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1.
J Immunol Methods ; 532: 113728, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39059746

RESUMO

Immunoassay relies on antibodies, but traditional antibodies such as monoclonal antibody (mAb) require animal immunization and complex procedures. Single-chain variable fragment (scFv) becomes a potential alternative to mAb with advantages of the low cost, rapid and easy prepared. In the present study, we prepared scFvs against dihydroartemisinin (DHA) based on E. coli and HEK293T cell expression system, named MBP-scFv and scFv-Fc, respectively. Their properties were compared with the parent mAb. The calculated affinity constants of mAb, MBP-scFv and scFv-Fc were 2.1 × 108 L mol-1, 2.2 × 107 L mol-1 and 1.6 × 108 L mol-1, respectively. The half inhibitory concentration (IC50) of mAb, MBP-scFv and scFv-Fc were 1.16 ng mL-1, 2.15 ng mL-1 and 6.57 ng mL-1, respectively. Both the scFv showed less sensitive than the mAb based on the IC50. The cross-reactivities of MBP-scFv for artemisinin and artesunate exhibited similarities to the mAb, yet the cross-reactivities of scFv-Fc for these compounds exceeded those of the mAb significantly. The stability of the scFvs was ascertained to be maintained for over 5 days at room temperature, and for more than a month at both 4 °C and - 20 °C. After that, the indirect competitive enzyme-linked immunosorbent assays (icELISAs) based on the scFv from E. coli were used to detect the DHA content in eight drug samples, and the result was consistent with ultra-performance liquid chromatography simultaneously. Although scFv can be used for quantitative determination of drugs, but it still cannot completely replace mAb in immunoassay without evolution and modification.


Assuntos
Anticorpos Monoclonais , Artemisininas , Anticorpos de Cadeia Única , Artemisininas/imunologia , Artemisininas/farmacologia , Anticorpos de Cadeia Única/imunologia , Humanos , Anticorpos Monoclonais/imunologia , Células HEK293 , Afinidade de Anticorpos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/imunologia , Reações Cruzadas/imunologia , Antimaláricos/imunologia , Ensaio de Imunoadsorção Enzimática , Especificidade de Anticorpos
2.
Anal Bioanal Chem ; 408(22): 6003-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26873200

RESUMO

Artesunate is a frontline antimalarial drug for treating Plasmodium falciparum malaria. To produce specific antibodies to artesunate, the carboxyl group of artesunate was directly conjugated to carrier protein as the immunogen. A specific monoclonal antibody (mAb) 3D82G6 against artesunate was obtained by high-throughput screening of positive hybridoma clones. This monoclonal antibody had 4.0, 0.5, and 0.9 % cross reactivities with artemisinin, dihydroartemisinin, and artemether, respectively. A dipstick immunoassay was developed, and the indicator range for artesunate was 1000-2000 ng mL(-1). No interference was observed with artemisinin, dihydroartemisinin, artemether, and other commonly used antimalarial drugs for up to 20,000 ng mL(-1). The dipsticks were used for determination of artesunate contents in commercial drugs, and the results were agreeable with those determined by high-performance liquid chromatography. This dipstick, with its specificity and sensitivity for artesunate and simplicity to use, makes it a potential point-of-care device for rapid quality evaluation of artesunate-containing antimalarial drugs. Graphical Abstract Specific monoclonal antibody-based lateral flow dipstick for artesunate.


Assuntos
Anticorpos Imobilizados/química , Anticorpos Monoclonais/química , Antimaláricos/análise , Artemisininas/análise , Imunoensaio/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Antimaláricos/imunologia , Artemisininas/imunologia , Artesunato , Coloide de Ouro/química , Humanos , Hibridomas , Limite de Detecção , Malária Falciparum/tratamento farmacológico , Camundongos , Fitas Reagentes/análise
3.
Anal Chem ; 88(5): 2701-6, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26822789

RESUMO

Artemisinin, extracted from Artemisia annua, and its derivatives are important frontline antimalarials. To produce specific antibodies for the detection and quantification of artemisinin, artemisinin was transformed to 9-hydroxyartemisinin by microbial fermentation, which was used to prepare a 9-succinate artemisinin hapten for conjugation with ovalbumin. A monoclonal antibody (mAb), designated as 3H7A10, was selected from hybridoma cell lines which showed high specificity to artemisinin. No competitive inhibition was observed with artesunate, dihydroartemisinin, and artemether for up to 20,000 ng mL(-1). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed a concentration causing 50% of inhibition (IC50) for artemisinin as 2.6 ng mL(-1) and a working range of 0.6-11.5 ng mL(-1). The icELISA was applied for the quantification of artemisinin in crude extracts of wild A. annua and the study of pharmacokinetics of artemisinin in rat serum after intraperitoneal injection. The results were highly correlated with those determined by HPLC-UV analysis (R(2) = 0.9919). In comparison with reported antiartemisinin mAbs which have broad cross-reactivity with other artemisinin derivatives, the high specificity of 3H7A10 for artemisinin will enable development of methods for quantification of artemisinin in Artemisia plants and antimalarial drugs such as Arco and for pharmacokinetic studies.


Assuntos
Anticorpos Monoclonais/imunologia , Artemisininas/análise , Animais , Artemisia annua/química , Artemisininas/sangue , Artemisininas/imunologia , Ensaio de Imunoadsorção Enzimática , Hibridomas , Camundongos Endogâmicos BALB C , Ratos Sprague-Dawley
4.
Int J Immunopathol Pharmacol ; 28(3): 411-4, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26157064

RESUMO

Artemisia vulgaris L and Artemisia annua L (Chinese: qinghao) are similar plants of the Asterbaceae family. Artesunate, a semi-synthetic derivate of artemisin which is the active principle extract of the plant qinghao, has antimalarial properties. Some cases of severe allergic reactions to artesunate have been described. The purpose of this study was to evaluate the association between positive skin tests to Artemisia vulgaris L allergen and a preparation of injectable artesunate. A total of 531 children were skin prick tested with inhalants (including Artemisia vulgaris L), foods, and artesunate. Among the 59 patients positive to Artemisia vulgaris L only one child was also positive to artesunate. No child was positive to artesunate in those negative to Artemisia vulgaris L. We conclude that Artemisia vulgaris L sensitization is not associated with sensitization to artesunate; consequently, skin test to artesunate should not be carried out before using the drug considering the rare allergic reactions.


Assuntos
Alérgenos/imunologia , Artemisia/imunologia , Artemisininas/imunologia , Hipersensibilidade/imunologia , Adolescente , Artesunato , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Testes Cutâneos/métodos
5.
ScientificWorldJournal ; 2012: 978913, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22593717

RESUMO

Most investigations into the antimalarial activity of African plants are centered on finding an indigenous equivalent to artemisinin, the compound from which current frontline antimalarial drugs are synthesized. As a consequence, the standard practice in ethnopharmacological research is to use in vitro assays to identify compounds that inhibit parasites at nanomolar concentrations. This approach fails to take into consideration the high probability of acquisition of resistance to parasiticidal compounds since parasite populations are placed under direct selection for genetic that confers a survival advantage. Bearing in mind Africa's long exposure to malaria and extensive ethnobotanical experimentation with both therapies and diet, it is more likely that compounds not readily overcome by Plasmodium parasites would have been retained in the pharmacopeia and cuisine. Such compounds are characterized by acting primarily on the host rather than directly targeting the parasite and thus cannot be adequately explored in vitro. If Africa's long history with malaria has in fact produced effective plant therapies, their scientific elucidation will require a major emphasis on in vivo investigation.


Assuntos
Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Plantas Medicinais/química , Plasmodium/efeitos dos fármacos , África , Antimaláricos/imunologia , Artemisininas/imunologia , Artemisininas/uso terapêutico , Quimioterapia Combinada , Etnobotânica/métodos , Etnobotânica/tendências , Humanos , Malária/imunologia , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Fitoterapia/métodos , Fitoterapia/tendências , Plasmodium/imunologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia
6.
Anal Chem ; 84(4): 2002-8, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22260329

RESUMO

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 µg/mL to 40 µg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Antimaláricos/imunologia , Artemisininas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Anti-Infecciosos/imunologia , Afinidade de Anticorpos , Artemisia/química , Artesunato , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química
7.
Eur J Pharmacol ; 668(1-2): 6-14, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21756901

RESUMO

Artemisinins in combination with other antimalarial drugs remain the mainstay of current antimalarial armamentarium. It is interesting to note that many traditional drugs with antiprotozoal background can wield immunomodulation on the recipient's immune system in a positive or negative direction. Artemisinins also attribute immunomodulatory distensions. For instance, they demonstrate predominant immunosuppressive traits toward different immune components by particularly regulating the cellular proliferation and cytokine release, which indicates that they possess some additional mechanisms and features demanding deliberate attentions. This article reviews the data-based immunomodulatory effects of artemisinins on different immune cells including neutrophils, macrophages, splenocytes, T and B cells in conjunction with their therapeutic prospective with regard to inflammation, autoimmunity and delayed-type hypersensitivity.


Assuntos
Artemisininas , Sistema Imunitário , Animais , Artemisininas/imunologia , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Autoimunidade/efeitos dos fármacos , Humanos , Hipersensibilidade Tardia/tratamento farmacológico , Hipersensibilidade Tardia/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Malária/tratamento farmacológico , Malária/imunologia
8.
Int Immunopharmacol ; 10(9): 1055-61, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20601187

RESUMO

BACKGROUND: Artemisinin (ART) is a sesquiterpene lactone. Possessing an endoperoxide bridge is unique among antimalarial drugs, and now much attention is focused on the anti-cancer properties of ART. In this study we aimed at the immunomodulatory effects of artemisinin in the treatment of breast cancer in comparison to the conventional anti-cancer drug, cyclophosphamide (CTX). METHODS: We examined delayed-type hypersensitivity, antibody and IL-4 and IFN gamma production, tumour volume, tumour infiltrated regulatory T cells (Treg) and spleen lymphocyte proliferation assay. Briefly three groups of five 4-6 week old female Balb/c tumour-bearing mice (mouse mammary tumour) were treated with 2.8 mg/kg ART and 20mg/kg CTX intraperitoneally for 20 consecutive days. Tumour volume was measured using a digital vernier calliper (with accuracy of 0.01). Mice were sacrificed and percentage of tumour infiltrating Tregs were obtained using flow cytometry (BD, USA). Proliferation of splenocytes was obtained using BrdU proliferation assay (Roche). RESULTS: Our results showed that ART can reduce the number of Tregs in tumour stroma (P-value or=0.05) and control. Furthermore ART increased IFN gamma/IL-4 ratio produced in splenocyte culture (P-value or=0.05). DISCUSSION: Cancer is a multi-factorial disease which needs a multi-approach treatment. Early accumulation of Treg cells in the tumour tissue correlates with tumour progression and is an indication of bad prognosis. According to the obtained results, ART can reduce the number of Tregs. We suggest using artemisinin, with its dual action mechanism. It can effectively kill cancer cells along with reducing the suppressive microenvironment.


Assuntos
Antineoplásicos/uso terapêutico , Artemisininas/uso terapêutico , Carcinoma Ductal de Mama/tratamento farmacológico , Ciclofosfamida/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Anticorpos Antineoplásicos/análise , Anticorpos Antineoplásicos/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Artemisininas/imunologia , Artemisininas/farmacologia , Carcinoma Ductal de Mama/imunologia , Ciclofosfamida/imunologia , Ciclofosfamida/farmacologia , Feminino , Interferon gama/análise , Interleucina-4/análise , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Reguladores/imunologia
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