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KMID : 0624620090420110731
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BMB Reports
2009 Volume.42 No. 11 p.731 ~ p.736
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Methods for rapid identification of a functional single-chain variable fragment using alkaline phosphatase fusion
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Lee Kyung-Woo
Hur Byung-Ung
Song Suk-Yoon
Choi Hyo-jung
Shin Sang-Hoon
Cha Sang-hoon
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Abstract
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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. 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.
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KEYWORD
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Alkaline phosphatase, BCKD-E2, Hybridoma, PhoA, scFv
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