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.

Integrated transcriptome and proteome analyses unravel a series of early defence responses in Sarcandra glabra against Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of Sarcandra glabra . However, the defence responses and underlying mechanisms remain unclear. Herein, we aimed to investigate the molecular reprogramming in S. glabra leaves infected with C. gloeosporioides . Leaf tissues at 0, 24 and 48h post-inoculation (hpi) were analysed by combining RNA sequencing and Tandem Mass Tag-based liquid chromatography with tandem mass spectrometry. In total, 18 441 and 25 691 differentially expressed genes were identified at 24 and 48hpi compared to 0hpi (uninoculated control), respectively. In addition, 1240 and 1570 differentially abundant proteins were discovered at 24 and 48hpi compared to 0hpi, respectively. Correlation analysis revealed that transcription and translation levels were highly consistent regarding repeatability and expression. Analyses using databases KEGG and iPATH revealed tricitric acid cycle, glycolysis/gluconeogenesis and phenylpropanoid biosynthesis were induced, whereas photosynthesis and tryptophan were suppressed. Enzymatic activity assay results were consistent with the upregulation of defence-related enzymes including superoxide dismutases, catalases, peroxidases and chitinases. The transcriptome expression results were additionally validated by quantitative real-time polymerase chain reaction analyses. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host-Colletotrichum interactions and breeding disease-resistant plants.

Dynamic changes in the levels of metabolites and endogenous hormones during the germination of Zanthoxylum nitidum (Roxb.) DC. Seeds.

Accumulating experimental data have shown that endogenous hormones play important roles in regulating seed dormancy and germination. Zanthoxylum nitidum is a medicinal plant that propagates via seeds, which require a long dormancy period for normal germination, and complex changes in metabolites occur during the germination process. However, the regulatory network of endogenous hormones and metabolites during the germination of Z. nitidum seeds remains unclear. This study investigated the dynamic changes in the levels of metabolites and endogenous hormones during the germination of Z. nitidum seeds. The results revealed an increase in the levels of gibberellin 3 (GA3), 12-oxophytodienoic acid (OPDA), 1-aminocyclopropane-1-carboxylic acid (ACC) and trans-zeatin (TZ) and decrease in the levels of abscisic acid (ABA), jasmonic acid (JA), N-[(-)-jasmonoyl]-(S)-isoleucine (JA-Ile) and trans-zeatin riboside (TZR). Overall, 112 differential metabolites (DAMs) were screened from 3 seed samples (Sa, Sb and Sc), most of which are related to primary metabolism. A total of 16 DAMs (including 3 monosaccharides, 3 phosphate lipids, 3 carboxylic acids, 1 amino acid, 2 pyrimidines, and 4 nucleotides) were identified in the three sample comparison pairs (Sa vs Sb, Sa vs Sc, and Sb vs Sc); these DAMs were significantly enriched in purine metabolism; glycerophospholipid metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism and pyruvate metabolism. OPDA, ACC and GAs were significantly positively correlated with upregulated metabolites, whereas ABA and JA were significantly positively correlated with downregulated metabolites. Finally, a hypothetical metabolic network of endogenous hormones that regulate seed germination was constructed. This study deepens our understanding of the importance of endogenous hormonal profiles that mediate seed germination.

Isolation of atrazine nanobodies enhanced by depletion of anti-carrier protein phages and performance comparison between the nanobody and monoclonal antibody derived from the same immunogen.

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC50) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules.

Integrated metabolome and transcriptome analysis identifies candidate genes involved in triterpenoid saponin biosynthesis in leaves of Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban is a well-known medicinal plant which has multiple pharmacological properties. Notably, the leaves of C. asiatica contain large amounts of triterpenoid saponins. However, there have only been a few studies systematically elucidating the metabolic dynamics and transcriptional differences regarding triterpenoid saponin biosynthesis during the leaf development stages of C. asiatica. Here, we performed a comprehensive analysis of the metabolome and transcriptome to reveal the dynamic patterns of triterpenoid saponin accumulation and identified the key candidate genes associated with their biosynthesis in C. asiatica leaves. In this study, we found that the key precursors in the synthesis of terpenoids, including DMAPP, IPP and ß-amyrin, as well as 22 triterpenes and eight triterpenoid saponins were considered as differentially accumulated metabolites. The concentrations of DMAPP, IPP and ß-amyrin showed significant increases during the entire stage of leaf development. The levels of 12 triterpenes decreased only during the later stages of leaf development, but five triterpenoid saponins rapidly accumulated at the early stages, and later decreased to a constant level. Furthermore, 48 genes involved in the MVA, MEP and 2, 3-oxidosqualene biosynthetic pathways were selected following gene annotation. Then, 17 CYP450s and 26 UGTs, which are respectively responsible for backbone modifications, were used for phylogenetic-tree construction and time-specific expression analysis. From these data, by integrating metabolomics and transcriptomics analyses, we identified CaHDR1 and CaIDI2 as the candidate genes associated with DMAPP and IPP synthesis, respectively, and CaßAS1 as the one regulating ß-amyrin synthesis. Two genes from the CYP716 family were confirmed as CaCYP716A83 and CaCYP716C11. We also selected two UGT73 families as candidate genes, associated with glycosylation of the terpenoid backbone at C-3 in C. asiatica. These findings will pave the way for further research on the molecular mechanisms associated with triterpenoid saponin biosynthesis in C. asiatica.

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.

Development of a direct competitive enzyme-linked immunosorbent assay for quantitation of sodium saccharin residue in food.

Sodium saccharin is a common artificial sweetener. However, due to its possible carcinogenic effects and causing metabolic disorders, many countries have strictly regulated its use in food. In the study, we prepared a specific monoclonal antibody (mAb 2H11) using the new hapten (6-carboxylsaccharin) and developed a direct competitive enzyme-linked immunosorbent assay (dcELISA) for the screening of sodium saccharin residue in food. The half-maximum inhibition concentration (IC50 ) and working range (IC20 -IC80 , the concentrations causing 20% and 80% inhibition by sodium saccharin) were 32.5 and 6.47 to 164 ng/mL, which was 6.5 times more sensitive than the previously reported immunoassay. The average recoveries of sodium saccharin in spiked food samples detected by dcELISA ranged from 82.1% to 117%. Among 70 food samples bought in the physical stores and online, sodium saccharin residues were only detected in four samples purchased online (one canned pineapple, two winter jujube, and one kimchi). The content measured by dcELISA agreed well with those determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The developed dcELISA was proved to be a sensitive and accurate method for determining sodium saccharin in food. PRACTICAL APPLICATION: Quantitation of sodium saccharin residue in food is very necessary and important for consumers and regulatory agencies.

First report of Corynespora cassicola causing black spot on Sarcandra glabra in China.

Sarcandra glabra is an important Chinese medicinal plant, which was widely cultivated under forest in south China. Guangxi province is the main producing areas of this herb. In June 2019, a serious leaf disease was found causing severe defoliation in the S. glabra plantation under bamboo forest in Rongan country, Guangxi province (109°13'N''E). About 70% of the plants in the plantation (300 ha) showed the similar symptoms. Initially, circular lesions appeared on young leaves as black spots (about 1 to 2 mm). Then, the spots gradually enlarged usually with an obvious yellowish margin (6 to 8 mm). Finally, the lesions coalesced and formed irregular, black, and large necrotic areas, resulting in the leaf abscission. For pathogen isolation, small pieces of tissue (5×5 mm) taken from 25 diseased leaves were sterilized with 75% ethanol for 30 s, subsequently, soaked in 0.1% HgCl2 for 2 min, rinsed three times in sterile distilled water, dried, and then placed aseptically onto the potato dextrose agar (PDA) plates, and incubated at 28 °C (12 h/12 h light/dark). Three days later, the isolates were placed on a new PDA plate for subsequent purification and sporulation. 20 pure fungal isolates were obtained from single spores. Of which, 15 isolates showed similar morphological characteristics.The colonies on PDA were round, dense, gray edge and dark gray in center area. Conidia in culture were appeared light brown, cylindrical in shape, with 0 to 8 septa, and 55 to 165 µm × 5.2 to 13.5 µm in size (mean = 106.2 µm × 8.6 µm, n = 30). These morphological characteristics resemble those of Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). A single-spore isolate (ZD5) was selected from the 15 fungal isolates for a subsequent molecular identification. The genes of internal transcribed spacer (ITS) of ribosomal DNA, ß-tublin, and actin were amplified with the primer pairs ITS-1/ITS-4 (White et al. 1990), ß-tubulin 2-Bt2a/Bt2b (Glass and Donaldson 1995), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. And the ITS, ß-tublin, and actin sequences were deposited in the GenBank database with the accession numbers MW362446, MW367029, and MW533122. Blast analysis and neighbor-joining analysis based on ITS, ß-tublin, and actin sequences using MEGA 6 revealed that the isolate was placed in the same clade as C. cassicola with 100% bootstrap support. Pathogenicity test was performed on the two-year-old potted S. glabra. Six-mm-diameter mycelial plugs were attached to the healthy leaves of S. glabra for co-culture, while the control group was attached with PDA. All plants were covered with plastic bags for 2 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 2 days after co-culture were identical to those observed in the field. The same fungus was re-isolated from the lesions, and further morphological characterization and molecular assays, as described above.The control leaves remained symptomless during the pathogenicity tests. According to the previous literatures, C. cassicola is a plant pathogenic fungus with a broad host range, which can damage diverse tropical plants including Salvia miltiorrhiza (Lu et al. 2019), Solanum americanum (Wagner and Louise 2019), Vitex rotundifolia (Yeh and Kirschner 2017), Cucumis sativus, Lycopersicon esculentum (Hsu et al. 2002), Carica papaya (Tsai et al. 2015),and so on. To our knowledge, this is the first report of C. cassicola causing leaf spot on S. glabra in China.

First Report of Round Leaf Spot on Sophora tonkinensis Caused by Didymella glomerata in China.

The root of Sophora tonkinensis Gagnep is an important medicinal material in China. An unknown foliar disease, first observed In July 2018, occurred over 240 ha of S. tonkinensis (totally cultured 600 ha) in Guangxi, China, in December 2019. The initial symptoms on leaf were seen as small, tawny spots (0.5 to 1.5 mm in diam.). As the disease progressed, the lesions enlarged into grayish and dark brown concentric rings (5 to 10.0 mm in diam.) resulting in black protuberances in the center of the spots. Severe infections would adversely affect plant growth, and cut the production by 30-40%. Symptomatic leaves were sampled and the surface was sterilized with 75% ethanol for 30 s and then soaked in 0.1% HgCl2 for 2 min. After three washes with sterile distilled water, the samples were dried, placed aseptically onto potato dextrose agar (PDA) plates, and incubated at 28°C. Three days later, the isolates were placed on new PDA medium for subsequent purification and sporulation. The fungus, SDG-1, was recovered from 85% of the total 40 isolates. Its colonies were whitish initially and then became olive green 7 days after incubation at 28 °C. The pycnidias were globose to subglobose, initially brown and darken at maturity, 85 to 300 × 70 to 280 µm in size. The conidia were colorless, single-celled, rounded to ellipsoidal and 3.5 to 6.6 × 1.5 to 3.8 µm. The chlamydospores were multicellular and brick trellised, usually forming branched or unbranched chains, light to dark brown in color and measured 28.5 to 44.5 × 8.2 to 16.5 µm. The morphological characteristics were consistent with Didymella glomerata. The rDNA-ITS, ß-tubulin and actin of strain SDG-1 were PCR amplified, and the DNA sequencing results were almost 100% identical to those of D. glomerata, respectively (GenBank database accession numbers MN 435377, MN447333 and MN447334, respectively). The multi-locus phylogenetic analysis was carried out with the obtained sequences, which revealed that the isolates clustered within D. glomerata with the similarity of 100% (Fig.3). Therefore, based on the morphology and phylogenetic tree, strain SDG-1 was identified as D. glomerata. For pathogenicity tests, the S. tonkinensis was cultured for two years, and the conidial suspension of SDG-1 (1 × 106 conidia /mL) was prepared by harvesting conidia from a 10-day-old culture on PDA. Conidia were sprayed onto the healthy leaves of S. tonkinensis for co-culture, while the control group was sprayed with sterile distilled water. Each experiment was performed three times. All plants were covered with plastic bags for 3 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 7 days after the leaves were inoculated with spores, which were identical to those observed in the field. The pathogenic fungus was re-isolated from the infected leaves and identified as previously described. The control leaves remained symptomless during the pathogenicity tests. According to the previous literature, D. glomerata is a plant pathogenic fungus with a wide host range, which can damage up to 100 species of woody and herbaceous plants (Aghapour et al. 2009, Lahoz et al. 2007). To our knowledge, this is the first report of D. glomerata causing round leaf spot on S. tonkinensis in China.

Ultrasensitive quantitation of imidacloprid in vegetables by colloidal gold and time-resolved fluorescent nanobead traced lateral flow immunoassays.

Imidacloprid is a highly effective insecticide, but its potential hazards to the environment and ecosystems have limited its use in many regions. A fast and sensitive analytical method would aid monitoring imidacloprid residues. The monoclonal antibody 4D9, colloidal gold and time-resolved fluorescent nanobeads (CGN and TRFN, respectively) were applied to develop a lateral flow immunoassay (LFIA) for imidacloprid detection in the present work. Both the optimized TRFN-LFIA and CGN-LFIA had a similar limit of detection of approximately 0.02 ng/mL. Use of the portable optical scanner with the TRFN-LFIA or CGN-LFIA could quantitate concentrations of imidacloprid in vegetable as sensitively as the enzyme-linked immunosorbent assay (ELISA). The LFIAs quantitative results of imidacloprid in commercial Chinese leeks were verified by the liquid chromatography-mass spectrometry with a R2 of 0.91. The LFIAs are portable and simple and thus could fully replace the ELISA for onsite quantitation of imidacloprid residues in vegetables.

Application of Immunoassays for Rapid Monitor of Carbofuran Residue in Vegetables.

Carbofuran residue in vegetables is a concern to human health. Direct competitive enzyme-linked immunosorbent assay (dcELISA) and dipstick immunoassay were developed in the present study. The dcELISA showed a 50% inhibition concentration (IC50 ) and working range of 1.3 and 0.2 to 7.5 ng/mL, respectively, while the cutoff value of dipstick immunoassay was 20 ng/mL. Applying the two immunoassays, we achieved the goal of rapid screening of carbofuran residue in commercial vegetables with a simple sample processing method. Among 46 leek, 39 potato, and 39 sweet potato samples, carbofuran residue was detected in 22% of the leek samples, and two samples exceeded the maximum residue limit of China (0.02 mg/kg). In addition, carbofuran residue was found at less than 2.5 ng/g in one potato and one sweet potato samples. The residual level of carbofuran measured by immunoassays agreed well with those determined by ultra-performance liquid chromatography tandem mass spectrometry. To ensure food safety and human health, it is greatly necessary and meaningful to monitor carbofuran residue in commercial vegetables. PRACTICAL APPLICATION: Rapid monitoring of carbofuran residue in vegetables is very necessary and important for consumers, regulatory agencies, and food industry.

Development of a lateral flow dipstick for simultaneous and semi-quantitative analysis of dihydroartemisinin and piperaquine in an artemisinin combination therapy.

Dihydroartemisinin (DHA) and piperaquine (PPQ) are two drugs used in an artemisinin-based combination therapy (ACT). The circulation of counterfeit antimalarial drugs demands the development of simple, point-of-care (POC) tests for monitoring drug quality. Here we aimed to design an antibody-based lateral flow dipstick assay for simultaneous quality control of DHA and PPQ. To obtain a monoclonal antibody (mAb) for PPQ, one structural unit of the symmetric PPQ molecule was used to derive a carboxylic acid for linkage to a carrier protein as immunogen. Screening of hybridoma cells identified an mAb 4D112B2 that reacted with the PPQ-based immunogen. A highly-sensitive icELISA was designed based on this mAb, which showed 50% inhibition concentration of PPQ at 1.66 ng/mL and a working range of 0.35 - 7.40 ng/mL. The mAb showed 10.2, 15.9 and 30.4% cross reactivity to hydroxychloroquine sulfate, chloroquine and amodiaquine, respectively. No cross reactivity was observed to lumefantrine, mefloquine artemisinin and its derivatives. Using our previous DHA dipstick design, a lateral flow dipstick for simultaneous analysis of PPQ and DHA was developed. The indicator ranges for PPQ and DHA were 2 - 5 µg/mL and 250 - 500 ng/mL, respectively. The dipstick was used to semi-quantitatively analyze PPQ and DHA content in commercial ACT drugs, which produced agreeable results to those determined by high-performance liquid chromatography. This combination dipstick makes it a potential POC device for quality control of the two active ingredients in a commonly used ACT.

Development of a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for analysing nobiletin in citrus and herb samples.

Nobiletin, a polymethoxyflavone mainly found in citrus fruits, have been reported to exhibit various beneficial biological activities for human health. It is an important bioactive compound in traditional Chinese medicine, Pericarpium Citri Reticulatae and Fructus Aurantii. To detect the contents of nobiletin in citrus and herb samples, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on monoclonal antibodies. It possessed a median inhibition concentration (IC50) of 2.43 ±â€¯0.19 ng/mL and a working range of 0.52-12.3 ng/mL. The assay exhibited the average recoveries of 72.5-85.3% in citrus peel, pulp and juice samples. Moreover, eleven citrus cultivars samples and four herb samples were also detected by the icELISA. The nobiletin content varied in different citrus cultivars samples and herb samples, which were confirmed by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS). These results indicated that the developed immunoassay was suitable for detecting nobiletin in citrus and herb samples.

Development of an enzyme-linked immunosorbent assay and a dipstick assay for the rapid analysis of trans-resveratrol in grape berries.

trans-Resveratrol, a natural polyphenol present in grapes, has many beneficial effects to human health. However, measurement of trans-resveratrol is technically complicated and time-consuming. In the present study, we obtained a sensitive and specific monoclonal antibody (mAb), namely 3C9, against trans-resveratrol. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, with 50% inhibitory concentration and working range of 1.0 ng/mL and 0.19-4.9 ng/mL, respectively. A lateral flow immunoassay (LFIA) based on 3C9 was also developed for the semiquantitative detection of trans-resveratrol in an indicator range of 50-100 ng/mL. Average recoveries of trans-resveratrol spiked in red and green grape berries samples were respectively 88-107% and 83-102% by icELISA. The icELISA and LFIA were applied for determination of trans-resveratrol in grape berries. The results were highly consistent with those determined by high-performance liquid chromatography analysis (R2 = 0.9997). Therefore, we conclude that the immunoassay methods are suitable for the large-scale screening of trans-resveratrol in grape quality breeding.

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for flubendiamide detection.

Flubendiamide (FD), the first commercial phthalic acid diamide that targets insect ryanodine receptor (RyRs), has played an important role in pest management. With its extensive worldwide application, a rapid and convenient method to detect its existence in the environment is necessary. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to analyse FD residue on environmental and food samples. The established icELISA showed a half maximal inhibition concentration (IC50) of 17.25 µg L-1, with a working range of 4.06-103.59 µg L-1 for FD, and showed no cross-reactivity with chlorantraniliprole, cyantraniliprole, and several FD analogues. Average FD recoveries from spinach, tap water, and soil samples were 89.3-112.3%, 93.0-102.1%, and 86.9-97.6%, respectively. Meanwhile, FD detection results of icELISA were compared with those of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The comparable results verified that icELISA was suitable for rapid detection of FD residue in environmental and agricultural samples.

The effects of mepiquat chloride on the lateral root initiation of cotton seedlings are associated with auxin and auxin-conjugate homeostasis.

BACKGROUND: Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC. RESULTS: In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type. CONCLUSIONS: These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation.

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

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 ACTs 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.

Development of a lateral flow dipstick immunoassay for evaluation of folate levels in maize.

Folates (vitamin B9) are essential for all organisms as cofactors for one-carbon metabolism. However, measurement of folates is technically complicated and time-consuming. In this study, we developed a dipstick immunoassay using a folate-specific monoclonal antibody (mAb), allowing rapid and low-cost detection of folates. The indicator range of the dipstick for 5-formylterahydrofolate (5-CHO-THF), 5-methyltetrahydrofolate (5-CH3-THF) and their polyglutamyl forms was 100-200 ng mL-1; moreover, no cross-reactivity was observed with tetrahydrofolate (THF) or 5,10-methenyltetrahydrofolate (5,10-CH=THF) at 500 ng mL-1, or with the folate precursors pterin-6-COOH, p-aminobenzoate (pABA), and L-glutamate, or with the folate analogues methotrexate and 10-formyltetrahydrofolate (10-CHO-THF) at up to 1000 ng mL-1. The dipstick immunoassay was tested in maize seeds; the results classified the seeds into those with low, moderate, and high levels of folates, and were in agreement with those of liquid chromatography-mass spectrometry. Thus, we conclude that the dipstick assay will provide a versatile tool to facilitate large-scale screening of maize rich in folates. Graphical Abstract The dipstick based immunoassay for analyzing folate level in maize.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of 6-benzylaminopurine and its ribose adduct in bean sprouts.

6-Benzylaminopurine (6-BA), a cytokinin plant growth regulator, has been banned for use in bean sprout production in China. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a specific monoclonal antibody (mAb 3E5). The assay showed a half-maximum inhibition concentration (IC50) and detection range of 18.9 ng/mL and 3.6-106 ng/mL, respectively. Recoveries of 6-BA spiked in home cultured bean sprout samples averaged from 75% to 89% with a correlation coefficient (R(2)) of 0.998 between the results determined by icELISA and those by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS). LC-ESI-MS showed that 6-BA had been partially metabolized to 6-benzylaminopurine riboside (6-BAR) in the positive samples. The content of 6-BA determined by icELISA was about 5-70 times higher than that of LC-ESI-MS because mAb 3E5 had 315% cross-reactivity with 6-BAR. Such icELISA being ultra-sensitive to 6-BAR would allow quick monitoring of 6-BA by detecting 6-BAR as a potential biomarker.

Rapid evaluation of artesunate quality with a specific monoclonal antibody-based lateral flow dipstick.

Artesunate is a frontline antimalarial drug for treating Plasmodium falciparum malaria. To produce specific antibodies to artesunate, the carboxyl group of artesunate was directly conjugated to carrier protein as the immunogen. A specific monoclonal antibody (mAb) 3D82G6 against artesunate was obtained by high-throughput screening of positive hybridoma clones. This monoclonal antibody had 4.0, 0.5, and 0.9 % cross reactivities with artemisinin, dihydroartemisinin, and artemether, respectively. A dipstick immunoassay was developed, and the indicator range for artesunate was 1000-2000 ng mL(-1). No interference was observed with artemisinin, dihydroartemisinin, artemether, and other commonly used antimalarial drugs for up to 20,000 ng mL(-1). The dipsticks were used for determination of artesunate contents in commercial drugs, and the results were agreeable with those determined by high-performance liquid chromatography. This dipstick, with its specificity and sensitivity for artesunate and simplicity to use, makes it a potential point-of-care device for rapid quality evaluation of artesunate-containing antimalarial drugs. Graphical Abstract Specific monoclonal antibody-based lateral flow dipstick for artesunate.