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BACKGROUND Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.

Pyrethroids are insecticides that are widely used in rural and urban areas worldwide. After entering the environment, pyrethroids are rapidly metabolized or degraded by various biological or abiotic methods. In this study, a single-chain variable fragment (scFv) which could simultaneously detect three pyrethroid metabolites was constructed based on a hybridoma raised against 3-phenoxybenzoic acid (3-PBA). By molecular docking, it showed that there were hydrogen bonds, hydrophobic interactions, CH-π interaction, and cation-π interaction between 3-PBA and its scFv. All the contact residues contributing to hydrogen bonds are located in VH-CDR2 or its neighboring region, and two of them were mutants of the closest germline sequence. Based on competitive ELISA, the half maximal inhibitory concentration (IC50) of the scFv for 3-PBA, 3-phenoxybenzaldehyde (PBAld), and 3-phenoxybenzyl alcohol (PBAlc) were calculated to be 0.55, 0.59, and 0.63 μgmL−1, respectively. The scFv also showed 23.91%, 13.41%, 1.15%, 1.00%, and 0.56% cross-reactivity with phenothrin, deltamethrin, fenvalerate, beta-cypermethrin, and fenpropathrin. The broad specificity of the scFv may be due to its hapten design. The scFv could be employed in class-specific immunoassays for pyrethroid metabolites with phenoxybenzyl (PB) group. It is also potentially used for characterizing degradation of pyrethroids or detecting PBAlc (PBAld) alone, and the detection results should be confirmed by other selective methods. • A scFv which can simultaneously detect 3-PBA, PBAlc, and PBAld was constructed. • Antibody informatics and binding mode of the scFv were obtained. • The reason for its broad specificity was discussed. • It could be used to monitor single or multi-pyrethroid metabolites with PB group.

Anti-idiotypic antibody technology has been widely developed. Here, we prepared the F(ab’)2 fragments of the Cry1F toxin polyclonal antibody as Ab1and screened an anti-idiotype nanobody (VHH) for Bt Cry1F toxin from the camelid naive antibody phage display library. After three cycles of panning, six clones were isolated and subjected to indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for the detection of the Cry1F toxin. A competition inhibition rate of 47.00% exists between the anti-idiotype nanobody with the highest activity (5B) and Cry1F toxin for F(ab’)2 fragments. The binding abilities between the anti-idiotypic nanobody 5B and brush border membrane vesicles (BBMV) of Ostrinia furnacalis (Guenée) are also good. The anti-idiotypic nanobody 5B expressed through the prokaryotic system has the same activity as in phage form, and mortality compared to controls could reach 23.3% (P

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Microcystin-LR (MC-LR) is one of the most worldwide harmful cyanobacterial toxins. A lots of antibodies against MC-LR have been generated and characterized. However, the knowledge about the epitopes of MC-LR was still limited. The objective of this study was to analyze the epitopes of MC-LR and demonstrate the binding mode of MC-LR with its antibody. The variable genes of a mouse hybridoma cell line (Mab5H1–3B3) raised against MC-LR have been cloned and assembled in a single chain variable fragment (scFv), and then soluble expressed in E.coli BL21. Based on the scFv, the IC50 and IC10 for MC-LR were determined to be 7.45 nM and 0.30 nM by competitive ELISA. And the scFv also showed 115% and 112% cross-reactivities to MC-RR and MC-YR, and 59% to MC-LA. By molecular docking, the binding mode between MC-LR and its scFv was demonstrated. A hydrogen bond interaction was observed between the carbonyl group of Adda5 residue of MC-LR and its scFv, and the guanidyl group of Arg4 residue and phenyl group of Adda5 residue of MC-LR were also involved in the interaction. These predicted epitopes were supported by antibody cross-reactivity data. By comparing the antibody informatics of MC-LR scFv with its predicted paratopes, VH-CDR1 was crucial for MC-LR binding, and its specificity could be tuned by engineering in Vκ-CDR1 and Vκ-CDR3. These information would be useful for the hapten design for microcystins or improving the properties of MC-LR scFv in vitro.

Staphylococcus aureus is a common food-borne pathogenic microorganism that poses a serious threat to food quality and safety, and can do harm to human health. In the past, researchers relied on antibiotics to control Staphylococcus aureus, though very effective, yet it was also worrying in the aspect of bio-safety. In fact, anti-idiotypic antibody (Anti-Id) shows its potential to mimic some of the structural and biological functions of antigens. Therefore, in this study, based on Anti-Id theory and technology, we expect to obtain the vancomycin Anti-Id which can mimic vancomycin against Staphylococcus aureus from a human phage display domain antibody library. After four rounds of bio-panning, a total of 18 positive Anti-Ids were obtained. Among them, two Anti-Ids named Anti-Id-2C12 and Anti-Id-1F5 were identified as "β" type Anti-Ids, and afterwards they were selected for gene cloning and protein expression in prokaryotic expression system. As a result, a concentration of purified proteins with 568.6 μg/mL (Anti-Id-2C12) and 602.3 μg/mL (Anti-Id-1F5) were successfully obtained, and their minimum inhibitory concentration (MIC) values for Staphylococcus aureus were 125 and 200 μg/mL, respectively. As they are human heavy-chain domain antibodies, which were theoretically harmless to humans, they have the potential application value as preservatives in food and edible agricultural products.

Vancomycin (Van) is a common antibiotic that pollutes food, feed and ecological environment. In the present study, an anti-Van polyclonal antibodies (pAbs) and Eu3+-labeled goat anti-rabbit IgG monoclonal antibody based indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for ultrasensitive determination of Van, and its half-maximum inhibition concentrations (IC50) was 13.74 ng/L and the limit of detection (LOD) was 1.07 ng/L. The IC-TRFIA showed strong cross-reactivity (CRs) of 36.2% for norvancomycin, and less than 0.1% for polymyxin B, kanamycin and ampicillin. It was found that the IC-TRFIA for Van spiked in different milk powder samples determination were with good accuracy, stability and repeatability, and its recoveries are 83.0–97.9%, also with its coefficient of variations (CVs) are 3.0–12.9% in intra-assay and inter-assay. The results showed that the established IC-TRFIA was promising for ultrasensitive determination of Van in food samples. Highlights Prepared high-quality Van-KLH/BSA conjugates. Obtained high activity and purify anti-Van polyclonal antibodies. Prepared high-quality Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. Firstly established an IC-TRFIA for Van based on the anti-Van polyclonal antibodies and Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. The IC-TRFIA can be used for ultrasensitive determination of Van in milk powder samples.

PhoX is an extracellular alkaline phosphatase that is widely found in cyanobacteria and plays an important role in the conversion of extracellular organophosphorus into soluble inorganic phosphorus. However, the phoX gene has not yet been experimentally confirmed to exist in bloom-forming Microcystis species. In this study, we identified a putative phoX gene (GenBank accession no. ARI79942.1) in M. aeruginosa PCC7806 and overexpressed it in Escherichia coli 21 (DE3). The expressed PhoX protein displayed phosphodiesterase and phosphomonoesterase activities. In contrast to other bacterial PhoX proteins, which are activated mainly by Ca2+, Microcysits PhoX was most strongly activated by Mg2+, followed by Co2+, Ca2+, Zn2+ and Mn2+, but it was inhibited by Ni2+. Sequence analysis showed that phoX was highly conserved in the Microcystis genus (DNA similarity > 96% between species). phoX expression responded significantly to different environmental phosphorus levels. When PCC7806 cells were cultured in phosphorus-deficient medium (BG11-P), phoX expression reached its highest level at 2 h and then decreased to a low level at 4 h. Organophosphate induced the expression of phoX; its expression reached the highest level at 4 h and was maintained at a high level at 6 h. Our results confirmed a putative phoX gene and demonstrated that the phoX gene of Microcystis is conserved.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

PhoX is an extracellular alkaline phosphatase that is widely found in cyanobacteria and plays an important role in the conversion of extracellular organophosphorus into soluble inorganic phosphorus. However, the phoX gene has not yet been experimentally confirmed to exist in bloom-forming Microcystis species. In this study, we identified a putative phoX gene (GenBank accession no. ARI79942.1) in M. aeruginosa PCC7806 and overexpressed it in Escherichia coli 21 (DE3). The expressed PhoX protein displayed phosphodiesterase and phosphomonoesterase activities. In contrast to other bacterial PhoX proteins, which are activated mainly by Ca(2+), Microcysits PhoX was most strongly activated by Mg(2+), followed by Co(2+), Ca(2+), Zn(2+) and Mn(2+), but it was inhibited by Ni(2+). Sequence analysis showed that phoX was highly conserved in the Microcystis genus (DNA similarity > 96% between species). phoX expression responded significantly to different environmental phosphorus levels. When PCC7806 cells were cultured in phosphorus-deficient medium (BG11-P), phoX expression reached its highest level at 2 h and then decreased to a low level at 4 h. Organophosphate induced the expression of phoX; its expression reached the highest level at 4 h and was maintained at a high level at 6 h. Our results confirmed a putative phoX gene and demonstrated that the phoX gene of Microcystis is conserved.

The key to determine the performance of fuzzy inference inversion is to select a reasonable domain. However, there is no universal method for selecting domain at present. According to the characteristics of heat flux of geothermal heat source and the research of fuzzy inference inversion process, this paper modified the fuzzy inference method from two aspects of domain setting and iteration termination condition. The recommended domain and selection scheme for solving the problem of geothermal heat flux are given, and the modified fuzzy inference inversion method is applied to Rucheng geothermal field to verify the method. The results showed that the modified fuzzy inference inversion method could overcome the problem that the solution of the traditional method fell into a cycle, and the relative error of the verification term was less than 5%. Compared with the traditional method, the modified method greatly improved the computational efficiency, and the number of iterations was reduced to only 7. This method has a good application prospect for geothermal heat source inversion and resource evaluation.

Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7–CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7–CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7–CR11 was constructed by homology modeling. Molecular docking results of CR7–CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7–CR11, while CR7–CR8 was the region of CR7–CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7–CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification.

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC50) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC10) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

Here, we firstly reported to generate panels of anti-idiotypic single-chain variable fragments (scFvs) of Bacillus thuringiensis Cry2Aa toxin by chain shuffling method. Light- and heavy-chain-shuffled libraries based on two parental clones were constructed. The signal of the light-chain-shuffled library was 4.2-fold higher than the heavy one. Solely after one round panning, eight mutants were rapidly isolated from the chain-shuffled libraries. The apparent affinity of the best clones was 2.65 × 106 M−1, a 2.59-fold improvement over the parental clone. The binding abilities between anti-idiotypic scFvs with brush border membrane vesicle of Plutella xylostella larvae were also determined. This work demonstrated that chain shuffling could rapidly generate panels of antibodies with similar binding abilities but of the distinct idiotypic composition. The serials of mutants can be used to map the binding epitope of the receptors in target insects instead of Cry2Aa toxin.

To assess the contamination of lettuce with norovirus in Nanjing city (China), two hundred and eighty one lettuce samples were collected between 2017 and 2018 from production sites (preharvest) and retail outlets (postharvest) and analyzed. The presence of norovirus was confirmed in 26.9% (71/264) of lettuce samples, with norovirus genogroup I detected more frequently (20.1%) than norovirus genogroup II. Norovirus was detected less frequently in lettuce samples from supermarkets (4.9%) than in samples from greengrocers or farmer markets (36.5%) and was detected both in field-grown (28.9%) and in hydroponic lettuce samples (30%). The norovirus occurrence and levels varied markedly by seasons. The norovirus was more frequently detected in the cold season, with evident correlations between norovirus contamination and ambient temperatures. Interestingly, the norovirus occurrence in loose lettuce was significantly higher (p

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL-1 and the medium inhibition of control (IC50) was 0.73 ng mL-1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab’s amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10−8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL−1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5–100 ng mL−1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products.

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC 50 ) were 0.727–0.892 μg/mL and detection limit (IC 10 ) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10 7 M −1 . The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.

Colony is a key to Microcystis becoming a dominant population and forming blooms. To find the mechanism of colony formation, we investigated cell wall structures of colonial and unicellular strains. Results showed that colonial strains had significant surface layer protein (S-layer) on the surface of cells than unicellular strains by transmission electron microscopy. Western blot showed colonial strains had more S-layer than the unicellular strains. When the S-layer gene (GenBank accession number CAO89090.1) of Microcystis aeruginosa PCC7806 was expressed in Synechocystis sp. PCC6803, PCC6803 aggregated into colonial morphology. The results indicated that the S-layer could promote colony formation in Microcystis. Based on the S-layer sequences of PCC6803 and PCC7806, nine S-layer genes in other Microcystis strains were screened from the GenBank. Sequence comparing showed that the S-layers conserved regions were all located in N-terminal. The S-layers contain repeats-in-toxin (RTX) sequences with Ca2+-binding site, and their amino acid composition, hydrophobicity, isoelectric point, etc. were consistent with the characteristics of RTX-type S-layer in bacteria.

Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AVHH-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AVHH-MVH template, a capacity of 8.99 × 105 CFU/mL of phage display AVHH-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AVHH-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AVHH-MVH for MC-LR. The AVHH-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AVHH-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 μg/L, which was far below the maximum residue limit standard of 1.0 μg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.

Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 107 CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli (E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 μg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 μg/L and a limit of detection (LOD) of 0.044 μg/L, which is below the maximum residue limit standard (MRLs) of 1.0 μg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2–106.3% with CVs of 2.62–10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.

Considering the uncertainties of wind and photovoltaic power output and cooling, heating and power load forecasting values in micro-grid, the cooling, heating and power balance and power rotation reserve constraints with uncertain variables are expressed as chance constraints in this paper. And they are solved by transforming them into corresponding clear equivalence classes. Besides, under the electricity market time-sharing tariff policy, a scheduling model of grid-connected microgrid combined cooling, heating and power system is established, which considers both the economy and environmental protection of dispatching and the benefits of cooling and heating. The multi-objective processing method based on satisfaction index takes into account economic and environmental goals. Finally, an example is given to illustrate the validity of the model by analyzing the micro-power output plan and the corresponding dispatching scheme of cooling, heating and power loads.

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05μg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation

Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5–6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 μg mL−1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 μg mL−1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.

The cultivation of genetically modified crops (GMCs) has greatly increased worldwide. Given the fact that over 10 Bacillus thuringiensis Cry toxins have been applied in GMCs, there is a need to develop an efficient and economically affordable detection method that simultaneously screen such compounds with similar structures in crops and foodstuff. Here we described an approach using a site-directed mutagenesis that enhances the generic specificity of single chain variable fragment (scFv). After three rounds of panning against mixed antigen (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F) from the constructed mutagenesis library, one mutant showed greater ability to bind to a range of Cry toxins. The phage mutant, named D9, was cloned into pET26b expression vector, and then induced and purified. The mutant D9 was used to develop an indirect competitive immunoassay that sucessfully recognizes five targeted Cry1 toxins with a working range from 0.20 to 2.22 μg mL −1 . The recoveries of five Cry toxins from spiked rice samples ranged from 86.67% to 96.67%, with a coefficient of variation less than 8%. The results suggest that the immunoassay based on a scFv is a promising approach for detection of Cry toxins with a broad specificity.

An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L −1 ), respectively.

The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6.37–11.35 ng mL −1 and 17.36–24.48 ng mL −1 , respectively. The recovery tests showed that the recoveries ranged from 78% to 110% within the quantitation range (LOQ-100 ng mL −1 ). The established DAS-ELISA can be a useful tool for monitoring the Cry1 toxins in agricultural products. Mixture immunization opens a new path for producing diverse mAbs simultaneously in a single immunization circle.

In this study, by use of synthesized polypeptides as haptens, a monoclonal antibody with broad recognition against seven major Cry1 toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) has been produced and characterized. First, by comparing the three-dimensional structures of seven Cry1 toxins, analyzing the conserved sequences, and considering the antigenicity and hydrophilicity, three polypeptides (T1, T2, and T3) have been chosen and coupled to keyhole limpet hemocyanin as immunogens for the generic monoclonal antibody (Mab) generation. Thereafter, a double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) was developed for simultaneous determination of seven Cry1 toxins. The results revealed that the haptens T1, T2, and T3 had different effects in the production of antibodies. Among them, the obtained Mab (strain 2D3) generated by T2 can recognize seven Cry1 toxins simultaneously. Equilibrium dissociation constant (KD) values for seven Cry1 toxins with Mab 2D3 were 1.198 × 10(-8) M for Cry1Aa, 2.197 × 10(-8) M for Cry1Ab, 1.367 × 10(-8) M for Cry1Ac, 2.092 × 10(-8) M for Cry1B, 5.177 × 10(-8) M for Cry1C, 4.016 × 10(-8) M for Cry1E, and 3.497 × 10(-8) M for Cry1F. For 2D3-based DAS-ELISA, the limits of detection (LOD) and limits of quantification (LOQ) can reach 15 and 30 ng·mL(-1) for each Cry1 toxin, respectively. Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cry1 toxins, and it will provide a new idea and technical routes for development of multidetermination immunoassays.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

ScFvs are composed of the variable regions of the heavy and light chains via a short linker that maintain the specific antigen binding abilities of antibodies. In this study, we constructed a naive mouse phage displayed library to generate scFvs against Cry1Ab toxin. After affinity panning, positive phage-scFvs were isolated, sequenced and characterized by ELISA. The best binding ability scFv-G9 was expressed and purified. SDS-PAGE indicated that the relative molecular mass of scFv was estimated at 28 kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008 μg mL(-1), a working range 0.018-6.23 μg mL(-1), and the linear curve displayed an acceptable correlation coefficient of 0.98. The cross-reactivity showed that scFv-G9 had strongly binding ability to Cry1Ac toxin, but not to Cry1B, Cry1C and Cry1F toxin. The average recoveries of Cry1Ab toxin from spiked leaf and rice samples were in the range 92.1-94.8%, and 91.6-98.6%, respectively, with a coefficient of variation (C.V) less than 5.0%. These results showed promising applications of scfv-G9 for detecting Cry1Ab toxin with new DAS-ELISA.

Single chain variable fragment antibody (scFv) is capable of binding its target antigens and is one of the most popular recombinant antibodies format for many applications. In this study, a large human synthetic phage displayed library (Tomlinson J) was employed to generate scFvs against Cry1B toxin by affinity panning. After four rounds of panning, six monoclonal phage particles capable of binding with the Cry1B were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Two of the identified novel anti-Cry1B scFvs, namely H9 and B12, were expressed in Escherichia coli HB2151 and purified by Ni metal ion affinity chromatography. Sodium dodecyl sulfate polyacrylamine gel electrophoresis (SDS-PAGE) indicated that the relative molecular mass of scFv was estimated at 30 kDa. The purified scFv-H9 was used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for Cry1B toxin. The linear range of detection for standards in this ic-ELISA was approximately 0.19–1.1 μg mL −1 and 50% inhibition of control (IC 50 ) was 0.84 μg mL −1 for Cry1B. The affinity of scfv-H9 was (1.95±0.12)×10 7 M −1 and showed cross-reactivity with Cry1Ab toxin and Cry1Ac toxin (8.53% and 7.58%, respectively), higher cross-reactivity (12.8%) with Cry1C toxin. The average recoveries of Cry1B toxin from spiked leaf and rice samples were in the range 89.5–96.4%, and 88.5–95.6%, respectively, with a coefficient of variation (C.V) less than 6.0%. These results showed promising applications of scfv-H9 for detecting Cry1B toxin in agricultural and environmental samples.