The Simultaneous Determination of Chlorpyrifos-Ethyl and -Methyl with a New Format of Fluorescence-Based Immunochromatographic Assay.

The improper and excessive use in agriculture of chlorpyrifos-methyl (CPSM) and chlorpyrifos-ethyl (CPSE) may affect the health of human beings. Herein, a fluorescence-based immunochromatographic assay (FICA) was developed for the simultaneous determination of CPSM and CPSE. A monoclonal antibody (mAb) with equal recognition of CPSM and CPSE was generated by the careful designing of haptens and screening of hybridoma cells. Instead of labeling fluorescence with mAb, the probe was labeled with goat-anti-mouse IgG (GAM-IgG) and pre-incubated with mAb in the sample. The complex could compete with CPS by coating antigen in the test line. The new format of FICA used goat-anti-rabbit IgG (GAR-IgG) conjugated with rabbit IgG labeled with fluorescence microspheres as an independent quality control line (C line). The novel strategy significantly reduced nonspecific reactions and increased assay sensitivity. Under the optimal conditions, the proposed FICA showed a linear range of 0.015-64 mg/L and limit of detection (LOD) of 0.015 mg/L for both CPSE and CPSM. The average recoveries of CPS from spiked food samples by FICA were 82.0-110.0%. The accuracy was similar to the gas chromatography-tandem mass spectrometry (GC-MS/MS) results. The developed FICA was an ideal on-site tool for rapid screening of CPS residues in foods.

Nanobodies for Accurate Recognition of Iso-tenuazonic Acid and Development of Sensitive Immunoassay for Contaminant Detection in Foods.

The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.

Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody.

Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 1013 pfu mL-1 phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL-1 and IC50 was 0.0588 ng mL-1 in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay.

Citrulline Accumulation Mechanism of Pediococcus acidilactici and Weissella confusa in Soy Sauce and the Effects of Phenolic Compound on Citrulline Accumulation.

Citrulline is one of the major precursors of ethyl carbamate in soy sauce, and the accumulation of citrulline is attributed to the metabolism of arginine by bacteria with the arginine deiminase (ADI) pathway. However, key strains and factors affecting citrulline accumulation are not yet clear. In this study, two key strains of Pediococcus acidilactici and Weissella confusa were isolated from soy sauce moromi, and the regularity of citrulline formation was studied. Results showed that the conversion rates from arginine to citrulline (A/C rate) and the citrulline accumulation ability of W. confusa and P. acidilactici significantly increased in the presence of different concentrations of NaCl, indicating that salt stress was the main factor for citrulline accumulation. The inconsistent expression of arc genes by salt stress was the reason for citrulline accumulation for P. acidilactici, but for W. confusa, it may be due to the influence of arginine/citrulline on the transportation system: the intracellular citrulline could neither transport to extracellular space nor convert into ornithine. Environmental factors greatly influenced citrulline accumulation of the two key bacteria; A/C rate and citrulline formation in both strains decreased at low temperature (15°C) under high salt stress, but opposite effects were observed for the two key strains under anaerobic light condition. Moreover, quercetin and gallic acid significantly decreased the A/C rate and citrulline accumulation ability of the two key strains. The optimal quercetin and gallic acid as suggested by simulation experiment were 100 and 10 mg/l, respectively, and the lowest A/C rate of 28.4% and citrulline level of 1326.7 mg/l were achieved in the simulation system. This study explored the main factors for citrulline formation by the two key strains and proposed a targeted way to control citrulline in soy sauce.

Prussian blue nanoparticles-enabled sensitive and accurate ratiometric fluorescence immunoassay for histamine.

Herein, a nanozyme-mediated ratiometric fluorescence immunoassay for histamine (HA) has been developed. Prussian blue nanoparticles (PBNPs) with outstanding peroxidase-like activity were labelled with goat anti-mouse IgG via a facile electrostatic adsorption to yield the nanozyme-antibody conjugate which acted as a bridge to link the ratiometric fluorescence readout with HA concentration. As substrate, o-phenylenediamine (OPD) was oxidized into 2,3-diaminophenazine (oxOPD) by H2O2 under the catalysis of PBNPs, producing a novel emission at 570 nm and quenching the fluorescence of carbon dots (CDs) at 450 nm simultaneously. Under optimal conditions, the ratio of fluorescence intensity at 570 nm and 450 nm (I570/I450) linearly correlated with HA concentration ranging from 1.6 ng/mL to 125 µg/mL, with a detection limit (LOD) of 1.2 ng/mL. In addition, analytical performances including specificity, accuracy and applicability were evaluated, which revealed that this ratiometric fluorescence immunoassay affords an effective platform for sensitive and accurate detection of HA.

Noni (Morinda citrifolia L.) Fruit Polysaccharides Regulated IBD Mice Via Targeting Gut Microbiota: Association of JNK/ERK/NF-κB Signaling Pathways.

Inflammatory bowel disease (IBD) is a disease characterized by intestinal inflammation with immune dysregulation and intestinal microecological imbalance. In a dextran sulfate sodium salt (DSS)-induced IBD mouse model, noni (Morinda citrifolia L.) fruit polysaccharides (NFP) with homogalacturonan and rhamnogalacturonan-I domain decreased the concentration of serum LPS, TNF-α, and IL-17 by 84, 42, and 65%, respectively. It was abolished when intestinal microbiota were depleted by antibiotics. Sequencing analysis of gut microbiota showed an attenuated disruption of the microbial composition in the DSS+NFP group. Targeted metabolomic analysis revealed that NFP upregulated the content of acetic acid, propionic acid, and butyric acid by onefold but reduced isobutyric acid and isovaleric acid contents. NFP also inhibited JNK, ERK, and NF-κB phosphorylation of IBD mice. Taken together, the mechanism of NFP alleviating IBD is related to the intestinal microecological balance to inhibit inflammatory signaling pathways. This study provides a basis for NFP as a cheap intervention for the prevention and treatment of IBD patients.

Enhanced Non-Toxic Immunodetection of Alternaria Mycotoxin Tenuazonic Acid Based on Ferritin-Displayed Anti-Idiotypic Nanobody-Nanoluciferase Multimers.

The non-toxic immunoassay for mycotoxins is being paid more attention due to its advantages of higher safety and cost savings by using anti-idiotype antibodies to substitute toxins. In this study, with tenuazonic acid (TeA), a kind of highly toxic Alternaria mycotoxin as the target, an enhanced non-toxic immunoassay was developed based on the ferritin-displayed anti-idiotypic nanobody-nanoluciferase multimers. First, three specific ß-type anti-idiotype nanobodies (AId-Nbs) bearing the internal image of TeA mycotoxin were selected from an immune phage display library. Then, the AId-Nb 2D with the best performance was exploited to generate a nanoluciferase (Nluc)-functionalized fusion monomer, by which a one-step non-toxic immunodetection format for TeA was established and proven to be effective. To further improve the affinity of the monomer, a ferritin display strategy was used to prepare 2D-Nluc fusion multimers. Finally, an enhanced bioluminescent enzyme immunoassay (BLEIA) was established in which the half maximal inhibitory concentration (IC50) for TeA was 6.5 ng/mL with a 10.5-fold improvement of the 2D-based enzyme-linked immunosorbent assay (ELISA). The proposed assay exhibited high selectivities and good recoveries of 80.0-95.2%. The generated AId-Nb and ferritin-displayed AId-Nb-Nluc multimers were successfully extended to the application of TeA in food samples. This study brings a new strategy for production of multivalent AId-Nbs and non-toxic immunoassays for trace toxic contaminants.

Development of a chemiluminescence immunoassay for detection of tenuazonic acid mycotoxin in fruit juices with a specific camel polyclonal antibody.

The natural mycotoxin tenuazonic acid (TeA) in foods is identified as the most toxic mycotoxin among the over 70 kinds of secondary toxic metabolites produced by Alternaria alternata. Some hapten-antibody-mediated immunoassays have been developed for TeA detection in food samples, but these methods show unsatisfactory sensitivity and specificity. In this study, a rationally designed hapten for TeA mycotoxin generated with computer-assisted modeling was prepared to produce a highly specific camel polyclonal antibody, and an indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) was established with a limit of detection of 0.2 ng mL-1 under optimized conditions. The cross-reactivity results showed that several analogs and some common mycotoxins had negligible recognition by the anti-TeA polyclonal antibody. The average recoveries spiked in fruit juices were determined to be 92.7% with an acceptable coefficient of variation, and good correlations between icCLEIA and liquid chromatography tandem mass spectrometry (LC-MS/MS) results were obtained in spiked samples. This developed icCLEIA for TeA detection with significantly improved sensitivity and satisfactory specificity is a promising alternative for environmental monitoring and food safety.

The Most Active Oxidase-Mimicking Mn2 O3 Nanozyme for Biosensor Signal Generation.

Oxidase-mimicking nanozymes are more desirable than peroxidase-mimicking ones since H2 O2 can be omitted. However, only a few nanomaterials are known for oxidase-like activities. In this work, we compared the activity of Mn2 O3 , Mn3 O4 and MnO2 and found that Mn2 O3 had the highest oxidase activity. Interestingly, the activity of Mn2 O3 was even inhibited by H2 O2 . The oxidase-like activity of Mn2 O3 was not much affected by the presence of proteins such as bovine serum albumin (BSA), but the physisorption of antibodies to Mn2 O3 was not strong enough to withstand the displacement by BSA. We then treated Mn2 O3 with 3-aminopropyltriethoxysilane to graft an amine group, which was used to conjugate antibodies using glutaraldehyde as a crosslinker. A one-step indirect competitive ELISA (icELISA) was developed for the detection of isocarbophos, and an IC50 of 261.7 ng/mL was obtained, comparable with the results of the standard two-step assay using horseradish peroxidase (HRP)-labeled antibodies. This assay has the advantage of significant timesaving for rapid detection of large amounts of samples. This work has discovered a highly efficient oxidase-mimicking nanozyme useful for various nano- and analytical applications.

Inhibition of ethyl carbamate accumulation in soy sauce by adding quercetin and ornithine during thermal process.

Ethyl carbamate (EC), a genotoxic and carcinogenic compound in soy sauce accumulated during thermal processes, has raised public health concern for its multipoint potential carcinogenic risk to human. In this work, based on the analysis of EC accumulation during thermal processes of soy sauce, ornithine and quercetin were added before thermal processes to reduce EC accumulation. A reduction rate of 23.7-63.8% in simulated solution was founded. Kinetic studies indicated that ornithine was a byproduct of alcoholysis reaction when EC formed, while quercetin could compete with the precursor ethanol and react with carbamyl compounds, which therefore preventedEC accumulation. A maximum of 47.2% decrease of EC in soy sauce was achieved, and no remarkable changes in volatile compounds profile and color of soy sauce were found. In conclusion, the addition of quercetin and ornithine before thermal processes may be preferable for the controlling of EC content in soy sauce.

Visual upconversion nanoparticle-based immunochromatographic assay for the semi-quantitative detection of sibutramine.

Immunochromatographic assay (ICA) has been used widely for the onsite monitoring of illegal additives due to its simplicity, speed, and low cost. However, a scanner is commonly required for ICA to achieve quantitative results. In this work, we developed a visual semi-quantitative ICA for sibutramine, a banned additive in diet foods, without the need for a scanner for measurement. Monoclonal antibodies specific for sibutramine were raised and conjugated with upconversion nanoparticles (UCNPs) as the luminescent tracer. ICA was developed by employing multiple test lines to achieve the semi-quantitative detection of sibutramine. Based on the optimal conditions, the cutoff levels (limit of quantitation, LOQ) of T1 line, T2 line, T3 line, and T4 line were 0.02 µg/mL, 0.15 µg/mL, 1.0 µg/mL, and 7.5 µg/mL, respectively, in buffer system. The ICA demonstrated a LOQ at 0.2 mg/kg for sibutramine in diet food samples. The assay (including pretreatment) can be finished within 30 min without the aid of other instruments, except a laser pen. No false positive or false negative results were observed. The results indicated that the proposed method was reliable, simple, and rapid for the screening of sibutramine abuse in diet food samples.

Fluorescent enzyme-linked immunoassay based on silane-doped carbon dots for sensitive detection of microcystin-LR in water and crucian samples.

In this work, a fluorescent nanoparticles labeling-free fluorescence enzyme-linked immunoassay (FELISA) has been established for the ultrasensitive detection of microcystin-LR (MC-LR) in water and fish samples. Polyclonal antibody against MC-LR was labeled with horseradish peroxidase (HRP) and used as signal probe for binding with analyte in sample or for coating antigen. After washing of the unbound antibody, the substrate system (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)/H2O2) was added. The oxidation product of ABTS (ox-ABTS) catalyzed by HRP effectively caused the fluorescence quenching of subsequently added silane-doped carbon dots (Si-CDs), and the change in fluorescence intensity of Si-CDs was used to realize the quantitative detection of MC-LR. Under the optimum conditions, the Si-CDs based FELISA method showed a good linear relationship from 0.001 to 3.20 µg L-1 (R2 = 0.994) and provided a low detection limit of 0.6 ng L-1, which was approximately 30-fold lower than that of traditional indirect competitive ELISA. Average recovery values from 79.9% to 109.2% was obtained from spiked water and crucian samples, suggesting its potential application on the monitoring of MR-LR at a trace level.

A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein.

Tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant which can be released to environment and cause long-term hazard. In this work, we developed a rapid and highly sensitive fluorescence enzyme-linked immunosorbent assay (FELISA) for monitoring of TBBPA in soil samples. TBBPA specific nanobody derived from camelid was fused with alkaline phosphatase to obtain the bi-functional fusion protein, which enable the specific binding of TBBPA and the generation of detection signal simultaneously. The assay showed an IC50 of 0.23 ng g-1, limit detection of 0.05 ng g-1 and linear range from 0.1 to 0.55 ng g-1 for TBBPA in soil samples. Due to the high resistance to organic solvents of the fusion protein, a simple pre-treatment by using 40% dimethyl sulfoxide (DMSO) as extract solvent can eliminate matrix effect and obtain good recoveries (ranging from 93.4% to 112.4%) for spiked soil samples. Good relationship between the results of the proposed FELISA and that of liquid chromatography tandem mass spectrometry (LC-MS/MS) was obtained, which indicated it could be a powerful analytical tool for determination of TBBPA to monitor human and environmental exposure.

Dislocation Engineered PtPdMo Alloy With Enhanced Antioxidant Activity for Intestinal Injury.

Radiotherapy is the mainstay for abdomen and pelvis cancers treatment. However, high energy ray would inflict gastrointestinal (GI) system and adversely disrupt the treatment. The anti-oxidative agents provide a potential route for protecting body from radiation-induced injuries. Herein, highly catalytic nanocubes with dislocation structure are developed for treatment of intestinal injury. Structural and catalytic properties show that Mo incorporation can enhance antioxidant activity by dislocation structure in the alloy. In vitro studies showed that PtPdMo improved cell survival by scavenging radiation-induced ROS accumulation. Furthermore, after animals were exposed to lethal dose of radiation, the survival was increased by 50% with the PtPdMo i.p. treatment. Radioprotection mechanism revealed that PtPdMo alleviated the oxidative stress in multi-organs especially the small intestine by inhibiting intestinal epithelium apoptosis, reducing DNA strands breaks and enhancing repairing ability. In addition, PtPdMo protected hematopoietic system by improving the number of bone marrow and peripheral blood cells.

The Constipation-Relieving Property of d-Tagatose by Modulating the Composition of Gut Microbiota.

d-tagatose, a monosaccharide as well as a dietary supplement, has been reported as having a wide range of applicability in the food industry, however, the prebiotic activity, anticonstipation effects, and related mechanisms are still unclear. In this study, using the loperamide-induced constipation Kunming mice as the animal model, the effects of d-tagatose for the prevention of constipation were evaluated by gastrointestinal transit experiment and defecation experiment. Furthermore, the underlying mechanism was clarified by evaluating the change of the biochemical indicators and analyzing 16S rRNA amplicon of gut microbiota among the different mice groups. The results showed that the gastrointestinal transit rate, fecal number, and weight in six hours were significantly enhanced after the administration of d-tagatose. In addition, d-tagatose significantly increased the serum levels of acetylcholine (Ach) and substance P (SP), whereas the serum levels of nitric oxide (NO) were significantly decreased. Moreover, the 16S rRNA sequencing analysis revealed that the changes in the gut microbiota caused by constipation were restored by d-tagatose treatment. In conclusion, this study indicated that the administration of d-tagatose as a dietary supplement can effectively prevent and relieve constipation in Kunming mice, and it is a promising prebiotic candidate with constipation-relieving properties.

High affinity antibody based on a rationally designed hapten and development of a chemiluminescence enzyme immunoassay for quantification of Alternariol in fruit Juice, maize and flour.

Alternariol (AOH), a secondary metabolites of the genus Alternaria, is a genotoxic mycotoxin. Because the previous "Mannich antigen" for AOH readily underwent unstable induction, a new AOH hapten was designed for inducing a high-quality antibody against AOH using a conjugate of the AOH carboxymethyl derivative with a carrier protein as the immunogen. A competitive indirect chemiluminescence enzyme immunoassay (ciCLEIA) for AOH was optimized and showed good sensitivity (limit of detection 0.068 ng/mL), a linear range (0.11-1.23 ng/mL), and negligible cross-reactivity with analogues. Average rates of recovery from spiked samples (juice and cereal) were between 72.7% and 115.8%, with coefficients of variation <14.3%. Results from ciCLEIA correlated well with those of high-performance liquid chromatography coupled with tandem mass spectrometry, meaning the proposed method might be an effective screening tool for detection of AOH in food samples.

Ultrasensitive immunosensor for acrylamide based on chitosan/SnO2-SiC hollow sphere nanochains/gold nanomaterial as signal amplification.

An electrochemical immunosensor for ultrasensitive detection of acrylamide (AA) in water and food samples was developed. SnO2-SiC hollow sphere nanochains with high surface area and gold nanoparticles with good electroconductivity were fabricated onto the surface of a glassy carbon electrode pre-coated with chitosan. The coating antigen (AA-4-mercaptophenylacetic acid-ovalbumin conjugate, AA-4-MPA-OVA) was immobilized on the electrode. Polyclonal antibody specific for AA-4-MPA was conjugated to gold nanorod (AuNR) as primary antibody (AuNR-Ab1). Horseradish peroxidase labelled anti-rabbit antibody produced in goat was conjugated to AuNR as secondary antibody (HRP-AuNR-Ab2). For detection, the analyte (AA-4-MPA) in sample competed with coating antigen for binding with AuNR-Ab1. After washing, HRP-AuNR-Ab2 was added to capture the AuNR-Ab1, and the electrical signal was obtained by addition of hydroquinone and H2O2. After investigation of the binding ability on nanomaterials and optimization of competitive immunoassay conditions, the proposed immunosensor exhibited a sensitive response to AA with a detection limit of 45.9 ±â€¯2.7 ng kg-1, and working range of 187 ±â€¯12.3 ng kg-1 to 104 ±â€¯8.2 µg kg-1 for drinking water samples. Recoveries of AA from spiked samples were ranged from 86.0% to 115.0%. The specificity, repeatability and stability of the immunosensor were also proved to be acceptable, indicating its potential application in AA monitoring.

Naturally-Derived PHA-L Protein Nanoparticle as a Radioprotector Through Activation of Toll-Like Receptor 5.

High energy ray in medical diagnosis and therapy can benefit to patients, but can also cause the significant damages to biomolecules such as DNA, as well as free radical generation, inevitably leading to numerous side effects. Small molecular radioprotectors provide an effective route to preserve the healthy tissue and whole body from ionizing radiation, but always have a short circulation time in body. Inorganic nanoparticles show major protection effect but their heavy metal components considerably jeopardize translational promise due to suboptimal biocompatibility. Herein, we report a novel protein nanoparticle that can overcome limitations of both small molecular and inorganic nanoparticle radioprotectors and can be used as a radioprotector with spontaneous biocompatibility, outstanding pharmacokinetics and improvement on survival rate under exposure to γ-ray irradiation. PHA-L protein nanoparticle serves to clear excessive reactive oxygen species in vivo, prevents radiation-induced hematopoietic and gastrointestinal damages and boosts the survival rate of irradiated mice to ∼70%. A detailed study of the mechanism shows PHA-L protein nanoparticle can target and activate the toll-like receptor 5 in vitro and in vivo, and thus protect irradiated cells by immune response. Importantly, the PHA-L protein nanoparticle can perform highly efficient clearance while eliciting negligible toxicological response.

A full-automated magnetic particle-based chemiluminescence immunoassay for rapid detection of cortisol in milk.

A sensitive full-automated micromagnetic particles (MMPs) based competitive chemiluminescent immunoassay (CLIA) was developed to detect cortisol in milk. Polyclonal antibody (pAb) with good specificity against cortisol was produced. The antigen (cortisol-OVA) was labeled with acridinium ester (cortisol-OVA-AE) as signal tracer. During the detection, the free cortisol in sample will compete with cortisol-OVA-AE for binding to pAb. To capture pAb, MMPs conjugated with goat anti-rabbit IgG was added. The whole immunoassay process (exclude sample pretreatment) was performed by automatic chemiluminescence immunoassay instrument, which could consume less test time (within 40 min) and avoid error from manual operation. The method showed a good detection limit of 0.12 ng/mL, a broad linear range from 0.42 to 72.27 ng/mL for cortisol detection, negligible cross-reactivity with related analogues and satisfied recovery (84.3%-102.3%) for spiked milk samples test. Simultaneously, since the results of proposed method had no significant difference with those of LC-MS/MS, the proposed method was confirmed to have a potential applicability for rapidly monitoring cortisol in the food.

Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.