No abstract

The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.

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.

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

A single-chain variable fragment antibody (scFv) against ginsenoside Re (G-Re) was constructed and applied to an enzyme-linked immunosorbent assay (ELISA) for determining the total concentration of ginsenosides in various ginsengs. The variable heavy and light chain genes were cloned directly from the cDNA of the 4G10 hybridoma cell line and assembled by means of splicing by overlapping extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (Gly(4)Ser)(3) between the variable heavy chain and light chain domains. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The recombinant scFv against G-Re (GRe-scFv) was expressed as a chimera protein containing the His6-tag at its N-termini, purified by immobilized metal ion affinity chromatography (IMAC), and refolded by a stepwise dialysis method. The yield of GRe-scFv after purification was 1.7 mg per liter of culture medium. Characterization of GRe-scFv revealed that it retained the characteristics of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10) which has wide cross-reactivity with 20(S)-protopanaxadiol- and 20(S)-protopanaxatriol-type ginsenosides. The detectable range for G-Re in ELISA using scFv antibody was 0.02-10 µg/ml. Based on validation analysis, the use of GRe-scFv in ELISA is a precise, accurate, and sensitive method. In light of the time-consuming and labor-intensive procedures for the preparation of MAb, speedy bacterial expression of GRe-scFv is a powerful alternative tool for producing MAb to use in ELISA for quantitative analysis of total ginsenoside concentrations.