Generation of anti-IL13 antibodies for diagnostic agent by LAE technique

• Main Authors: Shu-Ching Chang, 張淑菁
• Other Authors: none
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/22000376701714203324

碩士 === 國立中正大學 === 分子生物研究所 === 93 === Interleukin-13 (IL-13) is a cytokine produced by T helper-2 lymphocytes and plays an important role in regulating several immune responses. The serum concentration of IL-13 increased in asthma, allergy, atopy and inflammatory conditions. It can promote B cell proliferation, induce B cells to produce IgE, down-regulate the production of proinflamatory cytokines and enhance the expression of class II MHC antigens. All these clinical observations implied that the serum concentration of IL-13 could be used as a diagnostic indicator for immunological disorders. We proposed to produce the anti-IL13 antibody, which could detect sub-concentration of IL-13 and be utilized for rapid diagnosis. Base on structural modeling, several different regions that exposed at the molecular surface were selected as antigenic targets. The linear array epitope (LAE) technique was employed to generate DNA fragments encoding for LAE antigen in fusion with Pseudomonas exotoxin domain Ia (PEIa) or glutathione S-transferase (GST). The purified fusion proteins were used to immunize rabbits. Antibodies against IL-13 epitopes were proved to be specific from ELISA assays. So far, we already have generated antigen of IL-13 epitopes by LAE technique, and taken the antigen to immunize rabbit to get the polyclonal antibodies. In these antisera, we found that antibodies against IL13-A10, IL13-D7, and IL13-E9 recognized the minimum quantity of native form IL-13 was 0.5 ng. Furthermore, in order to generate antibodies with high titer and stability, we prepared to generate monoclonal antibodies against IL-13 with hybridoma technique, and accomplished the monoclonal antibody against PE-A10 and PE-E9. The hybridoma cell lines were kept by freezing. In the future, we’ll purify antibodies of ascites to be the material of the antiboby chip.