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1.
J Environ Sci Health B ; 55(3): 250-256, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31694450

RESUMO

Paclobutrazol (PBZ) is a plant growth regulator (PGR) widely used in fruit and vegetable cultivation. However, due to the severe toxicity of PBZ, a sub-ppm level maximum residue limit (MRL) was established worldwide. Therefore, it is significant to propose a rapid, sensitive and high throughput screening method for monitoring the PBZ residues in foods. In this study, a simple and sensitive indirect competitive Enzyme-linked immunosorbent assay (icELISA) was established for PBZ detection in fruits basing polyclonal antibody. For both economy and pollution prevention, a microwave-solvent-free method was used to synthesize the PBZ hapten with high efficiency. The detection conditions, such as coating antigen concentration, antibody concentration, organic reagent concentration, ionic strength and pH, were optimized. Under the optimized conditions, this method showed high sensitivity and specificity. The detection range is 1.27-138.23 ng/mL, half-maximum inhibition concentration (IC50) is 13.26 ng/mL, and the IC20 was lower than the reported ELISAs for PBZ. Additionally, this method had high accuracy and precision. The recoveries were ranged from 88.78% to 96.80% in PBZ spiked apple samples with RSD below 4%. All the results showed that the polyclonal antibody based icELISA could be useful for PBZ screening in fruit samples.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Triazóis/análise , Triazóis/imunologia , Animais , Anticorpos/imunologia , Reações Cruzadas , Feminino , Análise de Alimentos/métodos , Frutas , Haptenos/imunologia , Limite de Detecção , Malus/química , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade
2.
PLoS One ; 14(10): e0223483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31596901

RESUMO

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.


Assuntos
Haptenos/imunologia , Hipersensibilidade/imunologia , Memória Imunológica , Otite/imunologia , Adjuvantes Imunológicos , Animais , Dinitrofluorbenzeno/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade/complicações , Masculino , Otite/etiologia , Oxazolona/imunologia , Suínos
3.
Med Hypotheses ; 131: 109303, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31443747

RESUMO

The problems of immunoprotection from the environmental chemical carcinogens are discussed. The main experimental argument pro active immunization against carcinogens is a possibility of specific mucosal antibodies (Abs) to inhibit the penetration of carcinogens from environment and to stimulate its excretion with the following decreasing of carcinogen-DNA adducts levels. Hypothesis of cancer immunostimulation after active immunization against carcinogens is based on a high cancer risk in persons with high levels of serum Abs specific to environmental carcinogens coupled with high levels of Abs to endogenous steroids stimulating the proliferation of target cells, for example, Abs to benzo[a]pyrene together with Abs to estradiol. The active immunization could increase the cancer risk much more in those persons. The passive immunization could be an alternative safe approach to avoid this problem.


Assuntos
Carcinógenos Ambientais/toxicidade , Neoplasias/prevenção & controle , Vacinação , Animais , Anticorpos/sangue , Especificidade de Anticorpos , Autoanticorpos/imunologia , Carcinógenos/toxicidade , Carcinógenos Ambientais/farmacocinética , Linhagem Celular Tumoral , Cocarcinogênese , Adutos de DNA/imunologia , Feminino , Haptenos/imunologia , Humanos , Imunização Passiva , Masculino , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/prevenção & controle , Neoplasias Hormônio-Dependentes/induzido quimicamente , Neoplasias Hormônio-Dependentes/imunologia , Neoplasias Hormônio-Dependentes/prevenção & controle , Ratos , Ratos Endogâmicos , Risco , Esteroides/imunologia , Vacinação/efeitos adversos
4.
Analyst ; 144(17): 5172-5178, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31343645

RESUMO

Personalized medicine is pushing forward new diagnostic techniques to aid in controlling drug therapeutic levels and their toxic effects. This study aims to develop a high-throughput screening method for therapeutic drug monitoring (TDM) and occupational exposure of cyclophosphamide (CP), an alkylating agent used as a chemotherapeutic and immunosuppressive drug. In order to achieve this goal, an immunizing hapten that exposes the cyclophosphamide moiety has been designed for the first time. Antibodies produced against this hapten have been used to develop an indirect competitive ELISA for the quantification of CP with high specificity and low cross-reactivity with some metabolites and other anticancer drugs. The assay obtained showed a LOD of 22 ± 6 nM in serum samples, with concentrations much below the blood CP levels of patients treated with the drug. A new tool for the detection and quantification of CP is provided which could be relevant for future pharmacokinetic studies and for therapeutic index improvement.


Assuntos
Ciclofosfamida/sangue , Animais , Anticorpos/imunologia , Ciclofosfamida/imunologia , Monitoramento de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Haptenos/química , Haptenos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoensaio/métodos , Limite de Detecção , Fosforamidas/síntese química , Fosforamidas/imunologia , Coelhos
5.
Food Chem ; 293: 144-150, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151594

RESUMO

Nobiletin, a polymethoxyflavone mainly found in citrus fruits, have been reported to exhibit various beneficial biological activities for human health. It is an important bioactive compound in traditional Chinese medicine, Pericarpium Citri Reticulatae and Fructus Aurantii. To detect the contents of nobiletin in citrus and herb samples, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on monoclonal antibodies. It possessed a median inhibition concentration (IC50) of 2.43 ±â€¯0.19 ng/mL and a working range of 0.52-12.3 ng/mL. The assay exhibited the average recoveries of 72.5-85.3% in citrus peel, pulp and juice samples. Moreover, eleven citrus cultivars samples and four herb samples were also detected by the icELISA. The nobiletin content varied in different citrus cultivars samples and herb samples, which were confirmed by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS). These results indicated that the developed immunoassay was suitable for detecting nobiletin in citrus and herb samples.


Assuntos
Citrus/química , Flavonas/análise , Anticorpos Monoclonais/imunologia , Citrus/metabolismo , Ensaio de Imunoadsorção Enzimática , Flavonas/química , Flavonas/imunologia , Frutas/química , Frutas/metabolismo , Sucos de Frutas e Vegetais/análise , Haptenos/química , Haptenos/imunologia , Medicina Tradicional Chinesa
6.
Anal Bioanal Chem ; 411(20): 5255-5265, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31119346

RESUMO

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 µg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. Graphical abstract.


Assuntos
Anticorpos Monoclonais/imunologia , Crustáceos/química , Dietilestilbestrol/análise , Ensaio de Imunoadsorção Enzimática/métodos , Estrogênios não Esteroides/análise , Haptenos/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Feminino , Concentração Inibidora 50 , Limite de Detecção , Coelhos
7.
J Immunol ; 203(1): 21-30, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31101666

RESUMO

Drug allergies occur when hapten-like drug metabolites conjugated to serum proteins, through their interactions with specific IgE, trigger allergic reactions that can be life threatening. A molecule termed covalent heterobivalent inhibitor (cHBI) was designed to specifically target drug hapten-specific IgE to prevent it from binding drug-haptenated serum proteins. cHBI binds the two independent sites on a drug hapten-specific Ab and covalently conjugates only to the specific IgE, permanently inhibiting it. The cHBI design was evaluated via ELISA to measure cHBI-IgE binding, degranulation assays of rat basophil leukemia cells for in vitro efficacy, and mouse models of ear swelling and systemic anaphylaxis responses for in vivo efficacy. The cHBI design was evaluated using two separate models: one specific to inhibit penicillin G-reactive IgE and another to inhibit IgE specific to a model compound, dansyl. We show that cHBI conjugated specifically to its target Ab and inhibited degranulation in cellular degranulation assays using rat basophil leukemia cells. Furthermore, cHBIs demonstrated in vivo inhibition of allergic responses in both murine models. We establish the cHBI design to be a versatile platform for inhibiting hapten/IgE interactions, which can potentially be applied to inhibit IgE-mediated allergic reactions to any drug/small-molecule allergy.


Assuntos
Anafilaxia/prevenção & controle , Basófilos/imunologia , Hipersensibilidade a Drogas/tratamento farmacológico , Naftalenos/metabolismo , Alérgenos/imunologia , Anafilaxia/etiologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Hipersensibilidade a Drogas/complicações , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Feminino , Haptenos/imunologia , Humanos , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/síntese química , Penicilinas/imunologia , Ligação Proteica , Ratos
8.
Biosensors (Basel) ; 9(2)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987327

RESUMO

Conjugation chemistry does not always provide adequate spatial orientation of hapten in immunogens for the best presentation of generic or individual epitopes. In the present study, the influence of unique and multiple orientations of immunizing hapten on the immune response repertoire was compared to select generic recognition system. The glycopeptides, teicoplanin (TPL) and ristomycin (RSM), were conjugated to BSA to produce immunogens with unique and multiple orientations of haptens. Polyclonal antibodies generated against TPL conjugated through a single site were of uniform specificity and demonstrated selective TPL recognition, regardless of the coating conjugates design. The sensitivity (IC50) of 4 enzyme-linked immunosorbent assays (ELISAs) for TPL varied little within the 3.5-7.4 ng/mL, with a dynamic range of 0.2-100 ng/mL. RSM was coupled to BSA through several glycoside sites that evoked a wider repertoire of response. This first described anti-RSM antibody was selective for RSM in homologous hapten-coated ELISAs with IC50 values in the range 4.2-35 ng/mL. Among the heterologous antigens, periodate-oxidized TPL conjugated to gelatine was selected as the best binder of generic anti-RSM fraction. The developed ELISA showed group recognition of glycopeptides RSM, TPL, eremomycin, and vancomycin with cross-reactivity of 37-100% and a 10-10,000 ng/mL dynamic range. Thus, multiple presentations of immunizing hapten help expand the repertoire of immune responses and opportunities for the selection of the required fine-specificity agent.


Assuntos
Antibacterianos/imunologia , Técnicas Biossensoriais , Glicopeptídeos/imunologia , Haptenos/imunologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Ensaio de Imunoadsorção Enzimática , Glicopeptídeos/genética , Haptenos/genética , Estrutura Molecular , Coelhos
9.
Contact Dermatitis ; 81(3): 174-183, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30957232

RESUMO

BACKGROUND: Over 4000 small chemicals have been identified as allergens capable of inducing skin sensitization. Many sensitizers are hypothesized to act as haptens producing novel antigens, which can be presented to T cells by human leukocyte antigens (HLAs). Recent studies suggest that some chemical allergens use hapten-independent mechanisms. OBJECTIVE: To determine whether molecular docking can identify HLA molecules that bind skin-sensitizing chemical allergens. METHODS: Structural models of HLA molecules were used as the basis for molecular docking of 22 chemical allergens. Allergens predicted to bind HLA-B*57:01 were tested for their ability to stimulate T cells by the use of proliferation and interferon-gamma enzyme-linked immunospot assays. RESULTS: Chemical allergens that did not satisfy the criteria for hapten activity in vitro were predicted to bind more strongly to common HLA isoforms than those with known hapten activity. HLA-B*57:01, which is an HLA allele required for drug hypersensitivity reactions, was predicted to bind several allergens, including benzyl benzoate, benzyl cinnamate, and benzyl salicylate. In in vitro T cell stimulation assays, benzyl salicylate and benzyl cinnamate were found to stimulate T cell responses from HLA-B*57:01 carriers. CONCLUSIONS: These data suggest that small-molecule skin sensitizers have the potential to interact with HLA, and show that T cell-based in vitro assays may be used to evaluate the immunogenicity of skin-sensitizing chemicals.


Assuntos
Alérgenos/química , Dermatite Alérgica de Contato/imunologia , Antígenos HLA-B/química , Haptenos/química , Perfumes/química , Alérgenos/imunologia , Alérgenos/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Proliferação de Células , Células Cultivadas , Cinamatos/química , Cinamatos/farmacologia , Antígenos HLA-B/imunologia , Haptenos/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Perfumes/farmacologia , Salicilatos/química , Salicilatos/farmacologia , Linfócitos T/fisiologia
10.
Food Chem ; 286: 234-240, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30827601

RESUMO

Avermectins (AVMs) are a group of anti-parasitic agents that have been widely used in food-producing animals. To monitor the residue of the AVMs, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure. Conjugates of 4″-HS-IVM/AVM on three different proteins were used to raise a broad-spectrum monoclonal antibody (mAb), 6D4, that had IC50 values for avermectin, ivermectin, eprinomectin and emamectin of 7.2, 10.4, 19.8 and 20.8 µg L-1, respectively. The limit of detection and limit of quantitation of this method for AVMs in various matrix samples ranged from 0.5 to 5.4 µg L-1 and 1.0 to 10.3 µg L-1, respectively. The recoveries of the samples spiked with AVMs were in the range of 78.1-110.5% with coefficients of variation below 14%. The ic-ELISA was applied to monitor ivermectin in the milk samples, and the results showed a good correlation with that of HPLC-MS/MS.


Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ivermectina/análogos & derivados , Leite/química , Animais , Antígenos/química , Antígenos/imunologia , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Haptenos/química , Haptenos/imunologia , Ivermectina/análise , Ivermectina/imunologia , Ivermectina/normas , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Padrões de Referência
11.
Food Chem ; 283: 359-366, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30722884

RESUMO

Alternariol (AOH), a secondary metabolites of the genus Alternaria, is a genotoxic mycotoxin. Because the previous "Mannich antigen" for AOH readily underwent unstable induction, a new AOH hapten was designed for inducing a high-quality antibody against AOH using a conjugate of the AOH carboxymethyl derivative with a carrier protein as the immunogen. A competitive indirect chemiluminescence enzyme immunoassay (ciCLEIA) for AOH was optimized and showed good sensitivity (limit of detection 0.068 ng/mL), a linear range (0.11-1.23 ng/mL), and negligible cross-reactivity with analogues. Average rates of recovery from spiked samples (juice and cereal) were between 72.7% and 115.8%, with coefficients of variation <14.3%. Results from ciCLEIA correlated well with those of high-performance liquid chromatography coupled with tandem mass spectrometry, meaning the proposed method might be an effective screening tool for detection of AOH in food samples.


Assuntos
Farinha/análise , Sucos de Frutas e Vegetais/análise , Técnicas Imunoenzimáticas/métodos , Lactonas/análise , Zea mays/química , Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão , Haptenos/química , Haptenos/imunologia , Lactonas/imunologia , Limite de Detecção , Medições Luminescentes , Micotoxinas/análise , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Zea mays/metabolismo
12.
J Immunol ; 202(4): 1045-1056, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30617225

RESUMO

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive effector CD8 T cells producing IFN-γ and IL-17- and IL-4-producing CD4 T cells that cannot mediate CHS. Although CXCR2-dependent Ly6G+ (neutrophil) cell recruitment into hapten-challenged skin is required to direct effector CD8 T cell infiltration into the challenge site to elicit CHS, 2,4-dinitrofluorobenezene (DNFB) sensitization of CXCR2-/- mice and neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during DNFB sensitization of neutrophil-depleted WT mice treated with anti-IL-12 mAb or neutrophil-depleted IL-12-/- mice. Neutrophil depletion during DNFB sensitization of WT mice markedly increased IL-12-producing hapten-primed dendritic cell numbers in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G (CG)-induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to DNFB challenge. Induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by s.c. injection of active, but not inactivated, CG during sensitization. Thus, hapten skin sensitization induces neutrophil release of CG that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of hapten-reactive CD4 T cells to IFN-γ-producing CHS effector cells.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Catepsina G/metabolismo , Células Dendríticas/metabolismo , Haptenos/metabolismo , Interleucina-12/biossíntese , Neutrófilos/enzimologia , Pele/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Feminino , Haptenos/imunologia , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Pele/imunologia
13.
Food Chem ; 280: 20-26, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30642487

RESUMO

To monitor the abuse of antibacterial synergists, a hapten, trimethoprim carboxylic derivative (TMPCOOH), was designed by using molecular modelling technology. A broad-spectrum monoclonal antibody (mAb) TMP/2G1 was prepared, for which the IC50 values of trimethoprim, diaveridine, aditoprim, baquiloprim, ormetoprim, and brodimoprim were 0.232, 0.527, 1.479, 4.354, 0.965, and 0.119 µg L-1, respectively. Based on the broad spectrum mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to determine the residues of antibacterial synergists. The limit of detection regarding the developed ic-ELISA for antibacterial synergists ranged from 0.025 to 1.126 µg L-1 in milk, honey and edible animal tissues. The recoveries ranged from 81.4% to 107.7%, with a coefficient of variation less than 20%. A good correlation (R2 = 0.994) between the ic-ELISA and HPLC-MS/MS showed the reliability of the developed ic-ELISA.


Assuntos
Antibacterianos/análise , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Mel/análise , Carne/análise , Leite/química , Animais , Antibacterianos/imunologia , Haptenos/química , Haptenos/imunologia , Limite de Detecção , Pirimidinas/análise , Pirimidinas/imunologia , Trimetoprima/análogos & derivados , Trimetoprima/análise , Trimetoprima/imunologia
14.
Clin Exp Dermatol ; 44(4): 411-413, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30239029

RESUMO

Allergic contact dermatitis (ACD) is an increasing problem in children. We sought to investigate the extent of haptens or contact allergens present in baby cosmetic products. Computer programming scripts were written to web-scrape UK supermarkets and pharmacies. In total, 438 unique 'baby products' were identified, and constituent ingredient information was extracted. Data were cross-referenced against 10 standardized patch test reference series. We found that 88% of products had at least one reference contact allergen. There was a mean of 2.21 (range 1-15) reference allergens per product. The most abundant compounds were parabens, fragrances, cetyl/steryl alcohol, methylisothiazolinone, sodium lauryl sulfate and lanolin alcohol. Branded products and those marketed as 'sensitive/gentle/organic/fragrance-free' appeared to contain a greater number of reference allergens than those not marketed as such. This study highlights the increasing number of cosmetic products targeted to children in the UK, and the extent of potential allergens present in these products.


Assuntos
Alérgenos/imunologia , Cosméticos/efeitos adversos , Dermatite Alérgica de Contato/epidemiologia , Haptenos/imunologia , Dermatite Alérgica de Contato/imunologia , Desinfetantes/efeitos adversos , Humanos , Lactente , Parabenos/efeitos adversos , Conservantes Farmacêuticos/efeitos adversos , Prevalência , Tiazóis/efeitos adversos , Reino Unido/epidemiologia
15.
Bioorg Med Chem ; 27(1): 125-132, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497790

RESUMO

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine's performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin's psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.


Assuntos
Analgésicos Opioides/imunologia , Proteínas de Choque Térmico HSP70/química , Haptenos/imunologia , Heroína/imunologia , Imunoconjugados/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos/química , Compostos de Alúmen/química , Animais , Proteínas de Bactérias/química , Escherichia coli/genética , Haptenos/química , Imunoconjugados/química , Masculino , Camundongos , Mycobacterium tuberculosis/química , Proteínas Recombinantes/química , Vacinas/síntese química , Vacinas/química
16.
Biosens Bioelectron ; 126: 590-595, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500774

RESUMO

A new immunoassay format using thermally induced defragmentation of some nitro-explosives with a high degree of selectivity is reported. Specific antibodies against three widely used explosives, 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) were generated by designing suitable haptens using geometry optimization modules. These in-house generated antibodies were used in a newly developed thermal mediated immunochemical biosensing technique which involves the binding of specific antibodies to respective nitro-explosives on a microtiter strip, resulting in the formation of specific immunocomplex. Heating the specific immuno-complex formed on microtiter wells resulted in thermal lysis of nitro-explosives to generate nitrite ions. These ions react with Griess reagent to form a colored chromophore which correlates the concentration of individual explosive in the sample. The present work fulfills the need for an improved explosive detecting system that is highly specific and capable of quickly determining the presence of nitrate containing explosives from a mixture pool.


Assuntos
Técnicas Biossensoriais , Substâncias Explosivas/isolamento & purificação , Triazinas/isolamento & purificação , Trinitrotolueno/isolamento & purificação , Anticorpos/química , Substâncias Explosivas/química , Haptenos/química , Haptenos/imunologia , Temperatura , Triazinas/química , Triazinas/imunologia , Trinitrotolueno/química , Trinitrotolueno/imunologia
17.
Food Chem ; 276: 707-713, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30409651

RESUMO

In this study, a sensitive monoclonal antibody (mAb) against toltrazuril (Tol) was developed based on a novel hapten. The 50% inhibitory concentrations (IC50) of toltrazuril and its metabolites ranged from 2.19 ng/mL to 4.21 ng/mL. Based on this mAb, a colorimetric paper-based sensor was developed for the rapid screening of Tol and its metabolites in samples. The proposed assay has cutoff values of <20 µg/kg for Tol and 50 µg/kg for Tol sulfone when evaluated with the naked eye, and the results could be obtained in 15 min. Quantitative results were obtained with a strip scan reader, with limits of detection <2.60 µg/kg for Tol and its metabolites in real samples. The sensitivity of both qualitative and quantitative detection meets the European Union requirements. Therefore, this strip assay provides a useful tool for the on-site detection and rapid initial screening of Tol and its derivatives in feed, egg, and chicken samples.


Assuntos
Ração Animal/análise , Colorimetria/métodos , Ovos/análise , Contaminação de Alimentos/análise , Triazinas/análise , Animais , Anticorpos Monoclonais/imunologia , Galinhas , Colorimetria/instrumentação , Feminino , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Haptenos/imunologia , Camundongos , Sensibilidade e Especificidade , Triazinas/imunologia , Triazinas/metabolismo
18.
Free Radic Biol Med ; 131: 318-331, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552998

RESUMO

The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-spin trapping technique, which combine the specificity of the spin trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.


Assuntos
Anticorpos Monoclonais/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Haptenos/química , Detecção de Spin/métodos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Galinhas , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/imunologia , Radicais Livres/análise , Haptenos/imunologia , Soros Imunes/química , Limite de Detecção , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/imunologia , Pirróis/química , Pirróis/imunologia , Coelhos , Marcadores de Spin , Vacinação
19.
Fish Shellfish Immunol ; 84: 781-786, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30393175

RESUMO

Affinity maturation of the antibody response, a process of antibody affinity increasing over response, is one of the key features of the mammalian immune system. However, the process is incompletely understood in teleost, including channel catfish (Ictalurus punctaus). In this study, IgM affinity maturation in channel catfish was investigated by estimating the kinetics of antibody affinity using ELISA and ELISPOT assays. Fish were immunized with a T-cell dependent antigen (TNP-KLH), and individual serum IgM antibody titers and affinities, and IgM+ antibody-secreting cells (ASCs) in peripheral blood were analyzed over a period of 14 weeks. A detectable serum anti-TNP response developed by 2-weeks post-immunization, and the maximal antibody production was observed by 6-weeks post-immunization. The average affinity of anti-TNP serum antibody increased consistently and reached the maximum by 10-weeks post-immunization. The increase of antibody affinity beyond the point of optimal antibody titer revealed that the affinity maturation of IgM antibody response occurred in channel catfish. Dissection of dynamics of individual affinity subpopulations indicated that a significant proportion of low affinity subpopulations appeared at early response, and high affinity subpopulations appeared predominantly at later, resulting in a 100-fold increase in affinity over response. Additional, TNP+ IgM+ ASCs was detected by 2-weeks post-immunization and achieved the maximal number by 6-weeks post-immunization. Using an inhibition ELISPOT assay, the findings of a consistent increase in the average affinity of secreted IgM antibody by peripheral blood ASCs, as the immune response progressed, confirmed the occurrence of the affinity maturation. Taken together, the results of this study indicated that affinity maturation occurred in channel catfish following immunization with a TD antigen TNP-KLH.


Assuntos
Haptenos/administração & dosagem , Hemocianinas/administração & dosagem , Ictaluridae/imunologia , Imunoglobulina M/imunologia , Linfócitos T/imunologia , Vacinação/veterinária , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , Haptenos/imunologia , Hemocianinas/imunologia , Imunoglobulina M/sangue , Vacinação/métodos
20.
Artigo em Inglês | MEDLINE | ID: mdl-30481558

RESUMO

INTRODUCTION: The use of new psychoactive substances as drugs of abuse has dramatically increased over the last years. Hallucinogenic phenethylamines gained particular popularity as they have both stimulating and psychedelic effects. Although generally perceived as safe, these illicit drugs pose a serious health risk; they have been linked to cases of severe poisoning or even deaths. Therefore, simple, cost-effective and reliable methods are needed for rapid determination of abused hallucinogens. METHODS: For this purpose, two haptens derived from 2C-H were designed, synthesized and subsequently attached to a carrier protein. Polyclonal antibodies obtained from a rabbit immunized with one of the prepared immunogens were used for the development of two immunoassays. RESULTS: In this study, a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of 2C-B and related hallucinogenic phenethylamines in urine were developed. The presented LFIA is primarily suitable for on-site monitoring as it is simple and can provide a visual evidence of 2C-B presence within a few minutes. Its reasonable sensitivity (LODLFIA = 15 ±â€¯7 ng mL-1) allows detection of the drug presence in urine after acute exposure. For greater accuracy, highly sensitive ELISA (LODELISA = 6 ±â€¯3 pg mL-1) is proposed for toxicological quantitative analyses of positive samples captured by the LFIA. DISCUSSION: The comparison of the ELISA with the well-established UHPLC-MS-MS method shows excellent agreement of results, which confirms good potential of the ELISA to be used for routine analyses of 2C-B and related hallucinogenic phenethylamines of both main sub-families.


Assuntos
Dimetoxifeniletilamina/análogos & derivados , Alucinógenos/urina , Drogas Ilícitas/urina , Imunoensaio/métodos , Detecção do Abuso de Substâncias/métodos , Dimetoxifeniletilamina/química , Dimetoxifeniletilamina/imunologia , Dimetoxifeniletilamina/urina , Feminino , Alucinógenos/química , Alucinógenos/imunologia , Haptenos/química , Haptenos/imunologia , Voluntários Saudáveis , Humanos , Drogas Ilícitas/química , Drogas Ilícitas/imunologia , Imunoensaio/economia , Masculino , Reprodutibilidade dos Testes , Detecção do Abuso de Substâncias/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
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