Guiyu Tan

Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)-ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)-EDTA-BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)-EDTA-BSA-horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator-protein complexes such as EDTA-BSA and EDTA-BSA-HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.

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.

Development of a Specific Monoclonal Antibody-Based ELISA to Measure the Artemether Content of Antimalarial Drugs

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.

Development of a Specific Monoclonal Antibody for the Quantification of Artemisinin in Artemisia annua and Rat Serum

Artemisinin, extracted from Artemisia annua, and its derivatives are important frontline antimalarials. To produce specific antibodies for the detection and quantification of artemisinin, artemisinin was transformed to 9-hydroxyartemisinin by microbial fermentation, which was used to prepare a 9-succinate artemisinin hapten for conjugation with ovalbumin. A monoclonal antibody (mAb), designated as 3H7A10, was selected from hybridoma cell lines which showed high specificity to artemisinin. No competitive inhibition was observed with artesunate, dihydroartemisinin, and artemether for up to 20,000 ng mL(-1). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed a concentration causing 50% of inhibition (IC50) for artemisinin as 2.6 ng mL(-1) and a working range of 0.6-11.5 ng mL(-1). The icELISA was applied for the quantification of artemisinin in crude extracts of wild A. annua and the study of pharmacokinetics of artemisinin in rat serum after intraperitoneal injection. The results were highly correlated with those determined by HPLC-UV analysis (R(2) = 0.9919). In comparison with reported antiartemisinin mAbs which have broad cross-reactivity with other artemisinin derivatives, the high specificity of 3H7A10 for artemisinin will enable development of methods for quantification of artemisinin in Artemisia plants and antimalarial drugs such as Arco and for pharmacokinetic studies.

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

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.

Production of Monoclonal Antibody to Herbicide Fenoxaprop-ethyl

Fenoxaprop-ethyl is a selective aryloxyphenoxypropionate herbicide used widely to control annual and perennial grasses. A monoclonal antibody (MAb) against fenoxaprop-ethyl (FE), designated as 3E6B9C, was produced and had very low cross-reactivity with some of its structural analogs, such as clodinafop-propargyl, diclofop-methyl, lactofen, and quizalofop-p-ethyl. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The concentration of R-(+)-fenoxaprop-ethyl (R-FE) producing 50% of inhibition (IC(50)) and the working range of icELISA were 3.1 ng/mL and 0.6-29 ng/mL, respectively. This assay is also sensitive to R-fenoxaprop, S-(-)-fenoxaprop-ethyl, and metamifop with IC(50) of 3.4, 2.7, and 3.5 ng/mL, respectively. The recoveries of R-FE in soil samples with the icELISA were 86-102%.

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of cadmium ions in water, soil and rape samples

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.

Development of a Sensitive Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Analysis of Paclobutrazol Residue in Wheat Kernel

An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with monoclonal antibody (mAb) mAb6H73C9 recognizing the plant growth regulator paclobutrazol (PBZ). The icELISA had a half-maximum inhibition concentration (IC50) and working range of approximately 8.7 and 2.0-50.4 ng/mL, respectively. Average recoveries of PBZ in the wheat (Triticum aestivum) kernel samples were between 84.3 and 118.9% with relative standard deviations between 3.9 and 14.2%. As determined by the icELISA and further confirmed by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS) analysis, the maximum residue concentration was about 0.07 mg/kg in the kernel samples, which indicated that PBZ could transfer from PBZ- treated seedlings to the kernel samples. The correlation coefficient (R(2)) between icELISA and LC-ESI-MS results was 0.979, which manifested that the developed icELISA was sensitive enough for monitoring PBZ residues in wheat kernels.

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

HighlightsA new hapten for dihydroartemisinin (DHA) was obtained by microbial fermentation.Screening of hybridoma identified a DHA‐specific monoclonal antibody.Two immunoassays icELISA and dipsticks were developed for DHA detection.The dipsticks have the potential for DHA quality control in point‐of‐care settings. 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 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.

Functional and binding characterization of a single chain Fv antibody to abscisic acid and conjugated abscisic acid

ABSTRACT 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.