Development of single- and multiplex immunoassays for rapid detection and quantitation of amodiaquine in ACT drugs and rat serum.

Amodiaquine (AQ) is a commonly used antimalarial drug, and N-desethyl-AQ (N-DEAQ) is an active metabolite of AQ. Given the significance of drug quality in the management of malaria cases, this study aims to develop antibody-based assays for the detection and quantitation of AQ without the need for sophisticated equipment. Two monoclonal antibodies (mAbs) against AQ, designated as JUN7 and TE7, were selected, which showed 72.7% and 9.5% cross-reactivity to N-DEAQ, respectively. These mAbs showed <0.1% cross-reactivity to other commonly used antimalarial drugs. An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on JUN7 showed a 50% inhibitory concentration (IC50) of 0.16 ng/mL and a working range of 0.06-0.46 ng/mL. A lateral flow immunoassay (LFIA) based on JUN7 was also developed with a working range of 2.58-30.86 ng/mL. The icELISA and LFIA were applied for the quantification of AQ in commercial drugs, and the results were comparable to those determined using high-performance liquid chromatography. In addition, a combination dipstick for simultaneous, qualitative analysis of AQ and artesunate was developed. All immunoassays based on JUN7 can be applied for quality control of AQ-containing artemisinin-based combination therapies. As TE7 showed low cross-reactivity to N-DEAQ, an icELISA based on TE7 was developed with an IC50 of 0.38 ng/mL and a working range of 0.14-1.67 ng/mL. The TE7 icELISA was applied for the study of pharmacokinetics of AQ in rat serum after intragastric administration, and the results were consistent with those of previous studies.

Development and application of immunoassays for rapid quality control of the antimalarial drug combination artesunate-mefloquine.

Artesunate-mefloquine is one of the commonly-used artemisinin-based combination therapies (ACTs). Given the significance of drug quality in the management of malaria cases, the objective of this study was to develop antibody-based assays as the point-of-care (POC) tests for monitoring the quality of this ACT. Using mefloquine conjugated to a carrier protein as the immunogen, we selected a specific monoclonal antibody (mAb) against mefloquine with no cross-reactivity to other antimalarial drugs. Using this mAb, we developed a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA) to measure the mefloquine contents. The dcELISA for mefloquine showed a 50% inhibitory concentration (IC50) and a working range of 2.79 ng/mL and 0.58-16.37 ng/mL, respectively. With the aid of a portable optical scanner, the LFIA had a working range of 0.15-2.67 µg/mL for mefloquine. When used to measure mefloquine contents in commercial drugs, the dcELISA and LFIA results were compatible with those determined using high-performance liquid chromatography. Using the same LFIA format, we developed a combination LFIA, which correctly estimated the artesunate and mefloquine contents in commercial ACTs. Therefore, both LFIAs could be used as POC devices for rapid quality control of artesunate and mefloquine in ACTs.