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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 48 h 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 48 hpi compared to 0 hpi (uninoculated control), respectively. In addition, 1240 and 1570 differentially abundant proteins were discovered at 24 and 48 hpi compared to 0 hpi, 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. Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of the medicinal plant Sarcandra glabra. We explored the molecular changes during the early stages of this infection through systems biology. 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. [ABSTRACT FROM AUTHOR]

The safety of medicinal herbs is critical to human health, and the monitoring of pesticide residues in medicinal herbs is necessary. In the present study, a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of imidacloprid in medicinal herbs. Using the optimal conditions, the 50% inhibiting concentration and detection range (20–80% inhibiting concentration) of the established dc-ELISA were 0.08 and 0.03–0.29 ng/mL, respectively. Except for clothianidin (4.64%) and acetamiprid (2.52%), the cross-reactivity with analyte analogs was negligible. In purple perilla, cordate houttuynia, water lily, bitter melon, and mung bean, the average imidacloprid recoveries were from 62.97 to 134.09%, with relative standard deviations between 0.21 and 14.58%. Furthermore, the immunoassay results correlated well with spiked samples analyzed by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The established immunoassay is rapid, sensitive, and specific and suitable to monitor imidacloprid residues in medicinal herbs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In May 2019, leaf spots were found on Amomum villosum in Guangxi Province, China. Light yellow-green spots first appeared at the tips or edges of leaves. A fungus was isolated from the leaves and identified as Colletotrichum fructicola based on morphological characteristics and molecular identification using the DNA sequences from ITS, TUB2, ACT, and GAPDH sequences. Similar leaf spots developed after leaves were inoculated with the isolate. This is the first report that C. fructicola causes leaf spots on A. villosum. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the present research, we studied the binding characterization of a single chain fragment variable (scFv) antibody S12B8 with abscisic acid (ABA) and conjugated ABA by competitive enzyme-linked immunosorbent assay (ELISA) and in silico molecular docking. The nucleotide sequences of variable light (VL) and variable heavy (VH) were obtained from hybridoma cells (F12B8), which secrete monoclonal antibodies recognizing ABA and conjugated ABA. And then the genes of VL and VH were constructed to a scFvS12B8 with a nucleotide sequence of a flexible linker (G4S)3 by splicing overlap extension PCR (SOE-PCR). The fragment of S12B8 was cloned into pMAL-c2x with a maltose binding protein tag and expressed in Escherichia coli with a calculated molecular mass of 68 kDa. The results of ELISA showed dose-dependent inhibition of the purified recombinant protein S12B8 by ABA, (ABA-ME) and ABA glucose ester tetra acetyl (ABAGE tetra acetyl). The detection limits (10% inhibition) of ABA, ABA-ME and ABAGE tetra acetyl were 19.71, 8.06 and 3.95 μg/ml, respectively. On the other hand, we investigated the interactions between S12B8 and ligands by homology modeling using RosettaAntibody and molecular docking by Discovery studio 2.5 (DS 2.5). The binding energy of ABA, ABA-ME and ABAGE when fused to the S12B8 antibody was .37.1, .69.6 and .112.0 kcal/mol, respectively, which agreed well with our competitive ELSIA results. [ABSTRACT FROM AUTHOR]

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions. [ABSTRACT FROM AUTHOR]

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL−1 and a working range of 0.7–19 ng mL−1. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only. [ABSTRACT FROM AUTHOR]

Cadmium is classified as a probable human carcinogen. A monoclonal antibody (mAb) against Cd2+-ethylene-diamine-N,N,N′,N′-tetraacetic acid (EDTA) complex was generated by immunising mice with the conjugate of Cd2+-EDTA-aminobenzyl-ovalbumin. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and applied for the analysis of Cd2+ in water, soil and rape samples. The half-maximum inhibition concentration and detection range were 2.59 ng/ml and 0.20–40 ng/ml, respectively. The assay cross-reactivities with non-target metal-EDTA complexes were all below 1%, except Mn2+ (2.9%) and Hg2+ (1.6%). The average recoveries of Cd2+ in tap water, Jingmi Canal, Xiaoqing River (at concentrations of 2–20 ng/ml), soil (0.25–10 ug/g) and rape (0.1–1.2 ug/g) samples were 103.0%, 101.0%, 103.0%, 82.4% and 93.7%, respectively. Concentrations of Cd2+ measured by this immunoassay agreed well with those determined by graphite furnace atomic absorption spectroscopy (GFAAS) (k=1.1225, r 2=0.9873).The established icELISA was a quick, reliable and sensitive method to detect Cd2+ in environmental and vegetable samples. [ABSTRACT FROM PUBLISHER]

• Colloidal gold and time-resolved fluorescent nanobead for immunoassay. • On-site quantitation of imidacloprid in vegetable by two lateral flow immunoassays. • Comparison of two tracers for assay sensitivity at optimizing conditions. • Lateral flow immunoassay is an alternative to enzyme-linked immunosorbent assay. 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 R 2 of 0.91. The LFIAs are portable and simple and thus could fully replace the ELISA for onsite quantitation of imidacloprid residues in vegetables. [ABSTRACT FROM AUTHOR]

Highlights • The mAb3C9 against trans -resveratrol with a new hapten was sensitive and specific. • Using mAb 3C9, we developed icELISA and LFIA for trans -resveratrol analysis. • To identify trans -resveratrol levels in grapes, they can be screened by LFIA. Abstract 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. [ABSTRACT FROM AUTHOR]

• Hapten of nobiletin has been successfully prepared. • This is the first paper on mAb-based icELISA for nobiletin in citrus. • The developed ic-ELISA offers an advantage for detecting nobiletin in 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 (IC 50) of 2.43 ± 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. [ABSTRACT FROM AUTHOR]

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 2G 1 1G 4 , 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. [ABSTRACT FROM AUTHOR]

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 (IC 50 ) 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. [ABSTRACT FROM AUTHOR]