Cunzheng Zhang

Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay

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.

Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin

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 (IC₅₀) was 0.84 μg mL⁻¹ for Cry1B. The affinity of scfv-H9 was (1.95±0.12) × 10⁷ 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.

Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries

As bioinsecticides Bacillus thuringiensis Cry1C δ-endotoxins also have been used in genetically modified crops worldwide since last century. In this study, single chain variable fragments (scFvs), which could specifically recognize and detect Cry1C in food samples, were isolated from naive phage displayed human antibody libraries (Tomlinson I + J) by iterative affinity selection procedure instead of immunization process. With increasing selection pressure, after four rounds of panning, three individual scFvs were obtained and sequenced. The antibodies were characterized by enzyme-linked immunosorbent assay (ELISA). Thereafter, a conformed novel anti-Cry1C scFv, namely scFv-H6, was expressed in Escherichia coli (E. coli) HB2151 and purified by Ni metal ion affinity chromatography. An indirect competitive ELISA assay (ic-ELISA) of scFv-H6 was developed for the determination of Cry1C toxin in the range from 0.023 μg mL⁻¹ to 4.35 μg mL⁻¹, and 50% inhibition concentration (IC₅₀) was 0.39 μg mL⁻¹. This approach showed ignorable cross-reactivity with toxin Cry1Ac and Cry1B (3.51% and 7.28%, respectively). This ic-ELISA approach was exploited for the determination of Cry1C in spiked ground rice samples with a mean recovery rate of 92.5% and coefficient of variation (C.V.) less than 5.0%. This study proves that phage display libraries provide a valuable system for the low-cost, rapid and continuous generation of specific antibody fragments directed against toxin targets and develop a simple detection method. Our results show that anti-Cry1C scFv could be a valuable tool for detection of Cry1C in food and agricultural samples.

Established a new double antibodies sandwich enzyme- linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library

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 naïve 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.

Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten.

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.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 108 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFvs were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC50) was 11.56 ng/mL and the detection limit (IC10) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application

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.

Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mutagenesis and screening

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 2.22μgmL-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.

Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis

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.

Generation of panels of anti-idiotypic single-chain variable fragments mimicking Cry2Aa toxin using the chain shuffling technique

ABSTRACT 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.