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. Author manuscript; available in PMC: 2019 Feb 5.
Published in final edited form as: J Pharm Biomed Anal. 2018 Jun 25;159:66–72. doi: 10.1016/j.jpba.2018.06.051

Development of monoclonal antibody-based immunoassays for quantification and rapid assessment of dihydroartemisinin contents in antimalarial drugs

Xiangxue Ning 1, Weizhi Li 2, Mian Wang 1, Suqin Guo 1, Guiyu Tan 1, Baomin Wang 1,*, Liwang Cui 2,*
PMCID: PMC6363535  NIHMSID: NIHMS1009053  PMID: 29980021

Abstract

Dihydroartemisinin (DHA) is one of the artemisinin derivatives widely used in artemisinin-based combination therapies (ACTs) for malaria treatment. The availability of a point-of-care device for estimation of DHA quantity would allow a quick quality assessment of the DHA-containing drugs. In this study, 9-O-succinylartemisinin was obtained from microbial fermentation of artemisinin, which was hydrogenated to 9-O-succinyldihydroartemisinin as the hapten for DHA. A monoclonal antibody (mAb), designated as 2G11G4, was identified after screening the hybridoma library, which showed 52.3% cross reactivity to artemisinin, but low or no cross reactivity to artesunate, artemether, and several ACT partner drugs. Based on this mAb, a highly-sensitive, indirect competitive enzyme-linked immunosorbent assay was designed, which showed 50% inhibition concentration of DHA at 1.16 ng/mL, a working range of 0.26–4.87 ng/mL, and limit of detection of 0.18 ng/mL. In addition, a colloidal gold-based lateral flow immunoassay (dipstick) was developed with an indicator range (indicating sensitivity) of 50–100 ng/mL. This dipstick was evaluated for determination of DHA contents in commercial drugs and the results were highly agreeable with those determined by high-performance liquid chromatography.

Keywords: Dihydroartemisinin, dipstick, malaria, antibody, immunoassay

1. Introduction

Malaria is still a serious health problem in the world, especially in sub-Saharan Africa. According to the most recent estimates, there were 216 million malaria cases and approximately half a million malaria deaths in 2016. Artemisinin-based combination therapies (ACTs) are the front-line treatment for falciparum malaria. Good-quality ACTs are necessary for effective malaria case management, but the increasing proportions of poor-quality drugs in many endemic countries are disturbing [1]. Substandard drugs not only cannot deliver the needed clinical efficacy, but also promote the development of resistance to these drugs. Furthermore, falsified artemisinin family drugs with little or no active ingredient can be life-threatening [2]. Antimalarial drugs, especially ACTs, have been the targets of counterfeiting [3]. A review of the quality of antimalarial drugs collected from Southeast Asia and Africa showed that at least 35% of the antimalarials did not pass the chemical analysis and a large proportion was deemed falsified [4]. This problem calls for concerted action of both government and private sectors. Whereas quality control needs to be reinforced at all levels of the supply chain, enabling technology for the detection of falsified ACTs at the point of care, like the rapid diagnostic tests (RDTs) for malaria diagnosis, is urgently needed.

Among the artemisinin derivatives, dihydroartemisinin (DHA) (Fig. 1A) is included in one of the most widely used ACTs, DHA-piperaquine [5]. Methods for determining DHA contents include high-performance liquid chromatography (HPLC) [6, 7], HPLC-ultraviolet-visible spectroscopy [8], a colorimetric assay, and thin-layer chromatography (TLC) [9, 10]. TLC, in particular, implemented in the Minilab from Global Pharma Health Fund, is recommended by the World Health Organization for the detection of fake drugs [11]. However, these assays, often requiring expensive instruments and trained personnel, as well as toxic reagents, are not appropriate for rapid analysis of DHA drugs under field settings in resource-limited malaria-endemic areas. The widespread use of RDTs for point-of-care malaria diagnosis has motivated us to develop similar technologies for quality control of ACTs in remote endemic settings. Thus far, we have obtained specific monoclonal antibodies (mAbs) against artemisinin and two of its derivatives, artesunate and artemether, which enabled us to develop enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays for quantifying these artemisinins [12–15]. Our earlier attempt to screen for a DHA-specific mAb after immunization of mice with an immunogen consisting of a carrier protein conjugated to the carboxyl group of artesunate has led to the identification of a mAb that showed high cross reactivity with artemisinin, artesunate and DHA [15]. In this study, inspired by the transformation of artemisinin to DHA [16], we developed a mAb against DHA using a novel hapten 9-O-succinyldihydroartemisinin, which exhibited almost no cross-reactivity to artesunate and artemether. With this DHA antibody, an indirect competitive ELISA (icELISA) [13] and a colloidal gold-based lateral flow immunoassay were developed and evaluated for monitoring DHA content in ACTs.

Fig. 1.

Fig. 1.

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(A) Chemical structure of DHA. (B) A schematic for preparation of 9-O-succinyldihydroartemisinin. (C) A schematic for preparation of DHA hapten and protein-hapten conjugate (9-O-succinyldihydroartemisinin-BSA/OVA).

2. Materials and methods

2.1. Reagents

DHA, artesunate, artemisinin and artemether were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Lumefantrine, piperaquine, chloroquine and amodiaquine were purchased from Sigma (St. Louis, MO, USA). Cunninghamella elegans (ATCC 9245) was from the American Type Culture Collection. Cell culture medium (Dulbecco’s modified Eagle’s medium, DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Paisley, Scotland). All other chemicals and organic solvents used were of analytical grade and purchased from Sinopharm Chemical Reagent (Beijing, China). 1-(3-Dimethyl amine propyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), succinic anhydride, 4-dimethylamino-pyridine (DMAP), bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol 2000, dimethyl sulfoxide (DMSO), hypoxanthine, aminopterin, thymidine, penicillin, streptomycin, L-glutamine, horseradish-peroxidase-labeled goat anti-mouse IgG, complete and incomplete Freund’s adjuvant were purchased from Sigma. The antimalarial drugs used for testing specificity of the mAb were convenient samples purchased from Africa and Myanmar.

2.2. Preparation of 9-O-succinyldihydroartemisinin

Artemisinin was transformed to 9-hydroxyartemisinin by microbial fermentation in C. elegans culture, and the transformed products reacted with succinic anhydride to form 9-O-succinylartemisinin [13]. To prepare the DHA hapten, potassium borohydride was added to the solution of 9-O-succinylartemisinin (molecular ratio1.2:1) with calcium chloride in methanol at 5 °C. The reaction was ended when no more gas came out, and excess potassium borohydride was destroyed by adding concentrated hydrochloric acid at 0–6 °C until the pH reached 0.5–1. To precipitate the reaction product, water was added, and the pH of the reaction mixture was adjusted to 5–6 by quenching the excess hydrochloric acid with aqueous potassium hydroxide. Pure 9-O-succinyldihydroartemisinin was obtained after filtration, washed with water, and dried. The purity of 9-O-succinyldihydroartemisinin was verified by HPLC and high-resolution mass spectrometry.

2.3. Preparation of immunogen and coating antigen

The 9-O-succinyldihydroartemisinin was conjugated to BSA and OVA as the immunogen and coating antigen, respectively. In brief, 4.2 mg of EDC and 2.96 mg of NHS were added to 4 mg of 9-O-succinyldihydroartemisinin in 0.5 mL of DMSO. The reaction mixture was stirred overnight at 4 °C. Afterwards, the mixture was added dropwise to 46.0 mg of BSA or 29.5 mg of OVA dissolved in 4 mL of 0.01 M phosphate buffered saline (PBS), which was then stirred overnight at 4 °C. The mixture was dialyzed against 2 L of 0.01 M PBS (pH 7.5) containing 0.15 M NaCl for 3 days with six changes of the buffer, and the immunogen was stored at −20 °C.

2.4. Preparation of a mAb against DHA

To obtain a specific mAb against DHA, Balb/c mice were initially immunized with 100 μg of the immunogen emulsified with an equal volume of complete Freund’s adjuvant, and two booster immunizations with 100 μg of the immunogen in incomplete Freund’s adjuvant were given in two weeks interval. Three days after the final immunization, blood was collected and antibody titers against DHA were determined by icELISA. The mouse with the highest antibody titer was selected, and further boosted with 100 μg of the immunogen. Three days after the final boost, spleen cells were collected and fused with the murine SP2/0 myeloma cells. Hydridoma screening and mAb production followed the procedure described previously [13]. To exclude mAbs that cross-reacted with artemisinin and artemether, all positive hybridomas were counter-screened with artemisinin and artemether.

2.5. Development of icELISA for quantification of DHA

The optimum conditions for the icELISA (dilutions of 9-O-succinyldihydroartemisinin-OVA conjugate, DHA antibody, and HRP-conjugated goat anti-mouse IgG antibody) were determined by checkerboard titrations. Briefly, 96-well microtiter plates were coated with 100 μL coating antigen at 37 °C for 3 h. Afterwards, 50 μL of drug standard or sample at a known concentration or specified dilution, and 50 μL of diluted antiserum or antibody were successively added to each well. After incubation at 37 °C for 30 min, the plate was washed and 100 μL of goat anti-mouse IgG-HRP solution were added and incubated for another 30 min at 37 °C. After washing, 100 μL of freshly-prepared substrate solution were added, and optical density was measured with a microplate reader after 15 min of reaction.

2.6. Validation of icELISA for quantifying DHA in culture medium

To determine the potential uses of the icELISA for accurately determining DHA concentrations in biological samples, we used this assay to monitor the dynamics of DHA added into in vitro culture of the Plasmodium falciparum parasite. Malaria parasite was cultured in a complete medium at 37°C in a CO2 incubator as described earlier [17]. DHA was added at a final concentration of 200 nM to (A) culture medium only, (B) red blood cells (RBCs) in culture medium at 5% hematocrit, and (C) P. falciparum-infected RBCs in culture medium at 5% hematocrit and 5% parasitemia. An aliquot of the samples (1 ml) was removed at 2, 4, 6, 9, 12, 24, and 36 h and DHA in the culture medium was determined using the icELISA. The half-life of DHA in the culture medium was calculated using Origin Pro 9.0 (Origin Lab).

2.7. Preparation of drug samples for analysis

All drugs were powdered by a high-speed grinder and 5 mL of acetonitrile were added to the powder in tubes, incubated for 2 h at room temperature, and extracted by sonication using a Branson SB5200 sonicator (Shanghai, China) for 20 min. After centrifugation, the supernatant was stored at −20 °C until the icELISA and HPLC analyses. Further dilution of the extract with acetonitrile was based on the claimed amount from the packaging label. The final dilution for HPLC analysis was filtered through a 0.45 μm membrane.

2.8. HPLC analysis of DHA

HPLC analysis of DHA standards and drug samples was performed using the Waters 600E multisolvent delivery system and Waters 2487 dual λ absorbance detector (Milford, MA, USA). The mobile phase was 60% acetonitrile in H2O at a flow rate of 0.6 mL/min. UV absorption at 216 nm was recorded. The injection volume was 20 μL. The retention times for the two enantiomers of DHA were approximately 12 min and 17 min, respectively.

2.9. Development of colloidal gold-based dipstick assay for DHA

Colloidal gold with an average particle diameter of 30 nm (G30) was purchased from Shanghai Jieyi Biotechnology. Twenty microliters of aqueous solution of the anti-DHA mAb at 6 mg/mL was added to 2 mL of colloidal gold solution (pH 7.8). After gentle stirring for 10 min, 40 μL of 10 % (w/v) BSA was added to the solution. Then, the mixture was centrifuged at 9600 ×g for 20 min. The pellet was resuspended in 500 μL of 0.01 M PBS (pH 7.0) and stored at 4 °C. Finally, the conjugate pad was saturated with the gold-antibody conjugate and dried at room temperature. Goat anti-mouse IgG was used as a control capture reagent. 9-O-succinyldihydroartemisinin-BSA was used as the capture reagent immobilized at the test line on the membrane. 9-O-succinyldihydroartemisinin-BSA and goat anti-mouse IgG in the volume of 1 μL/cm were immobilized onto the nitrocellulose (NC) membrane. The distance between the control and test lines was 5 mm. The NC membrane was pasted onto the center of the PVC plate which served as the backing of the test strip. The colloidal gold-Ab conjugate pad was pasted on the plate by overlapping with the NC membrane. The absorption pad was pasted on the plate at the opposite end downstream from the control line, and the sample pad was pasted by overlapping with the conjugate pad. The card was cut to 3 mm width. Strips were then sealed in a plastic case with desiccant gel and stored at 4 °C.

2.10. Dipstick assay

DHA standard aqueous solutions at concentrations of 0, 6.25, 12.5, 25, 50 and 100 ng/mL were used to determine the sensitivity of the dipstick as described [15]. Sensitivity is expressed as the indicator range of the dipstick, i.e., the lowest concentration of the target analyte at which the test line was not visible. A number of commonly used antimalarial drugs including the artemisinin derivatives were tested for specificity. The stock solutions of these drugs were prepared at 1 mg/mL as described previously [15], and each sample was analyzed in triplicates. Interpretation of the dipstick assay results was according to the earlier description [18]. Briefly, for a valid assay with the control line visible, if the test line showed no color, the drug in the solution would be above the indicator range and the drug examined was considered to have passed the test. If the test line showed a dark or faint color, it suggested that the drug in the solution was below the indicator range and the drug content should be lower than the claimed amount on the label. Further testing at a concentration ten times higher was generally done to confirm the finding.

3. Results

3.1. Preparation of hapten and immunogen

To prepare the hapten for DHA, we used a microbial fermentation strategy and obtained 9-hydroxyartemisin, which was converted to 9-O-succinylartemisinin after reacting with succinic anhydride. 9-O-succinylartemisinin was then reduced to 9-O-succinyldihydroartemisinin, which was purified (Fig. 1B). The purity of 9-O-succinyldihydroartemisinin was verified by HPLC analysis, which showed a single peak (data not shown). High-resolution mass spectrometry analysis of the purified 9-O-succinyldihydroartemisinin showed a molecular size of 423.1624, consistent with the calculated size of C19H29O9 [M+Na]+ of 423.1626, confirming the purity and authenticity of 9-O-succinyldihydroartemisinin. Subsequently, 9-O-succinyldihydroartemisinin was conjugated to a carrier protein of BSA to generate the immunogen for immunization of mice or OVA to produce the coating antigen for ELISA (Fig. 1C).

3.2. Screening for an anti-DHA mAb

To obtain specific mAbs against DHA, five mice were immunized with an immunogen consisting of 9-O-succinyldihydroartemisinin linked to BSA. The one with the highest antibody titer against DHA was used for cell fusion. Screening of the library in five 96-well plates identified 10 positive hybridoma clones, which were selected and tested for specificity to DHA. One of the DHA-positive clones, 2G11G4, showed 52.3% cross reactivity to artemisinin and very low cross-reactivity with artesunate (1.6%), artemether (<0.02%) and other ACT partner drugs (Table 1). This hybridoma clone was further cloned twice by limiting dilution, followed by expansion for large-scale production of mAbs. This DHA mAb was purified and used for the development of a DHA icELISA.

Table 1.

Cross reactivity of the mAb with artemisinin derivatives.

Analytes IC50 (ng/mL) Cross reactivitya(%)
Dihydroartemisinin 1.16 100
Artemisinin 2.22 52.3
Artesunate 70.4 1.6
Artemether >20000 <0.02
Lumefantrine >20000 <0.02
Piperaquine >20000 <0.02
Chloroquine >20000 <0.02
Amodiaquine >20000 <0.02
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a

Cross-reactivity (%) = (IC50 of dihydroartemisinin/IC50 of the other compound) × 100

3.3. DHA icELISA

Using checkerboard titration, the optimum dilutions of 9-O-succinyldihydroartemisinin-OVA conjugate, DHA antibody, and goat anti-mouse IgG-HRP were determined to be 3000, 1000 and 1000 at the concentration of 1 mg/mL, respectively. Using this icELISA, the DHA concentration causing 50% of inhibition (IC50) was determined to be 1.16 ng/mL, and the working range defined as the DHA concentration causing 20–80% inhibition of the signal (IC20 – IC80) was 0.26–4.87 ng/mL (Fig. 2A). The limit of detection (LOD), defined as the IC15 [19], was 0.18 ng/mL.

Fig. 2.

Fig. 2.

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(A) A standard inhibition curve of DHA in the icELISA format. B0 and B are absorbance without and with a competitor, respectively. IC20, IC50 and IC80 are marked in the graph. The working range is defined as IC20 - IC80. (B) Time course of DHA concentrations in the culture medium only (blue), RBCs at 5% hematocrit (red), and P. falciparum culture at 5% hematocrit and 5% parasitemia (black). The starting concentrations of DHA were at 200 nM.

This icELISA was used to determine the time course of DHA degradation once it was added to the malaria culture. Our results showed that DHA concentrations declined rapidly under all culture conditions tested (Fig. 2B). Especially, in parasite culture at 5% parasitemia, DHA concentration decreased to <4 nM after 12 h of incubation. DHA had a half-life of 5.56 ± 0.87 h, 4.55 ± 0.69 h, and 3.64 ± 0.48 h under the A, B, and C culture conditions, respectively. As a comparison, an earlier study determined the half-life of DHA in a buffer solution (pH=7.4) and 50% plasma to be 5.5 h and 2.3 h, respectively [20]. The study of DHA pharmacokinetics in vivo showed that the half-life in the plasma was ranged from 1 to 2 h and was shorter than the culture conditions [21, 22].

3.4. Development of the colloidal gold-based dipstick for DHA

The mAb 2G11G4 was selected to develop a colloidal gold-based lateral-flow immunoassay. The sensitivity of the dipstick is dependent on the concentration of 9-O-succinyldihydroartemisinin-BSA conjugate coated on the membrane and the amount of colloidal gold-mAb. In order to obtain the highest sensitivity for DHA, the optimum concentrations of 9-O-succinyldihydroartemisinin-BSA, goat anti-mouse IgG antibody, and colloidal gold-mAb 2G11G4 were determined by the checkerboard analysis, which were 1 mg/mL, 0.4 mg/mL and 30 μg/mL, respectively. The color intensity of the dipsticks on the test line was dependent on the concentration of DHA in the testing solution. Using serially diluted standard DHA, the indicator range, defined as the lowest concentration of DHA at which the test line was not visible, was 50–100 ng/mL (Fig. 3A).

Fig. 3.

Fig. 3.

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(A) Determination of the LOD of the DHA dipsticks. DHA was applied to the dipsticks at increasing concentrations (0, 6.25, 12.5, 25, 50 and 100 ng/mL). Note the disappearance of the test line as DHA concentration reached 50–100 ng/mL, which is considered the limit of detection of the dipsticks. (B) Specificity of the DHA dipsticks for DHA. Artemisinin (ART), artesunate (ATS) and artemether (ATM) were all tested at 100 ng/mL. (C) Testing of commercial DHA-containing drugs using the DHA dipsticks. 1. D-artepp Lot No: SQ160101; 2. Ridmal; 3. P-Alaxin Lot No: 1040; 4. Darte-Q Cap. /Dtq Cap; 5. Duo-Cotecxin Lot No: 141117 6; D-Artepp Lot No: SQ130801; 7. Duo-Cotecxin Lot No: 130635; 8. Darplex Lot No: 12EA. Each sample was analyzed in triplicates and the figure shows representative pictures. Drugs 1–5 passed the test, whereas expired drugs 5–8 showed variable color intensities of the test line (T). The lines marked C are the control lines.

To determine the specificity of the dipstick for the artemisinin family drugs, artemether, artesunate and artemisinin standards were used for the evaluation of cross-reactivity. In the indicator range of the dipsticks (100 ng/mL), DHA completely prevented the appearance of the test line, whereas artemether, artesunate and artemisinin showed obvious color (Fig. 3B). At 500 ng/mL, artesunate and artemisinin showed different degrees of inhibition, while artemether still showed no inhibition. Further, DHA, artemether, artesunate and artemisinin were extracted from four commercial drugs and used for validation of the assay at concentrations of 50 and 100 ng/mL. The testing results agreed well with those of the standard drugs (not shown). Altogether, these results demonstrated that the DHA dipstick was specific for DHA, and at the indicator range it could discriminate between DHA and artemisinin and its derivatives (artesunate and artemether).

3.5. Semi-quantitative analysis of DHA-based ACT

The potential use of the dipstick as a quality control test for DHA-based ACTs was evaluated using 15 commercial ACT drugs. DHA contents in these drugs were first determined by HPLC, which found that five drugs had DHA contents consistent with those specified on the labels, whereas the DHA contents in 10 expired drugs ranged from 70 to 90% of the labeled content (Table. 2). For dipstick analysis, the stock solutions of drugs were diluted to 100 ng/mL and applied to the dipsticks. No color development was observed on the test line for the five qualified drugs, whereas the color intensity on the test line for the expired drugs developed color of different intensity (Fig. 3C), indicating that the tested concentrations of these drugs were less than 100 ng/mL. When these drugs were tested at ten times lower dilution, the test lines did not show up (data not shown).

Table 2.

Comparison between dipstick and HPLC results for the quantitation of DHA in commercial drugs.

Drug names SM/DC* Lot no. Region* Dipstick results Expected DHA content (mg/mL) HPLC results (mg/mL)
Ridmal Unknown Unknown Africa passed 2 2.16±0.02
P-Alaxin Bliss GVS Pharma Ltd 1040 Africa passed 2 1.93±0.03
Darte-Q Cap./Dtq Cap. The Power of God International Unknown Africa passed 2 2.03±0.01
D-Artepp Guilin Pharmaceutical Co., Ltd, China SQ130401 Bamaw# passed 2 1.85±0.08
SQ160101 Africa passed 2 2.03±0.04
SQ130801 Singu# failed 2 1.73±0.05
SQ140102 Moemauk failed 2 1.74±0.09
Duo-Cotecxin Zhejiang Holley Nanhu Pharmaceutical Co., Ltd., China 130635 Mandalay# failed 2 1.49±0.01
141117 Shwebo# failed 2 1.76±0.04
130421 Unknown# failed 2 1.62±0.03
13072010 Unknown# passed 2 1.89±0.02
140329 Pha-Kant# failed 2 1.76±0.02
130635 Moenyin# failed 2 1.76±0.02
Unknown Africa passed 2 2.03±0.04
DARPLEX Kunming Pharmaceutical Co., China 12EA Pathein# failed 2 1.44±0.01
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*

SM: Stated Manufacturer. DC: Distributing Company.

*

Except African samples, all other samples were collected in different regions of Myanmar.

#

At the time of the assay, these were expired drugs.

4. Discussion

Artemisinin and its derivatives such as DHA, artesunate and artemether differ only at position 12 in their chemical structures (Fig. 1A). This poses a major difficulty to develop highly specific mAbs that can distinguish artemisinin and its derivatives, since most methods for conjugating the artemisinin haptens to carrier proteins employed the position 12 in the chemical structures [14, 23, 24]. For example, the artemisinin antibody developed earlier showed 57% and 650% cross-reactivity with DHA and artesunate, respectively [14]. Another mAb 3D82G7 obtained later also showed strong cross-reactivity with DHA (330%) and artesunate (460%) [15]. Such cross reactivity to artemisinin and its derivatives would be considered acceptable or even favorable for measuring artemisinin family compounds in commercial antimalarial drugs, since each commercial ACT only contains one artemisinin family drug. However, highly specific mAbs for artemisinin and its derivatives are required for quantifying individual compounds, especially for following their dynamic changes in pharmacokinetic-pharmacodynamic (PK-PD) studies. Since artemisinin and its derivatives all are rapidly converted to the primary metabolite DHA after administration, specific mAbs that could differentiate between DHA and other artemisinin derivatives are essential for quantification of these compounds in human blood. Although mass spectrometry is sometimes used as the analytical tool for PK-PD studies [25], ELISA will be a rapid, sensitive and reliable analytical method, and more importantly without the requirement for expensive instrument.

Using different conjugation strategies, we have obtained specific mAbs to artemisinin and artemether [26]. Here, using a microbial fermentation and chemical modification strategy, we designed a new immunogen of artemisinin with the 12 position of the chemical structure exposed in the immunogen. Immunization of mice allowed us to identify a DHA mAb with almost no cross reactivity with two other artemisinin derivatives artesunate and artemether. However, the mAb developed in this way had 52.3% cross reactivity with artemisinin due to the minor structural difference at position 12 between DHA and artemisinin. Though this cross reactivity with artemisinin hinders the use of this mAb for PK-PD study of artemisinin, it should still be useful for PK-PD studies of DHA, artemether and artesunate.

Using the DHA mAb 2G11G4, we designed a highly sensitive icELISA with the working range of DHA concentrations as low as 0.26–4.87 ng/mL and the LOD of icELISA as 0.18 ng/mL. This level of sensitivity is as good as that of the standard HPLC method, and should be able to accurately measure the quantity of DHA in blood. We have shown that the icELISA based on the specific mAb for artemisinin was suitable for PK studies of artemisinin in rat serum [13]. Using this icELISA for DHA, we followed the degradation of DHA during in vitro culture of the malaria parasites. Our results confirmed rapid degradation of DHA added to the in vitro culture of P. falciparum, further confirming the results from an earlier study [20]. While our work validated the potential use of the DHA icELISA for quantifying DHA with high sensitivity, it would be worthwhile to further test whether combination of several specific icELISAs for artemisinin and its derivatives would allow us to perform PK-PD studies of these drugs in humans.

Dipsticks have been used widely for rapid tests of residues of antibiotics [27, 28] and veterinary drugs [29] in animal-origin food. To date, we have developed mAb-based dipsticks for rapid assessment of the content of artemisinin family compounds in commercial ACTs. Pilot testing of these simple dipsticks highlights the potential for future optimization and deployment of these assays as qualitative and semi-quantitative assays for rapid screening of counterfeit and substandard artemisinin drugs in endemic settings [18]. The usefulness of these dipsticks as a point-of-care tool of ACT drug quality has been further validated in a recent survey of artemisinin-containing antimalarial drugs in Myanmar [30]. Here we have developed a prototype dipstick for DHA based on the mAb 2G11G4. The DHA dipsticks had an indicator range of 50–100 ng/mL. It is noteworthy that the indicator range observed by naked eyes may differ slightly from individual to individual, which may affect accurate determination of drug concentrations with the dipsticks. Since the dipsticks are meant to provide a rough estimate of the drug content, slight variation in individual’s perception should not have a major impact on determining whether a drug is substandard or not, given more drug dilutions can be tested.

When these dipsticks were evaluated using commercial drugs, we found that the color on the test line became apparently visible by naked eyes when the DHA content had decreased to 70% of the claimed content on the label. When the DHA content was 80–100% of the labeled value, the test line color was very faint and almost invisible to naked eyes. Testing of these drugs at 10-fold higher concentrations allowed us to verify the substandard drugs. With the potential use of these mAb-based dipsticks for quality control of ACTs in remote endemic settings, further optimization of the dipsticks is needed. The shelf life of the product under different conditions needs to be tested; sufficient stability under ambient temperature is required since many endemic areas do not have a cold chain. With regard to the poor water solubility of the artemisinin, DHA and artemether, we have used organic solvents such as acetonitrile for extraction in the lab and recommend the use of pure ethanol to prepare stock drug extracts for field use. The product performance under these conditions will have to be evaluated in the future.

5. Conclusions

A specific hapten for DHA was designed using a microbial fermentation strategy, which allowed the selection of a mAb with high avidity for DHA and low cross reactivity to artesunate and artemether. An icELISA based on this mAb was highly accurate for measuring DHA concentration, with an IC50 to as low as 1.16 ng/mL, working range of 0.26–4.87 ng/mL and an LOD of 0.18 ng/mL. A dipstick was developed using this mAb, which has an indicator rang of 50–100 ng/mL for DHA. This dipstick did not cross-react with artesunate, artemether, artemisinin or other ACT partner drugs when the drug concentration was less than 100 ng/mL, and was useful for qualitative and semi-quantitative analysis of DHA in commercial antimalarial drugs. Test results of this dipstick for DHA in commercial drugs were agreeable with those determined by HPLC. Thus, this DHA dipstick has the potential to be developed as a point-of-care device for identifying substandard and falsified DHA-based antimalarial drugs.

Acknowledgments

This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (U19AI089672).

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