Journal
BIOLOGICAL & PHARMACEUTICAL BULLETIN
Volume 32, Issue 3, Pages 434-439Publisher
PHARMACEUTICAL SOC JAPAN
DOI: 10.1248/bpb.32.434
Keywords
single-chain variable fragment; plumbagin; cross-reactivity; enzyme-linked immunosorbent assay; Plumbago zeylanica
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Funding
- Japan Science Society
- Kyushu University Foundation
- Japan Society for the Promotion of Science Asian CORE Program
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- National Center for Genetic and Biotechnology (BIOTEC), Thailand
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We constructed a single-chain variable fragment (scFv) antibody against plumbagin (PL) with improved specific binding to PL. Variable heavy- and light-chain genes were cloned directly from the cDNA of hybridoma cell line 3A3 and assembled using the splice-overlap extension polymerase chain reaction (SOE-PCR) with specific primers including flexible peptide (Gly(4)Ser)(3) linker primers. The constructed scFv gene was ligated into the pET28a expression vector and transformed into Escherichia coli BL21 (DE3). The denatured protein expressed as inclusion bodies in E. coli was solubilized, purified, and refolded by a stepwise dialysis. Intriguingly, the refolded scFv against PL displayed higher PL-binding specificity than that of its parental monoclonal antibody, MAb 3A3, which suggests the possibility of improving the function by constructing the scFv antibody. These notable properties of the recombinant antibody against PL made it possible to develop an enzyme-linked immunosorbent assay (ELISA) for reliable determination of PL.
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