Methods for rapid identification of a functional single-chain variable fragment using alkaline phosphatase fusion.
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 branched-chain 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.
Lee
Kyung-Woo
KW
Division of Molecular & Medical Biotechnology, College of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Korea.
Hur
Byung-Ung
BU
Song
Suk-Yoon
SY
Choi
Hyo-Jung
HJ
Shin
Sang-Hoon
SH
Cha
Sang-Hoon
SH
D000469:Alkaline Phosphatase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007150:Immunohistochemistry; D051379:Mice; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D000469:Alkaline Phosphatase