RESUMO
This study investigated the immunotoxic effects of the mycotoxin nivalenol (NIV) using antigen-presenting cells and a mouse model of atopic dermatitis (AD). In vitro experiments were conducted using a mouse macrophage cell line (RAW 264.7) and mouse dendritic cell line (DC 2.4). After cells were exposed to NIV (0.19-5 µmol) for 24 h, the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα) was quantified. To further investigate the inflammatory cytokine production pathway, the possible involvement of mitogen-activated protein kinase (MAPK) pathways, such as ERK1/2, p-38, and JNK, in NIV exposure was analyzed using MAPK inhibitors and phosphorylation analyses. In addition, the pro-inflammatory effects of oral exposure to NIV at low concentrations (1 or 5 ppm) were evaluated in an NC/Nga mouse model of hapten-induced AD. In vitro experiments demonstrated that exposure to NIV significantly enhanced the production of TNFα. In addition, it also directly induced the phosphorylation of MAPK, indicated by the inhibition of TNFα production following pretreatment with MAPK inhibitors. Oral exposure to NIV significantly exacerbated the symptoms of AD, including a significant increase in helper T cells and IgE-produced B cells in auricular lymph nodes and secretion of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, compared with the vehicle control group. Our findings indicate that exposure to NIV directly enhanced the phosphorylation of ERK1/2, p-38, and JNK, resulting in a significant increase in TNFα production in antigen-presenting cells, which is closely related to the development of atopic dermatitis.
Assuntos
Citocinas , Dermatite Atópica , Tricotecenos , Animais , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Tricotecenos/toxicidade , Tricotecenos/administração & dosagem , Camundongos , Administração Oral , Citocinas/metabolismo , Células RAW 264.7 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Animais de Doenças , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Fosforilação , Masculino , Fator de Necrose Tumoral alfa/metabolismo , FemininoRESUMO
Aflatoxins (AFs) are known to be oncogenic mycotoxins. This study investigated the mitigation effects of lactic acid bacteria (LAB) isolated from four types of vegetable, cucumber, Chinese cabbage, Japanese radish and eggplant, which are used to make Japanese traditional fermented pickles, on AFs. Using aflatoxin M1 (AFM1) binding assay for screening, four representative strains were selected (one from each vegetable) from total 94 LAB strains, based on the highest binding ratio. The ranges of the binding ratio of these representative strains to aflatoxin B1 (AFB1), aflatoxin B2, aflatoxin G1, aflatoxin G2 and AFM1 were 57.5%-87.9% for the LAB strain derived from cucumber, 18.9%-43.9% for the LAB strain derived from Chinese cabbage, 26.4%-41.7% for the LAB strain derived from Japanese radish, and 15.0%-42.6% for the LAB strain derived from eggplant. The strains isolated from cucumber, Chinese cabbage, Japanese radish and eggplant were identified as Lactococcus lactis subsp. lactis, Weissella cibaria, Leuconostoc mesenteroides and Leu. mesenteroides, respectively. An in vitro binding assay of the four strains under acidic conditions showed that the number of living bacteria decreased, while the binding ratio increased in some strains, suggesting that the LAB maintained their capacity to bind aflatoxins even in an environment that imitated the stomach. An in vivo experiment using L. lactis subsp. lactis derived from cucumber revealed that the bacteria significantly inhibited the absorption of AFB1 into blood. These results showed that the LAB used for Japanese vegetable pickles was an effective binding agent of AFs and suggested that they might play a role in mitigating AF absorption.
Assuntos
Aflatoxinas , Lactobacillales , Weissella , VerdurasRESUMO
Deoxynivalenol (DON) contamination in food is a public health concern; however, the effect of DON exposure on immune disorders including allergies remains unclear. The aim of this study is to elucidate the effect of oral exposure to DON on pro-inflammatory and pro-pruritic responses in a mouse model of allergic dermatitis, which was generated by topical application of toluene-2,4-diisocyanate (TDI), a hapten that induces type-2 helper T cells. To evaluate acute exposure to DON, the mice were orally administered vehicle alone, 0.1 mg/kg DON, or 0.3 mg/kg DON 48, 24, and 1 h before the final challenge with TDI. To study subacute exposure, the mice were fed DON-contaminated rodent diet (0.3 ppm) during the experimental period. After the itch behavior and ear-swelling response were monitored, the serum, auricular lymph node, and skin tissue were collected for analyzing immunocyte differentiation, cytokine determination, and histological changes. Acute oral administration of DON significantly enhanced pro-inflammatory responses including ear-swelling response, immunocyte infiltration, and cytokine productions. Histological evaluation supported the occurrence of pro-inflammatory responses. In contrast, acute DON exposure only slightly increased itch behavior. Subacute oral exposure to DON significantly up-regulated the inflammatory responses, but showed almost no effect on pruritic response. In vitro evaluation in dendritic cells and keratinocytes indicated that DON pre-exposure induced a dose-dependent significant increase in cytokine production. Our results imply that both acute and subacute exposures to DON are associated with pro-inflammatory responses in cutaneous allergy.
Assuntos
Citocinas/metabolismo , Dermatite Alérgica de Contato/etiologia , Mediadores da Inflamação/metabolismo , Prurido/induzido quimicamente , Pele/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Tricotecenos/toxicidade , Administração Oral , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Dermatite Alérgica de Contato/patologia , Modelos Animais de Doenças , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Camundongos Endogâmicos BALB C , Prurido/imunologia , Prurido/metabolismo , Prurido/patologia , Pele/imunologia , Pele/metabolismo , Pele/patologia , Células THP-1 , Células Th2/imunologia , Células Th2/metabolismo , Tolueno 2,4-Di-Isocianato , Tricotecenos/administração & dosagemRESUMO
The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.
Assuntos
Infecções por Escherichia coli/diagnóstico , Antígenos O/imunologia , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/imunologia , Ressonância de Plasmônio de Superfície , Anticorpos Antibacterianos/imunologia , Diagnóstico Precoce , Humanos , Limite de Detecção , Sorogrupo , Sorotipagem , Toxina Shiga/análiseRESUMO
Easy-to-use commercial kit-based enzyme-linked immunosorbent assays (ELISAs) have been used to detect neonicotinoid dinotefuran, clothianidin and imidacloprid in Chinese chives, which are considered a troublesome matrix for chromatographic techniques. Based on their high water solubility, water was used as an extractant. Matrix interference could be avoided substantially just diluting sample extracts. Average recoveries of insecticides from spiked samples were 85-113%, with relative standard deviation of <15%. The concentrations of insecticides detected from the spiked samples with the proposed ELISA methods correlated well with those by the reference high-performance liquid chromatography (HPLC) method. The residues analyzed by the ELISA methods were consistently 1.24 times that found by the HPLC method, attributable to loss of analyte during sample clean-up for HPLC analyses. It was revealed that the ELISA methods can be applied easily to pesticide residue analysis in troublesome matrix such as Chinese chives.
Assuntos
Cebolinha-Francesa/química , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Inseticidas/análise , Neonicotinoides/análise , Cromatografia Líquida de Alta Pressão/métodos , Análise de Alimentos/métodos , Guanidinas/análise , Nitrocompostos/análise , Resíduos de Praguicidas/análise , Tiazóis/análiseRESUMO
A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of total amount of aflatoxin B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), using a mouse monoclonal antibody that shows similar reactivity to each of these AFs. The working range of the developed dc-ELISA was 50-230 pg/mL for AFB1, 50-270 pg/mL for AFB2, 60-390 pg/mL for AFG1 and 65-700 pg/mL for AFG2. The recovery of AFs from spiked roasted peanuts was 98ï¼ . Further, when 4 samples actually contaminated with AFB1, AFB2, AFG1 and AFG2 were examined, the results of dc-ELISA were highly correlated with the values assigned by the Food Analysis Performance Assessment Scheme. The developed dc-ELISA appears to be suitable for the determination of total AFs at concentrations around the maximum permitted level (10 µg/kg for all foods) in Japan.
Assuntos
Aflatoxinas/análise , Ensaio de Imunoadsorção Enzimática , Análise de Alimentos/métodos , Animais , Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos , Japão , CamundongosRESUMO
A surface plasmon resonance-based immunosensor (SPR-immunosensor) was developed for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to the O-antigen groups O26, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The polyclonal antibodies (PoAbs) generated against each of the STEC O-antigen types in rabbits were purified and were immobilized on the sensor chip at 0.5 mg/mL. The limit of detection for STEC O157 by the SPR-immunosensor was found to be 6.3 × 10(4) cells for 75 s. Each of the examined 10 O-antigens on the STECs was detected by the corresponding PoAb with almost no reaction to the other PoAbs. The detected STECs were sufficiently removed from the PoAbs using gelatin or agarose gel without deactivation of the PoAbs, enabling repeatable use of the sensor chip. The developed SPR-immunosensor can be applied for the detection of multiple STEC O-antigens. Furthermore, the new antigen removal technique using the gel displacement approach can be utilized with various immunosensors to improve the detection of pathogens in clinical and public health settings.
Assuntos
Escherichia coli O157/metabolismo , Imunoensaio , Antígenos O/análise , Ressonância de Plasmônio de Superfície , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Limite de Detecção , Análise em Microsséries , Antígenos O/imunologia , Toxina Shiga/metabolismoRESUMO
Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.
Assuntos
Anticorpos Monoclonais/química , Cromatografia de Afinidade/instrumentação , Imunoensaio/instrumentação , Ocratoxinas/análise , Acetonitrilas/química , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Cacau/química , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Hemocianinas/administração & dosagem , Hemocianinas/química , Imunização Secundária , Imunoensaio/métodos , Concentração Inibidora 50 , Metanol/química , Camundongos , Camundongos Endogâmicos BALB C , Ocratoxinas/administração & dosagem , Ocratoxinas/química , Fenômenos de Química Orgânica , Cloreto de Sódio/química , SolventesRESUMO
BACKGROUND: With the aim of expanding the applicability of a kit-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide thiamethoxam, the ELISA was newly applied to three kinds of agricultural samples (green pepper, eggplant and spinach). To offer the ELISA as a screening analysis for thiamethoxam residues, a rapid and simple method of extraction by hand-shaking was used, and speed-up and simplification of the sample treatment before the ELISA analysis were examined. Finally, the validity of the ELISA combined with the proposed extraction method was verified against a reference high-performance liquid chromatography (HPLC) method using real-world agricultural samples. RESULTS: There were no marked matrix effects derived from green pepper and eggplant extracts. On the other hand, although the effect due to a pigment in spinach extract on the assay performance was significant, it was effectively avoided by increasing the dilution level of the spinach extract. For thiamethoxam-spiked samples, acceptable recoveries of 97.9-109.1% and coefficients of variation of 0.3-11.5% were obtained. Inspection of the validity of the ELISA by comparison with the reference HPLC method showed that the two analytical results were very similar, and a high correlation was found between them (r>0.997). CONCLUSION: The evaluated ELISA combined with hand-shaking extraction provided a rapid and simple screening analysis that was quantitative and reliable for the detection of thiamethoxam in complex agricultural products.
Assuntos
Produtos Agrícolas/química , Contaminação de Alimentos/análise , Inseticidas/química , Nitrocompostos/química , Oxazinas/química , Resíduos de Praguicidas/química , Tiazóis/química , Ensaio de Imunoadsorção Enzimática , Neonicotinoides , TiametoxamRESUMO
Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food. A membrane-based yeast two-hybrid system using split-ubiquitin was successfully used to detect practically important concentration ranges of caffeine and aflatoxins using separated variable regions of an antibody. Moreover, enterohemorrhagic Escherichia coli O157 was detected using a specific single-chain antibody, in which Zymolyase was added to partially destroy the cell wall. The incorporation of secreted Cypridina luciferase reporter further simplified the signal detection procedures without cell lysis. The methodology is more cost-effective and faster than using mammalian cells. The ability to detect various targets renders Patrol Yeast a valuable tool for ensuring food and beverage safety and addressing other environmental and technological issues.
RESUMO
Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.
Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Guanidinas/análise , Inseticidas/análise , Nitrocompostos/análise , Tiazóis/análise , Cromatografia Líquida de Alta Pressão , Guanidinas/imunologia , Inseticidas/imunologia , Neonicotinoides , Nitrocompostos/imunologia , Tiazóis/imunologiaRESUMO
Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.
Assuntos
Fusarium , Micotoxinas , Tricotecenos , Camundongos , Animais , Micotoxinas/análise , Anticorpos Monoclonais , Tricotecenos/análise , Fusarium/metabolismoRESUMO
Previously, researchers have demonstrated that mycotoxin deoxynivalenol (DON) significantly enhances immunocyte activation. However, the interaction between DON exposure and immune disorders remains unclear. In this study, we aimed to investigate whether acute and subacute oral exposure to DON exacerbates the development of respiratory allergy using a mite allergen (Dermatophagoides farina, Derf)-induced mouse model of asthma. The direct relationship between DON exposure and asthma development was examined following acute oral DON administration (0, 0.1, or 0.3 mg/kg body weight), immediately before the final mite allergen challenge. Simultaneously, the influence of subacute oral exposure via low dose DON contaminated wheat (0.33 ppm) was evaluated using the same settings. To detect the proinflammatory effects of DON exposure, we examined the total and Derf-specific serum IgE levels, histology, number of immunocytes, and cytokine and chemokine secretion. Acute oral DON significantly enhanced the inflammatory responses, including cellular infiltration into bronchoalveolar lavage fluid, infiltration of immunocytes and cytokine production in local lymph nodes, and cytokine levels in lung tissues. Corresponding proinflammatory responses were observed in a mouse group exposed to subacute oral DON. In vivo results were validated by in vitro experiments using the human bronchial epithelial (BEAS-2B) and human eosinophilic leukemia (EOL-1) cell lines. Following exposure to DON, the secretion of interleukin (IL)-1ß, IL-6, IL-8, and/or tumor necrosis factor-α in BEAS-2B cells, as well as EoL-1 cells, increased significantly. Our findings indicate that DON exposure is significantly involved in the proinflammatory response observed in respiratory allergy.
Assuntos
Asma , Tricotecenos , Animais , Asma/induzido quimicamente , Citocinas , Dermatophagoides farinae , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Tricotecenos/toxicidadeRESUMO
Six pesticides, azoxystrobin, boscalid, chlorfenapyr, imazalil, isoxathion, and nitenpyram, were simultaneously detected by using a surface plasmon resonance (SPR) immunosensor. The working ranges were 3.5 - 19 ng/mL for azoxystrobin, 4.5 - 50 ng/mL for boscalid, 2.5 - 25 ng/mL for chlorfenapyr, 5.5 - 50 ng/mL for imazalil, 3.5 - 50 ng/mL for isoxathion, and 8.5 - 110 ng/mL for nitenpyram. They showed adequate recovery results in tomato samples: 104 - 116% for azoxystrobin, 94 - 101% for boscalid, 90 - 112% for chlorfenapyr, 96 - 106% for imazalil, 107 - 119% for isoxathion, and 104 - 109% for nitenpyram. The correlation coefficient with liquid chromatography (HPLC or LC-MS/MS) using vegetable samples also agreed well: 0.91 - 0.99 as R2 without strong bias, except for nitenpyram for which the SPR immunosensor sensitivity was too low. The SPR immunosensor will have high applicability for pesticide residue analyses in vegetable samples.
Assuntos
Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Praguicidas/análise , Técnicas Biossensoriais , Resíduos de Praguicidas/análise , Ressonância de Plasmônio de Superfície/métodosRESUMO
An immunosensor based on surface plasmon resonance was developed for detection of c-Kit expressed on a cell surface. The combination of the antibody solution modified with gelatin before immobilization to the sensor chip and its blocking with gelatin drastically decreased the nonspecific reaction. The condition may be useful for the detection of various cells by using antibody against cell surface marker including the c-Kit.
Assuntos
Regulação da Expressão Gênica , Imunoensaio/métodos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Proto-Oncogênicas c-kit/imunologiaRESUMO
An immunosensor based on surface plasmon resonance was applied to detect mast cells expressing c-Kit. Sufficient detection of the mast cells was achieved by covalent immobilization of gelatin firstly on the sensor surface and followed by covalent binding of the anti-c-Kit antibody to lysine residues in the gelatin molecules through bis(sulfosuccinimidyl)suberate (BS3) treatment. By using BS3, which is a homo-bifunctional reagent, the lysine residues of the anti-c-Kit antibody easily bound to the lysine residues of the gelatin in the physiological condition. The lower limit of detection was 104 cells/mL.
Assuntos
Anticorpos Imobilizados/química , Gelatina/química , Regulação da Expressão Gênica , Imunoensaio/métodos , Mastócitos/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Imobilizados/imunologia , Ácidos Decanoicos/química , Humanos , Limite de Detecção , Mastócitos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/imunologia , Succinimidas/químicaRESUMO
A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.
RESUMO
A simultaneous immunosensor based on surface plasmon resonance (SPR) was developed for determination of 3 pesticides -boscalid, clothianidin and nitenpyram- instead of the direct competitive enzyme-linked immunosorbent assays (dcELISAs) widely used as individual determination methods. Carboxy groups that introduced compounds to their pesticides were designed, and conjugates of them and bovine serum albumin were immobilized onto separate channels of the same sensor chip. When a mixture of 3 monoclonal antibodies reacted to each pesticide, and 3 pesticides were injected into the SPR immunosensor, each channel showed specific reactivity at 15 - 93 ng mL-1 for boscalid, 6.7 - 27 ng mL-1 for clothianidin, and 7.3 - 62 ng mL-1 for nitenpyram. Recovery tests using vegetables spiked with a mixture of 3 pesticides showed good results: 75 - 90%, 88 - 104%, and 72 - 105%, respectively, with a high correlation to results of the dcELISAs. The SPR immunosensor would be useful for the determination of pesticide residues in vegetables.
Assuntos
Compostos de Bifenilo/análise , Ensaio de Imunoadsorção Enzimática , Guanidinas/análise , Neonicotinoides/análise , Niacinamida/análogos & derivados , Resíduos de Praguicidas/análise , Tiazóis/análise , Verduras/química , Técnicas Biossensoriais , Estrutura Molecular , Niacinamida/análise , Ressonância de Plasmônio de SuperfícieRESUMO
The cell adhesion molecule (CADM) family of the immunoglobulin superfamily (IgSF) comprises four members, CADM1-CADM4, and participates in the formation of epithelial and synaptic adhesion through cell-cell homophilic and heterophilic interactions. To identify the partners that interact with each member of the CADM family proteins, we set up a platform for multiple detection of the extracellular protein-protein interactions using surface plasmon resonance imaging (SPRi) and analyzed the interactions between the CADM family proteins and 10 IgSF of their structurally related cell adhesion molecules. SPRi analysis identified a new interaction between CADM1 and CADM4, where this heterophilic interaction was shown to be involved in morphological spreading of adult T-cell leukemia (ATL) cells expressing CADM1 when incubated on CADM4-coated glass. Moreover, class-I MHC-restricted T-cell-associated molecule (CRTAM) was identified to show the highest affinity to CADM1 among its binding partners by comparing the dissociation constants calculated from the SPR sensorgrams. These results suggest that the SPRi platform would provide a novel screening tool to characterize extracellular protein-protein interactions among cell-surface and secreted proteins, including IgSF molecules.
RESUMO
This work describes the analytical performance of the enzyme-linked immunosorbent assay (ELISA) for the fungicide chlorothalonil to effectively exploit as a simple and rapid detection system for pesticide residue on the scenes of the agricultural production and distribution. This ELISA represents the satisfactory analytical characteristics (I50 value, 0.34 ng/g; limit of detection, 0.052 ng/g) to detect chlorothalonil at the regulatory values or thereabout in a sample. Noticeable cross-reactivities were shown with two fungicides, fthalide (58.8%) and pentachloronitrobenzene (quintozene) (20.0%), and some non-agrochemicals such as tetrachloroterephthalonitrile (96.8%) and tetrachlorophthalonitrile (68.3%). The influence of three organic solvents (methanol, acetone, and acetonitrile) used as extractants for chlorothalonil residue was evaluated, with the result that methanol was the most suitable solvent for the ELISA, and the final concentration in the well could be up to 5% (v/v) without any negative influence on the ELISA. It has been possible to directly analyze chlorothalonil residue only by giving dilution of each sample extract with water prior to the ELISA analysis. The average recovery values from the spiked samples by the ELISA were between 101.7 and 113.6% with the average coefficients of variation between 2.6 and 5.9%. Although the results obtained from the ELISA correlated well with those from the reference GC/MS methods for all agricultural samples (r>0.98), the linear function inclined to the ELISA results because of loss during complex sample preparations for GC/MS analysis. Nevertheless, the results demonstrated that the proposed ELISA is a reliable, cost-effective, and rapid quantitative method for chlorothalonil residue.