagnosis of lung cancer. Among men in Taiwan, lung cancer is the third most common cancer.It has non–specific symptoms of lung cancer. Cough is the main symptom, easy to be confused with other pulmonary diseases. Patients are not aware of this so that the lunge cancer cannot be found and treated early. Lung cancer is often so difficult to consciously and to enable find, So Patients cant early diagnosis and treatment . Alpha–enolase （ENO – 1） is a key glycolytic enzyme , that has been used as a diagnostic marker to identify human lung cancer. According to previous studies early stage of non–small cell lung cancer （NSCLC） patients with tumors expressing relatively higher ENO – 1 level were tightly correlated with poorer survival outcomes. Therefore, data strongly support a prognostic role of ENO – 1 in determining tumor malignancy of patients with NSCLC. This study is based on completion of L1–L6 anti–human ENO – 1 of monoclonal antibodies. We have previously isolated several anti–human ENO – 1 scFv antibodies from chicken. However, their binding affinity may not be satisfied for diagnostic/therapeutic application in clinic. Thus, this present study is to randomly combine the heavy and light chain variable region genes of L1 to L6 clones and to select any scFv antibodies with improved binding affinity to human ENO – 1 protein. To construct anti–Human alpha–enolase monoclonal antibodylibraries, overlap PCR was performed to generate a variable of DNA fragments containing heavy and light chain variable genes. These scFv DNA fragments were cloned into pComb3x VI vectors and then transfected into the XL–1 Blue E. coli following infected by VCSM13 phage to obtain recombinant phage. After four rounds panning by using recombinant phage, total DNA was transfected into TOP10F’ E. coli. We randomly select clones to express scFv fusion proteins and compare the sequence. And the scFv monoclonal antibody could bind to the Human alpha–enolase both in Western blot and ELISA analysis. Thus, we generated the anit–human alpha–enolase scFv monoclonal antibody with high protein expression and binding ability. The fragment of Human alpha enolase can be filtered out so that this can be helpful in the early detection or therapy of lung cancer in the clinical.