Jie-Xian Dong

Broad-Specificity Immunoassay for O,O-Diethyl Organophosphorus Pesticides: Application of Molecular Modeling to Improve Assay Sensitivity and Study Antibody Recognition

A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety.

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay.

A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning V(L) and V(H) genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified V(L) and V(H) regions were assembled using a linker (Gly(4)Ser)(3) by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv-AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISA(FP) exhibited good sensitivity and reproducibility.

Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli

Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.

Development of a sensitive time-resolved fluoroimmunoassay for organophosphorus pesticides in environmental water samples

Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL−1. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.

Indirect Competitive Chemiluminescence Enzyme Immunoassay for Furaltadone Metabolite in Metapenaeus Ensis

Abstract An indirect competitive chemiluminescence enzyme immonoassay (icCLEIA) for the quantitative analysis of furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone(AMOZ) in Metapenaeus ensis sample was established with the highly sensitive luminol-H 2 O 2 -HRP-4-iodophenol chemiluminescence reaction detection system. The icCLEIA method was based on a single-chain variable fragment (scFv) antibody against AMOZ derivative. The optimized assay conditions were as follows: 62.5 μg L −1 of coating antigen (5-morpholinomethyl-3-(glyoxalamino)-2-oxazolidone-ovalbumin, AMOZA-OVA) was used in the experiment, the dilute multiple of scFv antibody was 1:10, the immunoreaction time was 45 min, and the dilute multiple and incubation time of HRP-goat anti mouse IgG were 1:10000 and 50 min, respectively. The icCLEIA results showed that IC 50 value and limit of detection (LOD) were 1.38 μg L −1 and 0.09 μg L −1 , and linear range was in the range of 0.26–9.1 μg L −1 . Inter-assay and intra-assay RSD were both below 15%. The scFv antibody showed high specificity. The average recoveries of four spiked level of AMOZ were 72.2%, 73.4%, 72.6% and 78.6%, respectively. In comparison with HPLC-MS/MS, there was a good correlation between the two methods ( R 2 = 0.9997). The established icCLEIA method could be further used for detecting AMOZ in aquatic products samples rapidly and efficiently.

Bispecific Monoclonal Antibody-based Multi-analyte ELISA for Furaltadone Metabolite, Malachite Green and Leucomalachite Green in Aquatic Products

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.

Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris.

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.