RESUMEN
This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab')2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab')2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab')2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.
Asunto(s)
Bacteriófagos , Insecticidas , Lepidópteros , Anticuerpos de Cadena Única , Animales , Ratones , Biblioteca de Genes , Anticuerpos de Cadena Única/química , Endotoxinas/metabolismo , Anticuerpos Antiidiotipos , Biblioteca de PéptidosRESUMEN
On-chip integrated metasurface driven by in-plane guided waves is of great interests in various light-field manipulation applications such as colorful augmented reality and holographic display. However, it remains a challenge to design colorful multichannel holography by a single on-chip metasurface. Here we present metasurfaces integrated on top of a guided-wave photonic slab that achieves multi-channel colorful holographic light display. An end-to-end scheme is used to inverse design the metasurface for projecting off-chip preset multiple patterns. Particular examples are presented for customized patterns that were encoded into the metasurface with a single-cell meta-atom, working simultaneously at RGB color channels and for several different diffractive distances, with polarization dependence. Holographic images are generated at 18 independent channels with such a single-cell metasurface. The proposed design scheme is easy to implement, and the resulting device is viable for fabrication, promising plenty of applications in nanophotonics.
RESUMEN
Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.
Asunto(s)
Anticuerpos Monoclonales , Toxinas de Bacillus thuringiensis , Endotoxinas , Ensayo de Inmunoadsorción Enzimática , Proteínas Hemolisinas , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Conejos , Ratones , Endotoxinas/análisis , Endotoxinas/inmunología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/análisis , Proteínas Hemolisinas/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/análisis , Bacillus thuringiensis/química , Ratones Endogámicos BALB CRESUMEN
Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.
Asunto(s)
Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Endotoxinas , Proteínas Hemolisinas , Mariposas Nocturnas , Animales , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Mariposas Nocturnas/metabolismo , Mariposas Nocturnas/microbiología , Sitios de Unión , Bacillus thuringiensis/metabolismo , Control Biológico de Vectores , Dominios Proteicos , Helicoverpa armigeraRESUMEN
Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.
Asunto(s)
Bacteriófagos , Biblioteca de Péptidos , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoensayo , Anticuerpos MonoclonalesRESUMEN
Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.
Asunto(s)
Anticuerpos Monoclonales , Bacillus thuringiensis , Proteínas Bacterianas/análisis , Endotoxinas/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas HemolisinasRESUMEN
Chronic low-grade inflammation (CLGI) is closely associated with various chronic diseases. Researchers have paid attention to the comprehensive application and development of food materials with potential anti-inflammatory activity. Owing to their abundant nutrients and biological activities, coarse cereals have emerged as an important component of human diet. Increasing evidence has revealed their potential protective effects against CLGI in chronic conditions. However, this property has not been systematically discussed and summarized. In the present work, numerous published reports were reviewed to systematically analyze and summarize the protective effects of coarse cereals and their main active ingredients against CLGI. Their current utilization state was investigated. The future prospects, such as the synergistic effects among the active compounds in coarse cereals and the biomarker signatures of CLGI, were also discussed. Coarse cereals show promise as food diet resources for preventing CLGI in diseased individuals. Their active ingredients, including ß-glucan, resistant starch, arabinoxylan, phenolic acids, flavonoids, phytosterols and lignans, function against CLGI through multiple possible intracellular signaling pathways and immunomodulatory effects. Therefore, coarse cereals play a crucial role in the food industry due to their health effects on chronic diseases and are worthy of further development for possible application in modulating chronic inflammation.
Asunto(s)
Dieta , Grano Comestible , Humanos , Grano Comestible/metabolismo , Inflamación/metabolismo , Flavonoides/metabolismo , Enfermedad CrónicaRESUMEN
The local surface plasmon resonance (LSPR) effect has been widely used in various nanophotonic applications. However, because the LSPR effect is highly sensitive to the structure and geometry, it is desirable to efficiently search viable geometries for predefined local field enhancement spectrum. Herein we present a generative adversarial network-based LSPR nanoantenna design scheme. By encoding the antenna structure information into an red-green-blue (RGB) color image, the corresponding nanoantenna structure can be inverse-designed to achieve the required enhancement spectrum of the local field. The proposed scheme can accurately offer the multiple geometry layout for the customized specific spectrum in seconds, which could be beneficial for fast design and fabrication of plasmonic nanoantenna.
RESUMEN
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.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Toxinas de Bacillus thuringiensis/química , Endotoxinas/química , Proteínas Hemolisinas/química , Región Variable de Inmunoglobulina/química , Anticuerpos de Cadena Única/química , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Toxinas de Bacillus thuringiensis/inmunología , Toxinas de Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Simulación del Acoplamiento Molecular , Mariposas Nocturnas , Mutación , Biblioteca de Péptidos , Conformación Proteica , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
With the wide cultivation of transgenic plants throughout the world and the rising risk of resistance to Bacillus thuringiensis crystal (Cry) toxins, it is essential to design an adaptive resistance management strategy for continued use. Neuropeptide F (NPF) of insects has proven to be valuable for the production of novel-type transgenic plants via its important role in the control of feeding behavior. In this study, the gene encoding NPF was cloned from the diamondback moth, Plutella xylostella, an important agricultural pest. Real-time quantitative reverse transcription-polymerase chain reaction and in situ hybridization showed a relatively high expression of P. xylostella-npf (P. x-npf) in endocrine cells of the midgut of fourth instar larvae, and it was found to participate in P. xylostella feeding behavior and Cry1Ac-induced feeding inhibition. Prokaryotic expression and purification provided structure unfolded P. x-npf from inclusion bodies for diet surface overlay bioassays and the results demonstrated a significant synergistic effect of P. x-npf on Cry1Ac toxicity by increasing intake of noxious food which contains Cry toxins, especially quick death at an early stage of feeding. Our findings provided a potential new way to efficiently control pests by increasing intake of lower dose Cry toxins and a novel hint for the complex Cry toxin mechanism.
Asunto(s)
Toxinas de Bacillus thuringiensis , Endotoxinas , Proteínas Hemolisinas , Mariposas Nocturnas , Neuropéptidos , Animales , Toxinas de Bacillus thuringiensis/farmacología , Endotoxinas/farmacología , Conducta Alimentaria/fisiología , Expresión Génica , Genes de Insecto , Proteínas Hemolisinas/farmacología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/farmacología , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Mariposas Nocturnas/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Control de Plagas/métodosRESUMEN
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.
Asunto(s)
Microcistinas , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos , Toxinas Marinas , Ratones , Simulación del Acoplamiento MolecularRESUMEN
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. KEY POINTS: ⢠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.
Asunto(s)
Insecticidas , Piretrinas , Anticuerpos de Cadena Única , Simulación del Acoplamiento Molecular , Anticuerpos de Cadena Única/genéticaRESUMEN
The polarization-maintaining fiber-based Sagnac loop interferometer (PSLI) is frequently applied in directional torsion measurement, but may suffer from a large temperature cross talk. In this study, a novel method was proposed for fabrication of side-tapered PSLI by a two-step arc-discharge technique at the splicing point. The energy distribution of the taper region was characterized, and comprehensive tests were performed in terms of torsion and temperature. The experimental results showed that, through bias twisting, the measuring areas were effectively gapped and highly sensitive torsion and temperature responses were gained simultaneously. With a small wavelength shift, the torsion sensitivity reached 13.54 dB/rad in the range from -30 to 30 rad/m. Moreover, the temperature sensitivity was found to be -1.579nm/∘C, with a near-zero intensity fluctuation. Therefore, the sensor fabricated herein successfully achieves simultaneous measurement of directional torsion and temperature with high sensitivity and ultralow cross talk. The proposed scheme has the merits of practicality, low cost, and ease of operation, and is very promising for multiparameter engineering monitoring.
RESUMEN
In this paper, we report a novel in-fiber Mach-Zehnder interferometer based directional torsion sensor, in which a section of two-mode fiber is sandwiched between two single mode fibers by over core-offset splicing technique. The variety of fringe visibility demonstrates the strong dependence on offset and fiber length. For the first time the near zero visibility at 0° rotating state is obtained by fine offset-modulation. The experimental results show that, with 0° turning point, the counter-clockwise to the clockwise direction can be recognized by the reversal from peak to dip of fringes. Moreover, a competitive sensitivity of 21.485 dB/(rad/cm) is gained with high linearity and low-temperature crosstalk in the range from -40 rad/m to 40 rad/m. Without any pre-twisting, our fiber torsion sensor is small size, ease of fabrication, cost efficiency and very potential in the applications of industrial and artificial intelligence.
RESUMEN
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 (5-6â¯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⯵gâ¯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⯵gâ¯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.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/análisis , Endotoxinas/análisis , Proteínas Hemolisinas/análisis , Inmunoensayo/métodos , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/inmunología , Endotoxinas/inmunología , Oro/química , Proteínas Hemolisinas/inmunología , Límite de Detección , Nanopartículas del Metal/química , Plantas Modificadas Genéticamente/metabolismo , Reproducibilidad de los Resultados , Zea mays/metabolismoRESUMEN
In this paper, an in-line Mach-Zehnder interferometer embedded in a fiber Bragg grating (FBG) is fabricated by a tiny offset-core splicing technique and inserted into a fiber ring laser to effectively improve the detection limit and discrimination of sensing. Through fine power adjustment, the stable operation of dual-wavelength lasing is completed in a single FBG for the first time to our knowledge, and simultaneous measurement of refractive index (RI) and ambient temperature is achieved with high discrimination due to the RI immunity of the FBG. The experimental results indicate that the sensitivities of RI and temperature are -30.57 nm/RIU and 131.1 pm/°C, respectively, with very low intensity drift. Furthermore, the over 20 times improvement of the RI detection limit is obtained owing to the large extinction ratio (>30 dB) and small linewidth (≤0.13 nm).
RESUMEN
In this paper, a comprehensive passive torsion measurement is performed firstly in a 40-cm-long polarization maintaining fiber-based Sagnac interferometer (PMF-SI), and the non-linear torsion response is found and investigated. Then, a fiber laser torsion sensor (FLTS) with a dual-ring-cavity structure is proposed and experimentally demonstrated, in which the PMF-SI is utilized as the optical filter as well as the sensing unit. In particular, the highly sensitive linear range is adjusted through fine phase modulation, and owing to the flat-top feature of fringes, an ~83.6% sensitivity difference is effectively compressed by the generated lasing. The experimental results show that, without any pre-twisting, the ultra-wide linear response from -175 to 175 rad/m is gained, and the torsion sensitivities are 2.46 and 1.55 nm/rad with high linearity (>0.99) in the clockwise and anti-clockwise directions, respectively. Additionally, a high extinction ratio (>42 dB) and small line-width (~0.14 nm) are obtained in the proposed FLTS, and the corresponding detection limit reaches 0.015 rad/m.
RESUMEN
Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 107 CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL-1, respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.
Asunto(s)
Endotoxinas/metabolismo , Microbiología de Alimentos/métodos , Biblioteca de Péptidos , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/metabolismo , Animales , Endotoxinas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Ratones , Oryza/químicaRESUMEN
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.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Microcistinas/análisis , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Animales , Bacteriófagos , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Regulación de la Expresión Génica , Toxinas Marinas , Microcistinas/inmunología , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Bacteriophages (phages) are known to effectively kill extracellular multiplying bacteria. The present study demonstrated that phages penetrated bovine mammary epithelial cells and cleared intracellular Staphylococcus aureus in a time-dependent manner. In particular, phage vB_SauM_JS25 reached the nucleus within 3 h postincubation. The phages had an endocytotic efficiency of 12%. This ability to kill intracellular host bacteria suggests the utility of phage-based therapies and may protect patients from recurrent infection and treatment failure.