Tiegui Nan

Development of Protein A Functionalized Microcantilever Immunosensors for the Analyses of Small Molecules at Parts per Trillion Levels

Development of microcantilever biosensors for small molecules was exemplified with the beta-adrenergic agonist clenbuterol and the antibiotic chloramphenicol. In this paper, antibody sulfhydrylation and protein A were used to modify the microcantilever Au surface, and the antibody activities on the microcantilever were evaluated with direct competitive enzyme-linked immunosorbent assay (dcELISA). The activity of the antibodies immobilized on the microcantilever via protein A was 1.7-fold of that via the sulfhydrylation reagent 2-iminothiolane hydrochloride. A microcantilever immunosensor method with protein A as the functionalization reagent was established to detect the residues of clenbuterol and chloramphenicol at limits of detection (LOD) of approximately 0.1 and 0.2 ng/mL, respectively. Such LODs were better than that of the corresponding dcELISAs. The concentration of clenbuterol in a fortified feed sample detected with the microcantilever immunosensor after thorough extraction and purification agreed well with that detected with the dcELISA. Protein A showed to be simple and reproducible for functionalization of the antibodies on the Au surface and, thus, has common application values in microcantilever immunosensor development. The results suggest that microcantilever immunosensors be suitable for detection of small molecules, and the assay sensitivity is mainly related to the quality and activities of the antibodies.

Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants. In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples, a monoclonal antibody (MAb) (designated as MAb 3E(5)D(7)C(4)B(6)) against MeJA was derived from a JA-bovine serum albumin (BSA) conjugate as an immunogen. The antibody belongs to the IgG(1) subclass with a kappa type light chain and has a dissociation constant of approximately 6.07 x 10(-9) M. MAb3E(5)D(7)C(4)B(6) is very specific to MeJA. It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA), conventional and simplified indirect competitive ELISAs (icELISA). JA was derivatized into MeJA for the ELISA analysis. The IC(50) value and detection range for MeJA were, respectively, 34 and 4-257 ng/mL by the conventional icELISA, 21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA. The dcELISA was more sensitive than either the conventional or simplified icELISA. The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves. The increased jasmonates content indicated its role in response to the drought stress and pathogens.

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.

Mechanism and enhancement of the surface stress caused by a small-molecule antigen and antibody binding.

Generation of microcantilever bending from biochemical interactions can have wide applications, ranging from high-throughput molecular detection to bioactuation. However, the origin of the biochemically induced surface stress causing the bending is a subject of much scientific debate and interest. Unlike a compressive surface stress caused by biomacromolecule antigen and antibody binding, here we show that a small molecule antigen and antibody binding on the surface gives rise to a tensile stress. We propose that the tensile stress is induced by antibody conformational change which manifests itself as Fab arm motion that exposes the C1q binding site of the antibody due to antigen binding. A microcantilever immunosensor was developed for the detection of Chlorimuron-ethyl (CE). We found that antibodies with oriented immobilization induce a greater resultant surface stress than those with random immobilization. The length of linker between the surface and the antibody plays an important role on the stress transmission. The shorter the length, the greater the surface stress. These mechanism and principles will underpin the design of devices and coatings to significantly lower the small molecule detection limit and may also have an impact on our understanding of antigen and antibody binding.

Development of a lateral flow dipstick immunoassay for the rapid detection of glycyrrhizic acid

Abstract A sensitive antibody-based lateral flow dipstick was developed for glycyrrhizic acid (GL) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GL-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GL antibody. The visual detection limit was 20–50 ng ml−1 of GL and the reaction time was 10 min. The dipstick was used for the detection of GL in roots, leaves and stems of liquorice plant samples. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). After sample extraction with tap water for 30 min at room temperature, it also can be used for identifying liquorice roots and measuring their quality. The dipstick assay proved to be a sensitive and rapid tool for quality control of liquorice herbs.

Development of a Secondary Antibody Thio-Functionalized Microcantilever Immunosensor and an ELISA for Measuring Ginsenoside Re Content in the Herb Ginseng

Ginsenoside Re (GRe) is a major active component of the Chinese medicinal herb ginseng, Panax ginseng . A sensitive and specific monoclonal antibody (mAb), designated as mAb3D6, was generated with a GRe-bovine serum albumin conjugate as an immunogen. Microcantilever immunosensors (MCS), one modified with thiolated anti-GRe antibody and one modified with thiolated goat antimouse immunoglobulin G (IgG), were developed to detect the content of ginsenoside. The MCS immobilized with thiolated goat antimouse IgG had a better sensitivity than the MCS modified with thiolated anti-GRe antibody. The advantage of a secondary antibody thio-functionalized MCS was verified with the anti-paclitaxel mAb. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was also established with mAb3D6. The concentration of analyte producing 50% inhibition and the working range of icELISA were 1.20 and 0.15-16.1 ng/mL, respectively. The icELISA had a cross-reactivity of 89% with ginsenoside Rg1 and less than 3% with other ginsenosides. The icELISA and MCS with thiolated secondary antibody were applied for the determination of GRe in ginseng samples, and the results agreed well with those determined by high-performance liquid chromatography.

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 Sandwich Enzyme-linked Immunosorbent Assay for Phosphinothricin Acethl Transferrse Protein of Genetically Modified Crops

The phosphinothricin acethl transferase(pat) gene was cloned and constructed into a prokaryotic expression vectorpET30(+)-pat.The protein expressed by this gene was purified and used as immunogen to immunize NewZealand rabbits and mice for polyclonal antibody and monoclonal antibody separately.The polyclonal antibody titer was greater than 8000,the monoclonal antibody(mAb 2F6) titer was 5 ×104.The mAb was IgG1 isotype with κ light chains.pat protein sandwich enzyme-linked immunosorbent assay was developed with anti-pat protein mouse monoclonal antibody(mAb) and rabbit polyclonal antibody.The linear range of the method was 1.56-100.0 μg/L.The linear equation was y=0.6914x-2.572,and the determinative coefficient was 0.9951.The assay was used to detect the content of pat protein in the leaf of five transgenic Bt cotton and two non-GM cotton varieties.The results showed that,three varieties of transgenic Bt cotton were positive and the others were negative.