Novel hapten design, highly sensitive monoclonal antibody production, and immunoassay development for rapid screening of illegally added chloramphenicol in cosmetics.

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5-10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics.

Quantification of six types of cytokinins: Integration of an ultra-performance liquid chromatographic-electrospray tandem mass spectrometric method with antibody based immunoaffinity columns equally recognizing cytokinins in free base and nucleoside forms.

Cytokinins (CTKs) exist in various types in plants. The accurate quantification of free base and nucleoside types of cytokinins are helpful for better understanding their physiological role. In the present study, antibodies against trans-zeatin riboside (tZR) and N6-isopentenyladenine riboside (iPR) antibodies with equal recognition to free base and nucleoside cytokinins were developed. The cross-reactivity of tZR mAb 3G101G7 with tZR, trans-zeatin (tZ), dihydrozeatin riboside (DHZR), dihydrozeatin (DHZ), iPR, and N6-isopentenyladenine (iP) was 100.0%, 95.7%, 19.1%, 18.0%, 1.1%, and 0.7%, and that of iPR mAb 5C82F1 with above-mentioned 6 types of cytokinins was 1.5%, 1.4%, 5.7%, 3.1%, 100.0% and 92.6%, respectively. The obtained antibodies were used to prepare two immunoaffinity columns (IAC). The elution efficiencies of tZR 3G101G7-IAC for tZ and tZR, DHZ and DHZR and of iPR 5C82F1-IAC for iP and iPR were almost no difference with the same loading amount on their corresponding IACs. Subsequently, six types of cytokinins in mepiquat chloride (MC)-treated cotton (Gossypium hirsutum L.) roots were determined by IACs combined with ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). The contents of tZR, iPR and DHZR were increased by 9.3∼38.5%, 6.6∼23.5%, and 30.1∼110.0%, respectively, whereas those of tZ and iP were reduced by 5.3∼20.0% and 27.7∼32.1%, respectively. The decreased tZ and iP levels led to the ratio of auxin-to-active cytokinins increase to promote lateral root initiation in MC-treated cotton seeding. Integration of the IACs equally recognizing cytokinins in their free base and nucleoside forms with UPLC-ESI-MS/MS can accurately quantify different cytokinins in plant tissues.

Preparation and application of a specific single-chain variable fragment against artemether.

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC50) value and the working range based on IC20 to IC80 were 4.33 ng mL-1 and 1.05-22.65 ng mL-1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.

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.

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.

Rapid quantification of artemisinin derivatives in antimalarial drugs with dipstick immunoassays.

Substandard antimalarial drugs will result in unsatisfied therapeutic efficacy and increase the risk of resistance development. The point-of-care, qualitative, or semi-quantitative dipstick immunoassays cannot differentiate the substandard drugs with confidence. A rapid and quantitative analytical method that can be used under field conditions is needed. Here, three lateral flow immunoassays (LFIAs) based on colloidal gold nanobeads (CGN) as labels were developed for quantification of artemether, dihydroartemisinin and artesunate contents in antimalarial drugs with the aid of a portable optical scanner. Also, time-resolved fluorescent nanobeads (TRFN)-LFIA, coupled with a portable fluorescent lateral flow reader, was developed for quantification of artesunate. Commercial antimalarial drugs were used to validate these LFIAs with comparison to the gold standard high-performance liquid chromatography (HPLC) method. The drug contents estimated with these CGN-LFIAs were in the range of 85.5-109.3% of the contents determined by HPLC with a coefficient of variation (CV) of 4.5-13.0%. The TRFN-LFIA results were in the range of 93.7-108.4% of contents determined by HPLC with a CV of 5.2-8.9%. There were no significant differences between the results of CGN-LFIA and TRFN-LIFA (P = 0.5277, t-test). Both types of LFIAs with portable readers may be used for quantitation of active ingredients in antimalarial drugs and for screening substandard antimalarial drugs in resource-limiting settings.