RESUMEN
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.
Asunto(s)
Formación de Anticuerpos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Compuestos Organofosforados/aislamiento & purificación , Plaguicidas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Compuestos Organofosforados/efectos adversos , Compuestos Organofosforados/inmunología , Plaguicidas/efectos adversos , Plaguicidas/aislamiento & purificación , Proteínas Recombinantes/inmunologíaRESUMEN
A Love Wave (LW) immunosensor was developed for the detection of carbaryl pesticide. The experimental setup consisted on: a compact electronic characterization circuit based on phase and amplitude detection at constant frequency; an automated flow injection system; a thermal control unit; a custom-made flow-through cell; and Quartz /SiO2 LW sensors with a 40 µm wavelength and 120 MHz center frequency. The carbaryl detection was based on a competitive immunoassay format using LIB-CNH45 monoclonal antibody (MAb). Bovine Serum Albumin-CNH (BSA-CNH) carbaryl hapten-conjugate was covalently immobilized, via mercaptohexadecanoic acid self-assembled monolayer (SAM), onto the gold sensing area of the LW sensors. This immobilization allowed the reusability of the sensor for at least 70 assays without significant signal losses. The LW immunosensor showed a limit of detection (LOD) of 0.09 µg/L, a sensitivity of 0.31 µg/L and a linear working range of 0.14-1.63 µg/L. In comparison to other carbaryl immunosensors, the LW immunosensor achieved a high sensitivity and a low LOD. These features turn the LW immunosensor into a promising tool for applications that demand a high resolution, such as for the detection of pesticides in drinking water at European regulatory levels.
Asunto(s)
Acústica/instrumentación , Anticuerpos Monoclonales/inmunología , Carbaril/análisis , Inmunoensayo/instrumentación , Sistemas Microelectromecánicos/instrumentación , Plaguicidas/análisis , Contaminantes Químicos del Agua/análisis , Carbaril/inmunología , Diseño de Equipo , Análisis de Falla de Equipo , Plaguicidas/inmunología , Contaminantes Químicos del Agua/inmunologíaRESUMEN
Asthma and allergic diseases have increased worldwide; however, etilogic factors for this increase are still poor. Prenatal consumptions of fatty acids are hypothesized, although few clinical trials in developing countries have been performed. This study was designed to identify predictors of immunoglobulin E (IgE) levels in cord blood of Mexican newborns. Total IgE was measured in umbilical cord blood from 613 infants whose mothers participated in a double-blind randomized controlled trial of 400 mg of docosahexaenoic acid or placebo from 18 to 22 weeks gestation through delivery. During pregnancy, information on sociodemographic characteristics, environmental exposures, and perceived maternal stress were obtained; a maternal blood sample was also collected to determine atopy via specific IgE levels. Logistic regression models were used to identify the main prenatal predictors of detectable total IgE levels in cord blood. IgE was detectable in cord blood from 344 (53.7%) infants; the main predictors in multivariate analyses were maternal atopy (odds ratio [OR] = 1.69; 95% CI, 1.19-2.42; p < 0.05) and pesticide use in the home (OR = 1.49; 95% CI, 1.04-2.14; p < 0.05). When stratified by maternal atopy, season of birth was a significant predictor in the atopic group only (OR = 2.48; 95% CI, 1.00-6.16; p < 0.05), and pesticide use was a significant predictor for infants born to nonatopic mothers (OR = 1.64; 95% CI, 1.07-2.51; p < 0.05). No differences were seen in the proportion of infants with detectable IgE by treatment group. Prenatal supplementation with omega-3 polyunsaturated fatty acid did not alter the detectable cord blood IgE levels. Maternal atopy and pesticide use during pregnancy are strong predictors of cord blood IgE levels in newborns. Clinical trial NCT00646360, www.clinicaltrials.gov.
Asunto(s)
Alérgenos/metabolismo , Biomarcadores/metabolismo , Epítopos/metabolismo , Hipersensibilidad/diagnóstico , Plaguicidas/metabolismo , Adulto , Alérgenos/inmunología , Animales , Ácidos Docosahexaenoicos/administración & dosificación , Epítopos/inmunología , Femenino , Sangre Fetal/metabolismo , Humanos , Hipersensibilidad/etiología , Hipersensibilidad/terapia , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Recién Nacido , Masculino , México , Plaguicidas/inmunología , Valor Predictivo de las Pruebas , Embarazo , Pronóstico , Adulto JovenRESUMEN
A glassy carbon substrate was covalently modified with a mixed layer of 4-aminophenyl and phenyl via in situ electrografting of their aryldiazonium salts in acidic solutions. Single-walled carbon nanotubes (SWNTs) were covalently and vertically anchored on the electrode surface via the formation of amide bonds from the reaction between the amines located on the modified substrate and the carboxylic groups at the ends of the nanotubes. Ferrocenedimethylamine (FDMA) was subsequently attached to the ends of SWNTs through amide bonding followed by the attachment of an epitope, i.e., endosulfan hapten to which an antibody would bind. Association or dissociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. Antibody-complexed electrodes were exposed to samples containing spiked endosulfan (unbound target analyte) in environment water and interrogated using the square wave voltammetry (SWV) technique. The modified sensing surfaces were characterized by atomic force microscopy, XPS, and electrochemistry. The fabricated electrochemical immunosensor can be successfully used for the detection of endosulfan over the range of 0.01-20 ppb by a displacement assay. The lowest detection limit of this immunosensor is 0.01 ppb endosulfan in 50 mM phosphate buffer at pH 7.0.
Asunto(s)
Técnicas Electroquímicas/métodos , Endosulfano/análisis , Nanotubos de Carbono/química , Plaguicidas/análisis , Contaminantes Químicos del Agua/análisis , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Carbono/química , Dimetilaminas/química , Electrodos , Endosulfano/inmunología , Compuestos Ferrosos/química , Límite de Detección , Metalocenos , Plaguicidas/inmunología , Agua/análisis , Contaminantes Químicos del Agua/inmunologíaRESUMEN
BACKGROUND: Epidemiologic studies support the hypothesis that reduced microbial exposure in westernized societies promotes atopy. Dichlorophenols are widely used as pesticides and for chlorination of water. They have a strong bactericidal effect that could affect microflora in the environment. However, it is unknown whether their use is associated with a higher prevalence of allergies. OBJECTIVE: To test the association between exposure to environmental pesticides represented by dichlorophenols and allergic sensitization measured by allergen-specific serum IgE levels in a US nationally representative sample of 2,211 persons 6 years and older in the National Health and Nutrition Examination Survey 2005-2006. METHODS: Exposure to dichlorophenols was defined as high if their levels in urine were present at the 75th percentile and above. Association of the high exposure to dichlorophenols with sensitization to food and environmental allergens was assessed in logistic regression models after adjustment for sample weights and potential confounders. RESULTS: Sensitizations to 1 or more food allergens were more common in those with exposure to 2 dichlorophenol metabolites. After multivariable adjustment, urine dichlorophenol levels at the 75th percentile and above were associated with the presence of sensitization to foods (odds ratio, 1.8; 95% confidence interval, 1.2-2.5; P = .003). No significant association was found between dichlorophenol exposure and sensitization to aeroallergens alone. CONCLUSION: High urine levels of dichlorophenols are associated with the presence of sensitization to foods in a US population. Excessive use of dichlorophenols may contribute to the increasing incidence of food allergies in westernized societies.
Asunto(s)
Hipersensibilidad/epidemiología , Hipersensibilidad/inmunología , Plaguicidas/inmunología , Plaguicidas/envenenamiento , Fenoles/inmunología , Fenoles/envenenamiento , Adulto , Alérgenos/efectos adversos , Alérgenos/inmunología , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Alimentos/efectos adversos , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/etiología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Encuestas Nutricionales/métodos , Estados Unidos/epidemiologíaRESUMEN
Magnetic Fe(3)O(4) particle aggregates were prepared by cross-linking Fe(3)O(4) nanoparticles bearing surface carbonyl groups with poly-L-lysine. Upon further coupling with antiparaoxon methyl polyclonal antibody, the resultant particle aggregate-based probes were used in a lateral flow immunochromatographic assay (LFIA) of pesticide residue of paraoxon methyl. The results were compared with that achieved by using the mother Fe(3)O(4) nanoparticles. More quantitative results on the signal amplification effect endowed by the controlled aggregation of Fe(3)O(4) nanoparticles were extracted by relative optical density analysis. Under optimized conditions, a detection limit of 1.7 ng/mL for paraoxon methyl was achieved by using the particle aggregates, which is almost 40-fold lower than that based on the mother Fe(3)O(4) nanoparticles.
Asunto(s)
Cromatografía de Afinidad/métodos , Óxido Ferrosoférrico/química , Nanopartículas de Magnetita/química , Plaguicidas/análisis , Anticuerpos/inmunología , Colorantes/química , Nanopartículas de Magnetita/ultraestructura , Paraoxon/análisis , Paraoxon/inmunología , Plaguicidas/inmunología , Polilisina/químicaRESUMEN
The sensitive detection of pesticides in complex environment is important but still challenging in presence of organic-rich water sample and food matrix. Herein, we reported a nitrile-mediated SERS immunosensor for sensitive and optical anti-interference determination of imidacloprid. Raman tag contained CN bond could provide a sharp characteristic peak in the Raman-silent spectral window (1800 ~ 2800 cm-1), which could resist the optical noises from the fingerprint region (<1800 cm-1). Aucore-Agshell bimetallic nanocuboid (AuNR@Ag) connected with antigen and Raman tag was used as Raman probe, while Fe3O4 magnetic nanoparticle functionalized with anti-imidacloprid antibody was applied as signal enhancer. Owing to the specific recognition ability between antigen and antibody, the competitive system with imidacloprid was formed. Under the optimal condition, the linear relationship was developed in the range of 10-400 nM. Finally, the SERS immunosensor was successfully applied to determine imidacloprid in real samples with recoveries from 96.8% to 100.5%.
Asunto(s)
Análisis de los Alimentos/métodos , Inmunoensayo/métodos , Neonicotinoides/análisis , Nitrocompuestos/análisis , Espectrometría Raman/métodos , Contaminación de Alimentos/análisis , Oro/química , Inmunoensayo/instrumentación , Nanopartículas Magnéticas de Óxido de Hierro/química , Neonicotinoides/inmunología , Nitrocompuestos/inmunología , Plaguicidas/análisis , Plaguicidas/inmunología , Sensibilidad y Especificidad , Espectrometría Raman/instrumentaciónRESUMEN
A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Modelos Moleculares , Compuestos Organofosforados/análisis , Plaguicidas/análisis , Animales , Especificidad de Anticuerpos , Unión Competitiva , Bovinos , Femenino , Haptenos/química , Haptenos/inmunología , Ratones , Conformación Molecular , Compuestos Organofosforados/inmunología , Plaguicidas/inmunología , Relación Estructura-Actividad CuantitativaRESUMEN
BACKGROUND: The aim of the present study was to assess possible health effects of airway exposures to Bacillus thuringiensis (Bt) based biopesticides in mice. Endpoints were lung inflammation evaluated by presence of inflammatory cells in bronchoalveolar lavage fluid (BALF), clearance of bacteria from the lung lumen and histological alterations of the lungs. Hazard identifications of the biopesticides were carried out using intratracheal (i.t.) instillation, followed by an inhalation study. The two commercial biopesticides used were based on the Bt. subspecies kurstaki and israelensis, respectively. Groups of BALB/c mice were i.t instilled with one bolus (3.5 × 105 or 3.4 × 106 colony forming units (CFU) per mouse) of either biopesticide. Control mice were instilled with sterile water. BALFs were collected and the inflammatory cells were counted and differentiated. The BALFs were also subjected to CFU counts. RESULTS: BALF cytology showed an acute inflammatory response dominated by neutrophils 24 hours after instillation of biopesticide. Four days after instillation, the neutrophil number was normalised and inflammation was dominated by lymphocytes and eosinophils, whereas 70 days after instillation, the inflammation was interstitially located with few inflammatory cells present in the lung lumen.Half of the instilled mice had remaining CFU recovered from BALF 70 days after exposure. To gain further knowledge with relevance for risk assessment, mice were exposed to aerosols of biopesticide one hour per day for 2 × 5 days. Each mouse received 1.9 × 104 CFU Bt israelensis or 2.3 × 103 CFU Bt kurstaki per exposure. Seventy days after end of the aerosol exposures, 3 out of 17 mice had interstitial lung inflammation. CFU could be recovered from 1 out of 10 mice 70 days after exposure to aerosolised Bt kurstaki. Plethysmography showed that inhalation of Bt aerosol did not induce airway irritation. CONCLUSIONS: Repeated low dose aerosol exposures to commercial Bt based biopesticides can induce sub-chronic lung inflammation in mice, which may be the first step in the development of chronic lung diseases. Inhalation of Bt aerosols does not induce airway irritation, which could explain why workers may be less inclined to use a filter mask during the application process, and are thereby less protected from exposure to Bt spores.
Asunto(s)
Bacillus thuringiensis/inmunología , Exposición por Inhalación/efectos adversos , Pulmón/inmunología , Plaguicidas/efectos adversos , Animales , Bacillus thuringiensis/fisiología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Femenino , Humanos , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Control Biológico de Vectores , Plaguicidas/inmunología , Sistema Respiratorio/inmunología , Sistema Respiratorio/microbiologíaRESUMEN
New rat monoclonal antibodies (mAbs) for DDT [1,1,1 -trichloro-2,2-bis (4-chlorophenyl) ethane], namely DDT 7C12, DDT 1C1, and DDT 1B2, were developed, characterized, and applied in ELISA both in coating antigen and in enzyme-tracer format. The latter used horseradish peroxidase (HRP) or glucose oxidase as enzymes. The lowest concentration of p,p'-DDT was determined with mAb DDT 7C12 and DDT-hapten HRP, with a test midpoint (IC50) of 0.5 +/- 0.2 microg/L (n=10) in 40 mM PBS (phosphate-buffered saline). The mouse anti-rat immunoglobulin lambda-light chain mAb LA1B12 was used as capture mAb. The best IC50 for o,p'-DDT in 40 mM PBS was 1.0 +/- 0.3 microg/L (n=12) and was obtained with mAb DDT 1C1 and DDT-hapten HRP, whereas mAb DDT 1B2 was very selective for p,p'-DDT with an IC50 of 4.2 + 1.6 microg/L (in 40 mM PBS, n=9). An optical immunosensor was optimized and applied for the analysis of DDT (or DDT equivalents). This immunosensor consists of a bench-top optical readout device and disposable sensor chips, which include the fluidic system. Evanescent field excitation and emission of the fluorophore Oyster-645 was used. An IC50 for p,p'-DDT [in 5% (v/v) isopropanol in 40 mM PBS] of 4 microg/L was obtained using DDT 7C12-Oyster-645. ELISA and immunosensor were used for the analysis of p,p'-DDT in unspiked and spiked surface water samples. Within the working ranges of these immunotechniques, recoveries ranged from 80 to 120%.
Asunto(s)
Técnicas Biosensibles/instrumentación , DDT/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Plaguicidas/análisis , Animales , Anticuerpos Monoclonales , Reacciones Cruzadas , DDT/química , DDT/inmunología , Ensayo de Inmunoadsorción Enzimática/normas , Colorantes Fluorescentes , Glucosa Oxidasa , Haptenos , Peroxidasa de Rábano Silvestre , Inmunoquímica , Isomerismo , Ratones , Dispositivos Ópticos , Plaguicidas/química , Plaguicidas/inmunología , Ratas , Contaminantes Químicos del Agua/análisisRESUMEN
Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.
Asunto(s)
Clonación Molecular/métodos , Fragmentos de Inmunoglobulinas/genética , Ratones/genética , Ratones/inmunología , Biblioteca de Péptidos , Conejos/genética , Conejos/inmunología , Animales , Especificidad de Anticuerpos , Atrazina/inmunología , Secuencia de Bases , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Vectores Genéticos , Datos de Secuencia Molecular , Plaguicidas/inmunología , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/genética , Sulfonamidas/inmunologíaRESUMEN
The mechanism of specific recognition in pesticide immunochemistry was investigated by computer-based strategy, and a rapid method for the identification of antibody specificity was developed. Based on the previously produced anti-parathion monoclonal antibody (mAb), the DNA sequence was analyzed by polymerase chain reaction (PCR). From the translated amino acid sequences, a three-dimensional structure of the antibody was constructed by homology modeling method, and then it was coordinated by 1 ns molecular dynamics under the explicit solvent condition. The stereochemical property and folding quality were further assessed by Procheck and Profile-3D. The self-compatibility score for the antibody model was 98.7, which was greater than the low score 46.2 and close to the top score 102.6. In addition, parathion and several structural analogues were docked to the constructed antibody structure. The docking results showed that the interaction energy (-40.54 kcal/mol) of antibody-parathion complex was the lowest among all the tested pesticides, which accounted for the high specificity of the antibody to parathion and perfectly matched with the experimental data. Moreover, three residues, Phe165, Asp107 and Thr100 were recognized as the most important residues for antibody reacting with parathion. The interaction energy negatively correlated with the antibody specificity.
Asunto(s)
Anticuerpos/química , Compuestos Organofosforados/química , Paratión/química , Plaguicidas/química , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/inmunología , Especificidad de Anticuerpos , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Paratión/inmunología , Plaguicidas/inmunología , Unión Proteica , Relación Estructura-ActividadRESUMEN
A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22â¯pmolâ¯L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000â¯pmolâ¯L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.
Asunto(s)
Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Nanopartículas del Metal/química , Neonicotinoides/análisis , Nitrocompuestos/análisis , Plaguicidas/análisis , Anticuerpos Inmovilizados/inmunología , Anticuerpos Monoclonales/inmunología , Armoracia/enzimología , Bencidinas/química , Citrullus/química , Agua Potable/análisis , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Contaminación de Alimentos/análisis , Oro/química , Peroxidasa de Rábano Silvestre/química , Límite de Detección , Solanum lycopersicum/química , Neonicotinoides/inmunología , Nitrocompuestos/inmunología , Plaguicidas/inmunología , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/inmunologíaRESUMEN
Using a simple test for rapid identification and quantification of pesticide multiresidues in food and environmental samples is a long-cherished approach for practical monitoring purposes. Here two gold-based lateral-flow strips (strip A and strip B) were investigated for simultaneous detection of carbofuran and triazophos. For the strip A format, a bispecific monoclonal antibody (BsMcAb) against both carbofuran and triazophos was employed to prepare the immunogold probe. For the strip B format, anti-carbofuran monoclonal antibody (McAb) and anti-triazophos McAb separately labeled with colloidal gold were combined as detector reagents. By comparison of visual results from pesticide standard tests between the two formats, the strip B assay manifested higher sensitivities for both pesticides. Analysis of spiked water samples by the preferable strip indicated that the detection limits for carbofuran and triazophos were 32 and 4 microg/L, respectively. The strength of the portable one-step strip assay was in the simultaneous screening for two pesticides within a short time (8-10 min) without any equipment.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Carbofurano/análisis , Inmunohistoquímica/métodos , Organotiofosfatos/análisis , Plaguicidas/análisis , Triazoles/análisis , Contaminantes Químicos del Agua/análisis , Agua/análisis , Carbofurano/inmunología , Organotiofosfatos/inmunología , Plaguicidas/inmunología , Triazoles/inmunología , Contaminantes Químicos del Agua/inmunologíaRESUMEN
OBJECTIVE: To develop an immunoassay for multiple residues of organophosphorus pesticides. METHODS: The generic hapten O, O-dimethyl-O-(4-carboxyphenyl)ester (HP) was synthesized by the reaction between O, O-Dimethyl phosphorochloridothionate and 4-hydroxybenzoic acid methyl ester, and then conjugated to bovine serum albumin (BSA) by active ester method for immunogens and ovalbumin (OVA) by mixed anhydride reaction for coating antigens. RESULTS: The hapten was synthesized successfully, which was characterized by MS. The coupling ratios were 15.6:1 (HP-BSA) and 16.2:1(HP-OVA), respectively, calculated by UV spectrophotometry. CONCLUSION: suggested that the artificial antigens could be used for production of broad specificity anti-organophosphorous pesticides antibody.
Asunto(s)
Antígenos/química , Haptenos/química , Compuestos Organofosforados/química , Plaguicidas/química , Haptenos/inmunología , Compuestos Organofosforados/inmunología , Ovalbúmina/química , Plaguicidas/inmunología , Albúmina Sérica Bovina/químicaRESUMEN
The immunostimulating and therapeutic properties of Ginkgo biloba (GB) have always been the focus of traditional medicine over thousands of years. During last decade, special attentions were paid to use of GB in aquaculture to enhance fish health and survival. In the present study, we investigated for the first time the immunogenic effects of dietary GB against oxidative and toxicity induced by organophosphate pesticide, diazinon. In non-diazinon-exposed fish, the plasma total immunoglobulin, lysozyme activity, and peroxidase activity significantly elevated after 60-day experiment in fish supplemented with 1 and 2 g GB/kg diet (p < 0.05). The respiratory burst activity and complement activity significantly increased only in groups supplemented with 0.5 g GB/kg diet (p < 0.05). Furthermore, the peroxidase activity, total immunoglobulin, and lysozyme activity significantly declined in groups supplemented with 4 g GB/kg diet during feeding trial (p < 0.05). There were no significant differences in expression of interleukin 1 beta (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) genes in kidney between control group (non-GB-supplemented fish) and GB-supplemented fish (p > 0.05). In diazinon-exposed fish, all immunity components significantly decreased during exposure in control and those fed 0.5 and 4 g GB/kg diet (p < 0.05). In fish fed 1 and 2 g GB/kg diet, no alternations were found in immunity components during exposure period (p > 0.05). In addition, diazinon induced the expression of IL-1ß and TGF-ß1 genes in control and fish fed 0.5 and 4 g GB/kg diet (p < 0.05). No significant changes were observed in expression of IL-1ß and TGF-ß1 genes in fish supplemented with 1 and 2 g GB/kg (p > 0.05). In conclusion, the results of the present study suggest an immunogenic role for dietary GB at optimum dietary levels (1-2 g GB/kg diet) against toxicity induced by diazinon. Nevertheless, GB at high dietary levels (4 g GB/kg diet) showed immunosuppressive effects, which makes it necessary to optimize its levels in diet.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Diazinón/antagonistas & inhibidores , Enfermedades de los Peces/inducido químicamente , Ginkgo biloba/química , Oncorhynchus mykiss/inmunología , Plaguicidas/antagonistas & inhibidores , Extractos Vegetales/farmacología , Alimentación Animal , Animales , Acuicultura , Diazinón/inmunología , Suplementos Dietéticos , Enfermedades de los Peces/inmunología , Inmunidad Innata/efectos de los fármacos , Oncorhynchus mykiss/genética , Plaguicidas/inmunología , Plaguicidas/farmacologíaRESUMEN
Diethyl (carboxymethyl) phosphonate (DECP) was used as the hapten to develop an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for detecting organophosphorus pesticides (OPs). Conjugator of DECP with bovin serum albumin (BSA) was used as the immunogen for producing the polyclonal antibodies (PcAbs). Three antisera were obtained after the immune procedure. Characterization studies of the PcAbs indicated that the titer of antiserum-1 was highest in 3 antisera, and antiserum-1 had high affinity and specificity to the parathion, dichlorvos and pirimiphos. The IC-ELISA showed an IC50 of 0.428 micro g/mL with a detection limit of 0.0125 micro g/mL to parathion. The assay also indicated that the IC50 values of pirimiphos and dichlorvos were 0.331 micro g/mL and 1.25 micro g/mL respectively, and the detection limits of pirimiphos and dichlorvos were 0.0116 micro g/mL and 0.048 micro g/mL respectively. Recoveries of parathion, pirimiphos and dichlorvos spiked into water samples ranged from 90% to 160%. The results indicated that the ELISA could be a convenient and supplemental analytical tool for monitoring OPs residues in environmental water samples.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Compuestos Organofosforados/análisis , Plaguicidas/análisis , Contaminantes Químicos del Agua/análisis , Anticuerpos/inmunología , Unión Competitiva , Haptenos/inmunología , Dosificación Letal Mediana , Compuestos Organofosforados/inmunología , Compuestos Organofosforados/aislamiento & purificación , Plaguicidas/inmunología , Plaguicidas/aislamiento & purificación , Sensibilidad y Especificidad , Albúmina Sérica Bovina/inmunología , Contaminantes Químicos del Agua/inmunología , Contaminantes Químicos del Agua/aislamiento & purificaciónRESUMEN
The effect of melatonin, a major secretory product of the pineal gland, in attenuation of propoxur (2-isopropoxy phenyl N-methyl carbamate)-induced modulation of cell-mediated immune (CMI) response was studied in rats. Male Wistar albino rats were exposed to propoxur (a widely used pesticide) orally (10 mg/kg) and/or melatonin (10 mg/kg) orally for 4 weeks. CMI was measured by delayed-type hypersensitivity (DTH), leucocyte and macrophage migration inhibition (LMI and MMI) responses and estimation of cytokines TNF-alpha and IFN-gamma levels. Rats exposed to propoxur for 4 weeks showed significant decrease in DTH, LMI and MMI responses. Propoxur also suppressed TNF-alpha and IFN-gamma production significantly. Administration of melatonin alone caused a significant increase in DTH response. Although there were no changes in the LMI and MMI response, the cytokine levels were significantly increased, as compared to control. Co-administration of melatonin along with propoxur significantly nullified the effect of the pesticide on the CMI response, except DTH and reversed levels of cytokines to near control/normal values. Thus, melatonin treatment considerably attenuated immunomodulation caused by sub-chronic treatment of propoxur in experimental animals.
Asunto(s)
Antioxidantes/farmacología , Inmunidad Celular/efectos de los fármacos , Leucocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Melatonina/farmacología , Plaguicidas/antagonistas & inhibidores , Propoxur/antagonistas & inhibidores , Administración Oral , Animales , Antioxidantes/administración & dosificación , Citocinas/inmunología , Citocinas/metabolismo , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/metabolismo , Inmunidad Celular/fisiología , Leucocitos/inmunología , Leucocitos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Melatonina/administración & dosificación , Plaguicidas/inmunología , Glándula Pineal/química , Propoxur/inmunología , Ratas , Ratas Wistar , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Previous research suggests that farmers may have an increased risk of developing autoimmunity and that exposure to certain pesticides may alter immune function. Little is known, however, about the immunologic effects of farming and pesticide exposures. As part of the Prairie Ecosystem Study, associations between detection of antinuclear antibodies (ANA), an autoimmunity indicator, and exposure to the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) were investigated in a cross-sectional study of 208 residents (94 women, 114 men) of a cereal-producing region in Saskatchewan, Canada, during spring herbicide application, 1996. The ANA were assayed in serum by indirect immunofluorescence on HEp-2 cells. Bromoxynil was measured in plasma by gas chromatography/mass spectrometry analysis. Associations were explored between ANA detection and detection of bromoxynil in plasma, self-reported use of bromoxynil and other pesticides, farming exposures, gender, age, body mass index (BMI), and residency. The mean age (SD) of the participants was 50.8 (13.6) yr [women: 49.7 (13.5) yr, men: 51.6 (13.6) yr]. ANA prevalence was 37.5% (women: 39.4%, men: 36%,) at 1:40 serum dilution, 17.3% (women: 20.2%, men: 14.9%) at 1:80, and 10.1% (women: 13.8%, men: 7%) at 1:160. In the multiple-variable Generalized Estimating Equation (GEE) logistic regression analyses, female gender was a positive predictor of ANA detection and gender differences were observed in the relative importance of other study factors. None of the variables examined in the multiple-variable GEE analysis were statistically significant predictors of ANA detection for women. For many of these variables, however, the point estimates for women are similar to those seen in men. For men, with adjustment for age, ANA presence was inversely associated with detection of concentrations of bromoxynil in winter or spring samples and recent occupational use of 2,4-dichlorophenoxyacetic acid, and the positive ANA predictors included having a BMI in the obese (BMI > 30.04 kg/m2) category, recent occupational use of trifluralin or fungicides, and current exposure to oilseed, poultry, or dairy production. The inverse association between ANA detection and bromoxynil exposure observed in farmers in this study is consistent with earlier empirical observations that certain pesticides may suppress immune function. Further research is needed to examine whether these findings are confirmed in other populations and to elucidate the biological mechanisms involved.
Asunto(s)
Agricultura , Anticuerpos Antinucleares/sangre , Enfermedades Autoinmunes/sangre , Nitrilos/sangre , Plaguicidas/sangre , Salud Rural , Anticuerpos Antinucleares/inmunología , Enfermedades Autoinmunes/epidemiología , Enfermedades Autoinmunes/inmunología , Estudios Transversales , Exposición a Riesgos Ambientales , Monitoreo del Ambiente/métodos , Monitoreo Epidemiológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nitrilos/inmunología , Plaguicidas/inmunología , Saskatchewan/epidemiología , Estaciones del Año , Estudios SeroepidemiológicosRESUMEN
The parasitic fungus, Metarhizium anisopliae, is non-pathogenic to humans and licensed for indoor control of cockroach infestation. An important reason for the elimination of this vermin is that sensitisation to cockroaches is associated with asthma. Previously M. anisopliae has been shown to cause allergic- and asthma-like responses in mice and in the present study we have examined the adjuvant activity of M. anisopliae on the allergic response to the model allergen ovalbumin (OVA) in a mouse model. Levels of OVA-specific IgE, IgG1 and IgG2a in serum were measured and the weight and cell number of the excised popliteal lymph node were determined. Mice primed with mycelium+OVA and boosted with OVA had increased anti-OVA IgE and IgG1 levels compared with mice primed with OVA alone or mycelium. Priming with M. anisopliae (as mycelium or MACA) increased weight or cell number of the excised PLNs. These results suggest that M. anisopliae has the ability to increase an allergic response to an allergen and consequently, may worsen allergy in susceptible individuals.