Methods for rapid identification of a functional single-chain variable fragment using alkaline phosphatase fusion

Kyung-Woo Lee, Byung-Ung Hur, Suk-Yoon Song, Hyo-Jung Choi, Sang-Hoon Shin, Sang-Hoon Cha
2009 BMB Reports  
The generation of functional recombinant antibodies from hybridomas is necessary for antibody engineering. However, this is not easily accomplished due to high levels of aberrant heavy and light chain mRNAs, which require a highly selective technology that has proven complicated and difficult to operate. Herein, we attempt to use an alkaline phosphate (AP)-fused form of single-chain variable fragment (scFv) for the simple identification of a hybridoma-derived, functional recombinant antibody.
more » ... mbinant antibody. As a representative example, we cloned the scFv gene from a hybridoma-producing mouse IgG against branchedchain keto acid dehydrogenase complex-E2 (BCKD-E2) into an expression vector containing an in-frame phoA gene. Functional recombinant antibodies were easily identified by conventional enzyme-linked immunosorbent assay (ELISA) by employing scFv-AP fusion protein, which also readily serves as a valuable immuno-detective reagent. [BMB reports 2009; 42(11): 731-736] Rapid identification of a functional scFv Kyung-woo Lee, et al.
doi:10.5483/bmbrep.2009.42.11.731 pmid:19944014 fatcat:tf34emtohrbfvp4llfhij56lcy