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1.
Food Chem ; 371: 131071, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34537613

RESUMO

A growing number of ß-agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many ß-agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 ß-agonists and analogues. Holographic and three-dimensional quantitative structure-activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on ß-agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert-butyl at C-2' epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of ß-agonist haptens.


Assuntos
Haptenos , Relação Quantitativa Estrutura-Atividade , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos , Imunoensaio
2.
Vet Dermatol ; 32(6): 605-e161, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34796565

RESUMO

BACKGROUND: In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain. OBJECTIVES: To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis. ANIMALS: Seven healthy beagles without skin problems. METHODS AND MATERIALS: Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR. RESULTS: The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin. CONCLUSION AND CLINICAL RELEVANCE: Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.


Assuntos
Dermatite de Contato , Doenças do Cão , Terapia Ultravioleta , Animais , Dermatite de Contato/veterinária , Doenças do Cão/radioterapia , Cães , Haptenos , Pele , Linfócitos T , Raios Ultravioleta/efeitos adversos , Terapia Ultravioleta/efeitos adversos , Terapia Ultravioleta/veterinária
3.
Environ Sci Technol ; 55(19): 12984-12993, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34551520

RESUMO

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 µg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.


Assuntos
Haptenos , Microcistinas , Animais , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Camundongos , Peptídeos Cíclicos
4.
J Agric Food Chem ; 69(34): 9957-9967, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34410117

RESUMO

We previously found that the immune response to haptens is positively correlated with molecular hydrophobicity. The antibodies used in immunoassays for capsaicinoids (CPCs) in waste oil suffer from low affinity and loose recognition to structural analogues. To address this issue, four new haptens (hapten1-4), maximally exposing the hydrophobic alkane chain (noncommon moiety of CPCs), were designed and expected to produce antibodies with high affinity and accurate recognition to CPCs based upon our findings. The assumption was first evidenced by computational chemistry and animal immunization successively. Compared with four reported haptens (hapten5-8) that expose the hydrophilic vanillyl amide moiety (common structure of CPCs and other vanillin alkaloids), antisera from hapten1-4 showed an approximately 1000-fold increase in affinity and significantly improved recognition profiles for CPCs. The molecular recognition study showed that the high affinity of the antibody from new haptens mainly originated from hydrophobic forces. An indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody from hapten1 was developed and exhibited limits of detection as low as 0.73-3.29 µg/kg for four CPCs in oils and with insignificant cross-reactivities for other eight vanillin alkaloids, which have been never achieved in previous reports.


Assuntos
Química Computacional , Haptenos , Animais , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Interações Hidrofóbicas e Hidrofílicas , Imunoensaio
5.
J Pharmacol Toxicol Methods ; 112: 107116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34403747

RESUMO

The high throughput method using dansyl cysteamine (HTS-DCYA™) is a sensitive and rapid in chemico approach to characterize skin sensitizers' thio-reactivity. The direct quantification of fluorescent hapten-DCYA adducts facilitates the rapid testing of pure chemicals as well as mixtures. Poor solubility in acetonitrile was occasionally observed and can represent a limitation. To enable the range of solvent options compatible with the testing, the effect of binary solvent systems on thio-reactivity and the HTS-DCYA classification was explored. The method's robustness was validated using five different solvent modifiers: water, DMSO, methanol, ethanol, and tetrahydrofuran. Some modifiers, viz., water and methanol, resulted in unexpected DCYA depletion, negatively affecting the thio-reactivity and classification of potential sensitizers. This undesirable, non-specific depletion was circumvented by optimizing the original HTS-DCYA™ method's workflow, resulting in a more robust and reliable thio-reactivity and hence classification with a binary solvent system. The results were validated for both pure compounds and plant extracts as examples of complex test samples. Based on the obtained results, the modified HTS-DCYA optimal conditions in the various solvent systems were established. Concentrations of modifiers up to 10% DMSO, 40% water, 40% EtOH, 60% MeOH, or 60% THF in acetonitrile were found acceptable for the modified protocol, with results comparable to the original method. The improved workflow with binary solvent systems provides significant advantages by expanding the applicability of the HTS-DCYA to a wider array of chemicals poorly soluble in acetonitrile.


Assuntos
Cisteamina , Pele , Haptenos , Solubilidade , Solventes
6.
Analyst ; 146(15): 4744-4747, 2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34226908

RESUMO

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.


Assuntos
Corantes Fluorescentes , Microscopia , Antígenos , Linfócitos B , Haptenos
7.
Front Immunol ; 12: 653102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34267746

RESUMO

Allergic reactions to drugs and chemicals are mediated by an adaptive immune response involving specific T cells. During thymic selection, T cells that have not yet encountered their cognate antigen are considered naive T cells. Due to the artificial nature of drug/chemical-T-cell epitopes, it is not clear whether thymic selection of drug/chemical-specific T cells is a common phenomenon or remains limited to few donors or simply does not exist, suggesting T-cell receptor (TCR) cross-reactivity with other antigens. Selection of drug/chemical-specific T cells could be a relatively rare event accounting for the low occurrence of drug allergy. On the other hand, a large T-cell repertoire found in multiple donors would underline the potential of a drug/chemical to be recognized by many donors. Recent observations raise the hypothesis that not only the drug/chemical, but also parts of the haptenated protein or peptides may constitute the important structural determinants for antigen recognition by the TCR. These observations may also suggest that in the case of drug/chemical allergy, the T-cell repertoire results from particular properties of certain TCR to recognize hapten-modified peptides without need for previous thymic selection. The aim of this review is to address the existence and the role of a naive T-cell repertoire in drug and chemical allergy. Understanding this role has the potential to reveal efficient strategies not only for allergy diagnosis but also for prediction of the immunogenic potential of new chemicals.


Assuntos
Dermatite de Contato/imunologia , Hipersensibilidade a Drogas/imunologia , Epitopos de Linfócito T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Reações Cruzadas , Epitopos de Linfócito T/imunologia , Haptenos/imunologia , Haptenos/metabolismo , Humanos , Peptídeos/imunologia , Peptídeos/metabolismo , Linfócitos T/metabolismo
8.
Methods Mol Biol ; 2350: 267-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331291

RESUMO

The UltraPlex method for multiplexed two-dimensional fluorescent immunohistochemistry is described, in which hapten tags conjugated to primary antibodies facilitate multiplexed imaging of four or more antigens per tissue section at once. Anti-hapten secondary antibodies labeled with fluorophores provide amplified signal for detection, which is accomplished using a standard fluorescent microscope or digital slide scanner. The protocol is rapid and straightforward and utilizes conventionally prepared tissue samples. The resulting staining is highly sensitive and specific, enabling high-resolution imaging of multiple cellular subtypes within tissue samples. Tumor cells and tumor-infiltrating lymphocytes are presented as examples. Multiple 4-plex-stained tissue samples can be digitally overlaid to create 8-plex (or more) high-content images, enabling visualization of distribution of complex cellular subtypes across tissues.


Assuntos
Imunofluorescência , Haptenos , Imuno-Histoquímica/métodos , Biomarcadores , Biomarcadores Tumorais , Análise de Dados , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Coloração e Rotulagem
9.
J Food Sci ; 86(7): 2851-2860, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34146404

RESUMO

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC50 value of 6.24 ng/ml in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and displayed no cross-reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23 µg/kg. The recoveries in spiked chicken were 79.2%-107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. PRACTICAL APPLICATION: RBV is a broad-spectrum antiviral drug, which is commonly used illegally in poultry farms. A high-affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken.


Assuntos
Anticorpos Monoclonais/imunologia , Galinhas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Haptenos/biossíntese , Ribavirina/análise , Animais , Anticorpos Monoclonais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Ribavirina/imunologia , Ribavirina/metabolismo
10.
J Toxicol Sci ; 46(5): 235-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33952800

RESUMO

There has been an increased demand to eliminate animal experiments and to replace the experiments with alternative tests for assessing the safety of cosmetics. The SH test is an in vitro skin sensitization test that evaluates the protein binding abilities of a test substance. Skin sensitization must be evaluated by multiple test methods. The SH test uses the same cell line and measuring instruments as the human Cell-Line Activation Test (h-CLAT), which is one of the test methods used to evaluate different key events and is listed in the OECD test guidelines. There are cost advantages to usher the SH test into facilities that are already running the h-CLAT. The SH test is conducted only at a facility that has developed the SH test because studies on the between-facility reproducibility and validity have not been performed. Therefore, to verify the transferability of the SH test and the between-facilities reproducibility, we evaluated the reproducibility of the SH test results at three facilities, including the development facility. After an initial round of testing, the protocol was refined as follows to improve reproducibility among the three facilities: i) determine the optimum pH range, ii) change the maximum applicable concentration of water-soluble substances, and iii) define the appropriate dispersion conditions for evaluating hydrophobic substances. These refinements markedly enhanced the between-facility reproducibility (from 76.0% to 96.0%) for the 25 substances evaluated in this study. This study confirmed that the SH test is an effective skin sensitization test method with high technical transferability and between-facility reproducibility.


Assuntos
Dermatite Alérgica de Contato , Haptenos/toxicidade , Laboratórios/normas , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Alternativas aos Testes com Animais/métodos , Alternativas aos Testes com Animais/normas , Linhagem Celular , Humanos , Reprodutibilidade dos Testes
11.
Artigo em Inglês | MEDLINE | ID: mdl-33938398

RESUMO

Phosphodiesterase type 5 (PDE-5) inhibitors are commonly used to treat erectile dysfunction. There is a problem with synthesis and illegal use of a wide range of analogues of the licenced drugs and a simple class-wide analytical method is required. In this work, based on structural modelling, we developed an immunological method using norneovardenafil as a hapten as it contains only the general sub-structure and the common features of sildenafil-like adulterants, such as hydrophobic centres, hydrogen-bond donor atoms and hydrogen-bond acceptor atoms. Thus theoretically it could induce production of antibody which could recognise multiple sildenafil-like adulterants. By immunising rabbits, a group-specific polyclonal antibody was obtained with the desired broad-spectrum molecular recognition performance against sildenafil-like adulterants. Then, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the detection of sildenafil-like adulterants in herbal spirit drinks. Under the optimised conditions, the icELISA method showed broad linear ranges for acetildenafil, sildenafil and vardenafil respectively of 0.7 to 27.7 µg/kg, 1.0 to 70.7 µg/kg and 1.5 to 22.7 µg/kg, with half-maximal inhibition concentration (IC50) values of 4.5 µg/kg, 8.3 µg/kg and 5.7 µg/kg, respectively. For eleven herbal spirit drinks, there was good agreement between total levels of sildenafil-like adulterants measured by icELISA and levels of each of four individual adulterants determined by LC-MS/MS. In short, the developed icELISA can be employed for rapid and simple screening for adulteration of herbal spirit drinks with sildenafil-like compounds.


Assuntos
Anticorpos/química , Bebidas Adoçadas Artificialmente/análise , Suplementos Nutricionais/análise , Aditivos Alimentares/análise , Contaminação de Alimentos/análise , Citrato de Sildenafila/química , Animais , Técnicas Biossensoriais , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Haptenos/química , Humanos , Limite de Detecção , Modelos Moleculares , Coelhos , Espectrometria de Massas em Tandem
12.
Bioorg Med Chem ; 41: 116225, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34034147

RESUMO

Unintentional overdose deaths related to opioids and psychostimulants have increased in prevalence due to the adulteration of these drugs with fentanyl. Synergistic effects between illicit compounds and fentanyl cause aggravated respiratory depression, leading to inadvertent fatalities. Traditional small-molecule therapies implemented in the expanding opioid epidemic present numerous problems since they interact with the same opioid receptors in the brain as the abused drugs. In this study, we report an optimized dual hapten for use as an immunopharmacotherapeutic tool in order to develop antibodies capable of binding to fentanyl-contaminated heroin in the periphery, thus impeding the drugs' psychoactive effects on the central nervous system. This vaccine produced antibodies with nanomolar affinities and effectively blocked opioid analgesic effects elicited by adulterated heroin. These findings provide further insight into the development of chemically contiguous haptens for broad-spectrum immunopharmacotherapies against opioid use disorders.


Assuntos
Overdose de Drogas/prevenção & controle , Fentanila/imunologia , Haptenos/imunologia , Heroína/efeitos adversos , Heroína/química , Vacinas/imunologia , Animais , Contaminação de Medicamentos , Overdose de Drogas/mortalidade , Fentanila/efeitos adversos , Fentanila/química , Humanos , Camundongos , Transtornos Relacionados ao Uso de Opioides
13.
J Med Chem ; 64(8): 4947-4959, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33825469

RESUMO

Hapten-specific endogenous antibodies are naturally occurring antibodies present in human blood. Herein, we investigated a new strategy in which small-molecule haptens were utilized as naturally occurring antibody binders for peptide half-life extension. The glucagon-like peptide 1 receptor agonist exendin 4 was site-specifically functionalized with the dinitrophenyl (DNP) hapten at the C-terminus via sortase A-mediated ligation. The resulting Ex4-DNP conjugates retained GLP-1 receptor activation potency in vitro and had a similar in vivo acute glucose-lowering effect comparable to that of native Ex4. Pharmacokinetic studies and hypoglycemic duration tests demonstrated that the Ex4-DNP conjugates displayed significantly elongated half-lives and improved long-acting antidiabetic activity in the presence of endogenous anti-DNP antibodies. In chronic treatment studies, once-daily administration of optimal conjugate 7 demonstrated more beneficial effects without prominent toxicity compared with Ex4. This strategy provides a new approach and represents an alternative to the well-established peptide-Fc fusion strategy to improve the peptide half-life and the therapeutic efficacy.


Assuntos
Anticorpos/sangue , Exenatida/química , Haptenos/química , Hipoglicemiantes/síntese química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Glicemia/análise , Diabetes Mellitus Experimental/tratamento farmacológico , Dinitrobenzenos/química , Dinitrobenzenos/imunologia , Desenho de Fármacos , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Teste de Tolerância a Glucose , Meia-Vida , Haptenos/imunologia , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL
14.
Food Chem ; 356: 129710, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33836353

RESUMO

We developed a sensitive and rapid lateral flow immunochromatographic (LFI) assay for the simultaneous detection of fipronil and its metabolites in eggs and cucumbers using gold nanoparticle (GNP)-labeled monoclonal antibodies (mAbs). Anti-fipronil mAbs (1B6) were produced using two haptens and identified by heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) with half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 0.46 ± 0.07 and 0.05 ± 0.01 ng mL-1, respectively. The developed LFI strip showed high sensitivity and specificity in the detection of fipronil with cut-off and visual limit of detection (vLOD) values of 10 and 0.25 ng mL-1, respectively. Furthermore, the application of LFI in the detection of fipronil-spiked egg and cucumber samples was validated by liquid chromatography tandem mass spectrometry (LC-MS/MS). Our developed LFI assay is suitable for detection of fipronil and its metabolites in real samples.


Assuntos
Análise de Alimentos/métodos , Imunoensaio/métodos , Pirazóis/análise , Animais , Cromatografia Líquida , Ovos/análise , Ouro/química , Haptenos/química , Limite de Detecção , Nanopartículas Metálicas/química , Pirazóis/metabolismo , Espectrometria de Massas em Tandem , Fatores de Tempo
15.
Anal Chim Acta ; 1161: 238180, 2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-33896564

RESUMO

Enzyme-linked immunosorbent assays (ELISAs) are essential for monitoring various biomarkers. Competitive and noncompetitive (sandwich) assay formats are used to determine hapten and macromolecule levels, respectively. Both formats require more sensitive detection of reporter enzymes for greater assay sensitivities. We previously reported the utility of wild-type Gaussia luciferase (wtGLuc) as a fusion partner with antibody single-chain Fv fragments (scFvs) for developing sensitive luminescent ELISAs. Here, we evaluated utility of NanoLuc luciferase (NLuc), a recently developed luciferase, as fusion partner with scFvs from the view of comparison with wtGLuc and a mutant of alkaline phosphatase (ALP'). Thyroxine (T4) and T4-labeled albumin were chosen as model haptenic and macromolecular antigens, respectively. An in-house-prepared anti-T4 scFv was fused with NLuc, wtGLuc, or ALP'. The scFv-NLuc fusion protein showed 47-fold and 29-fold lower limit of detection [LOD; 59 zmol (per assay)] than the wtGLuc- and ALP'-fusions, respectively. In a competitive T4 ELISA, the NLuc-fusion showed 9.3- and 6.3-fold lower LOD, (0.67 pg) than the wtGLuc- and ALP'-fusions, respectively, with a higher specificity in clinical applications. A typical colorimetric ELISA using a peroxidase-labeled second antibody showed 70-fold higher LOD than NLuc-based ELISA. Another advantage of the NLuc-fusion was shown in the sandwich assays; the LOD of T4-labeled albumin (5.0 fmol) was >6-fold lower than that of the other luminescent ELISAs. In an additional sandwich assay developed to count bacteriophage particles, NLuc enabled more sensitive determination than wtGLuc, whereas ALP' showed nearly equivalent performance. Its slowest alteration rate for light intensity after starting the enzyme reaction should enable robust batch-by-batch assay operations. Thus, we concluded that scFv-NLuc fusions serve as suitable probes in various types of immunoassays and may facilitate higher sensitivities with practical specificities.


Assuntos
Haptenos , Fragmentos de Imunoglobulinas , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Luciferases/genética , Proteínas Recombinantes de Fusão/genética
16.
Bioconjug Chem ; 32(4): 649-654, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33819023

RESUMO

Pretargeted imaging and radioimmunotherapy approaches are designed to have superior targeting properties over directly targeted antibodies but impose more complex pharmacology, which hinders efforts to optimize the ligands prior to human applications. Human embryonic kidney 293T cells expressing the humanized single-chain variable fragment (scFv) C825 (huC825) with high-affinity for DOTA-haptens (293T-huC825) in a transmembrane-anchored format eliminated the requirement to use other pretargeting reagents and provided a simplified, accelerated assay of radiohapten capture while offering normalized cell surface expression of the molecular target of interest. Using binding assays, ex vivo biodistribution, and in vivo imaging, we demonstrated that radiohaptens based on benzyl-DOTA and a second generation "Proteus" DOTA-platform effectively and specifically engaged membrane-bound huC825, achieving favorable tumor-to-normal tissue uptake ratios in mice. Furthermore, [86Y]Y-DOTA-Bn predicted absorbed dose to critical organs with reasonable accuracy for both [177Lu]Lu-DOTA-Bn and [225Ac]Ac-Pr, which highlights the benefit of a dosimetry-based treatment approach.


Assuntos
Engenharia Celular , Haptenos , Radioimunoterapia/métodos , Compostos Radiofarmacêuticos/química , Animais , Autorradiografia , Células HEK293 , Humanos , Camundongos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Biochemistry ; 60(19): 1447-1458, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33930269

RESUMO

Antibody recruiting molecules (ARMs) represent an important class of "proximity-inducing" chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten-specific serum antibody (Ab) (e.g., anti-dinitrophenyl (DNP)) and a cancer cell surface protein, enforcing their proximity. ARM anticancer efficacy depends on the formation of ARM:Ab complexes on the cancer cell surface, which activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes, namely, intrinsically low binding affinity and/or low concentrations of anti-hapten antibodies in human serum. To address this potential limitation, we previously developed a covalent ARM (cARM) chemical tool that eliminates the ARM:antibody equilibrium through a covalent linkage. In the current study, we set out to determine to what extent maximizing the stability of ARM:antibody complexes via cARMs enhances target immune recognition. We observe cARMs significantly increase target immune recognition relative to ARMs across a range of therapeutically relevant antibody concentrations. These results demonstrate that ARM therapeutic function can be dramatically enhanced by increasing the kinetic stability of ARM:antibody complexes localized on cancer cells. Our findings suggest that a) high titres/concentrations of target antibody in human serum are not neccessary and b) saturative antibody recruitment to cancer cells not sufficient, to achieve maximal ARM therapeutic function.


Assuntos
Anticorpos/química , Imunoterapia/métodos , Neoplasias/imunologia , Anticorpos/uso terapêutico , Formação de Anticorpos , Haptenos/química , Haptenos/imunologia , Humanos , Imunoglobulinas , Cinética , Neoplasias/tratamento farmacológico , Ligação Proteica/imunologia
18.
Toxicol In Vitro ; 74: 105154, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33774146

RESUMO

The human cell line activation test (h-CLAT) is an OECD approved (Test No. 442E) assay to identify novel skin sensitizers. h-CLAT simulates dendritic cell activation in the skin sensitization pathway and is based on the measurement of CD54 and CD86 overexpression on monocytic, leukemic THP-1 cells. However, the current h-CLAT markers show inconsistent results with moderate and weak sensitizers. Moreover, these markers have accessory roles in cell adhesion and signaling rather than a direct role in cellular inflammation. Therefore, we have explored other inflammation-related markers in this study. PBMCs comprises a mixture of cells that resemble the complex immunological milieu in adults and were primarily used to identify markers. PBMCs (n = 10) and THP-1 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB, strong) and NiCl2 (Ni, moderate) sensitizers or DMSO (control) and incubated for 24 h. The samples were subjected to RNA sequencing to obtain log2fold change in gene expression. DNCB and NiCl2 significantly upregulated 80 genes in both cell types. Of these, CD109, CD181, CD183, CLEC5A, CLEC8A & CD354 were experimentally validated. DNCB and Ni but not isopropyl alcohol (non-sensitizer) significantly induced the expression of all novel markers except CLEC8A. Moreover, the percentage induction of all novel markers except CLEC8A satisfied the OECD acceptance criteria. In summary, we identified five novel markers that may supplement the current repertoire of h-CLAT markers.


Assuntos
Alérgenos/toxicidade , Haptenos/toxicidade , Antígenos CD/genética , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas Tipo C/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Receptores de Superfície Celular/genética , Testes Cutâneos , Células THP-1
19.
Arch Toxicol ; 95(5): 1647-1657, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33715048

RESUMO

IL-1 functions as an essential pro-inflammatory mediator for the sensitisation of allergic contact dermatitis (ACD). However, studies conducted to date have typically used a limited number of haptens and examined their effects only on murine ACD or murine dendritic cells (DCs). It therefore remains unclear whether IL-1α and/or IL-1ß is produced in ACD induced by haptens other than those commonly used in mouse ACD models, and whether they are essential for sensitisation leading to ACD in humans. In addition, it is unclear whether human DCs also produce IL-1α or IL-1ß after stimulation by haptens in general. Here, we first demonstrated that 10 haptens (3 extreme, 1 strong, 3 moderate and 3 weak) increased both IL-1α mRNA and IL-1ß mRNA expression by the human monocyte cell line THP-1, a commonly used surrogate of DCs in in vitro skin sensitisation tests. Next, we constructed an in vitro skin sensitisation test using a stable IL-1ß reporter cell line, THP-G1b, and evaluated whether 88 haptens and 34 non-haptens increase IL-1ß reporter activity. We found that 94% of 77 haptens evaluated after considering their applicability domain and solubility in the chosen media stimulated reporter activity. These studies demonstrated that most haptens, irrespective of their potency, increased IL-1ß mRNA expression by THP-1 cells, confirming that human DCs also produce IL-1ß after stimulation by most haptens. The luciferase assay using THP-G1b cells is thus another skin sensitisation test based on the adverse outcome pathway with reasonable performance.


Assuntos
Interleucina-1alfa/metabolismo , Luciferases/metabolismo , Alérgenos , Alternativas aos Testes com Animais , Animais , Linhagem Celular , Células Dendríticas , Dermatite Alérgica de Contato/diagnóstico , Haptenos , Humanos , Técnicas In Vitro , Interleucina-8 , Camundongos , Monócitos , Regiões Promotoras Genéticas , Pele , Testes Cutâneos , Células THP-1
20.
Food Chem ; 355: 129598, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33765482

RESUMO

Derivatization is usually employed in immunoassay for detection of metabolites of nitrofurans and avoiding derivatization could be preferable to achieve an efficient screening. In the study, we designed four haptens of 4-hydroxybenhydrazide (HBH), the nifuroxazide metabolite. The effect of hapten structures on antibody affinity were evaluated and one monoclonal antibody was produced by using the Hapten C with a linear alkalane spacer arm. After optimization, an enzyme linked-immunosorbent assay (ELISA) was established with an 50% inhibition concentration of 0.25 ng mL-1 for HBH, which could ensure the direct detection of HBH without derivatization. The limit of detection of the ELISA for HBH was 0.12 µg kg-1 with the recoveries of 90.1-96.2% and coefficient of variation (CV) values lower than 9.1%. In conclusion, we produced several high affinity antibodies to HBH with new designed hapten and developed an icELISA for the direct detection of HBH without derivatization in chicken.


Assuntos
Anticorpos Monoclonais/imunologia , Galinhas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Hidroxibenzoatos/análise , Hidroxibenzoatos/metabolismo , Nitrofuranos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Formação de Anticorpos , Haptenos/química , Haptenos/imunologia , Concentração de Íons de Hidrogênio , Hidroxibenzoatos/imunologia , Camundongos , Concentração Osmolar , Temperatura
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