title: Preparation of a single-chain variable fragment and a recombinant antigen-binding fragment against the anti-malarial drugs, artemisinin and artesunate, and their application in an ELISA.
abstract: Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Madan K
authors.initials: MK
authors.lastname: Paudel
authors.forename: Ayako
authors.initials: A
authors.lastname: Takei
authors.forename: Junichi
authors.initials: J
authors.lastname: Sakoda
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Juengwatanatrakul
authors.forename: Kaori
authors.initials: K
authors.lastname: Sasaki-Tabata
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity; D000962:Antimalarials / Q000276:immunology*; D019306:Artemisia / Q000737:chemistry; D037621:Artemisinins / Q000276:immunology*; D000077332:Artesunate; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology*
chemical_list: D000890:Anti-Infective Agents; D000911:Antibodies, Monoclonal; D000962:Antimalarials; D037621:Artemisinins; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D000077332:Artesunate; C031327:artemisinin