Surface Modification of Bamboo Charcoal as a Template of High-through-put Cell Sorter for Monoclonal Antibody Production
Date Issued
2009
Date
2009
Author(s)
Chang, Yu-Chung
Abstract
George Kohler and Cesar Milstein employed fusion theory to produce monoclonal antibody in 1975. An attempt was made in order to increase the mAb production by a high-through-out cell sorter in subcloning (selection step). A siphon bioreactor (cytoflow-bioreactor) and the new material (bamboo charcoal) combined as a cell sorter called “high-through-put cell sorter”. The advantage of the high-through-put cell sorter was reduced time-comsuming, labor-intensive, cost-effective and animal-reducing for monoclonal antibody production. The feasibility of the high-through-put cell sorter was tested with A549 cell as the model antigen, due to the high expression of the “epidermal growth factor receptor, EGFR” on the membrane of the A549 cell. The results of Raman spectrum revealed the oxidation of bamboo charcoal might be because of the usage of nitric acid or acid mixture (nitric acid/sulfuric acid: 1/3). From the FT-IR, we could further analyze the introduction of carboxyl group on the surface of bamboo charcoal. The IFA confirmed that we could conjugate purified human EGFR on the surface of the acid-treated bamboo charcoal by forming an amide bond using EDC/NHS and those results were further confirmed by TGA. In SEM, the conjugation of purified human EGFR on the acid-treated bamboo charcoal surface captured the splenocytes (B cells) from immunized mice. The results of ELISA monitored the IgG secretion from the supernatant of the bamboo charcoal binding with hybridoma cell cultured medium. Thus it was confirmed that it could secrete anti-human EGFR antibody in the supernatant of the cultured bamboo charcoal binding with hybridoma cell from IFA. In the future, this “high-through-put cell sorter” could be also employed to harvest antibody-secreting B cells from the peripheral blood without sacrificing the animals and also expected to produce humanized monoclonal antibody.
Subjects
bamboo charcoal, cytoflow-bioreactor, siphon bioreactor, bioreactor, surface modification, monoclonal antibody, hybridoma cell, epidermal growth factor receptor, EGFR, anti-EGFR antibody, cell sorter
Type
thesis
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