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.

Synthetic vitamin K3 (VK3, 2-methyl-1,4-naphthoquinone, or menadione) has been shown to have anticancer activity in various human cancer cells. We developed an enzyme-linked immunosorbant assay (ELISA) for determination of VK3 in fetal calf serum (FCS) without any pretreatment. The monoclonal antibody against VK3, which was secreted from an established hybridoma cell line (3A3) has been proven to have highly specific binding to VK3 in cross-reactivity analysis. The full measuring range of the assay extends from 0.39 to 50 µg/ml of VK3. Based on validation analysis, this immunological analysis is a precise, accurate, and sensitive method for determination of VK3. This simple immunological method would deserve to be applicable clinically.

Abstract:We prepared a monoclonal antibody (mAb 1C1) showing specificity for artemisinin (AM) and artesunate (AS), and we developed an indirect competitive enzymelinked immunosorbent assay (icELISA) using this novel mAb. Moreover, we prepared a recombinant antibody derived from mAb 1C1 in order to overcome insufficient mAb production by hybridoma culture. A recombinant antigen-binding fragment (Fab) was easily constructed using antibody manipulation technologies and was produced by microorganisms in high yield. We herein review immunochemical approaches for analysis of the antimalarial drugs AM and AS that were able to yield analysis results for multiple samples in a short period of time using simple and reliable protocols.

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