Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Front Immunol ; 14: 1190404, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37342321

RESUMO

Background: Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT). Methods: The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT. Results: We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed. Conclusion: The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.


Assuntos
Vacinas contra Difteria, Tétano e Coqueluche Acelular , Difteria , Tétano , Estados Unidos , Humanos , Toxina Pertussis , Hemaglutininas , Reprodutibilidade dos Testes , Anticorpos Antibacterianos , Imunoglobulina G
2.
J Med Microbiol ; 68(5): 741-747, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30990403

RESUMO

PURPOSE: Serological analysis is an essential tool for the diagnosis of pertussis or whooping cough, disease surveillance and the evaluation of vaccine effectiveness against Bordetella pertussis. Accurate measurement of anti-pertussis toxin (anti-PT) IgG antibody levels in sera is essential. These measurements are usually performed using immunological methods such as ELISA and multiplex immunoassays. However, there are a large number of different assay systems available, and therefore standardization and harmonization between the methods are needed to obtain comparable data. METHODOLOGY: In collaboration with ECDC, the EUPert-LabNet network has organized three External Quality Assessment (EQA) schemes (2010, 2012 and 2016), which initially identified the diverse range of techniques and reagents being used throughout Europe. This manuscript discusses the findings of each of the EQA rounds and their impact on the participating laboratories. RESULTS: The studies have shown an increasing number of laboratories (from 65% to 92%) using only the recommended coating antigen, purified PT, in immunoassays, as this allows exact quantification of serum anti-PT IgG and since PT is only produced by Bordetella pertussis this prevents cross-reactivity with other species. There has also been an increase in the numbers of laboratories (from 59% to 92%), including a WHO reference serum in their assays, which allows anti-PT IgG concentrations to be measured in International Units, thus enabling the comparison of results from different methods and laboratories. In addition, manufacturers have also considered these recommendations when they produce commercial ELISA kits. CONCLUSION: The three EQA rounds have resulted in greater harmonization in methods among different laboratories, showing a significant improvement of the ELISA methods used for serodiagnosis of pertussis.


Assuntos
Bordetella pertussis/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Colaboração Intersetorial , Coqueluche/diagnóstico , Anticorpos Antibacterianos/sangue , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Ensaio de Imunoadsorção Enzimática/normas , Europa (Continente) , Humanos , Imunoensaio , Imunoglobulina G/sangue , Internacionalidade , Controle de Qualidade , Kit de Reagentes para Diagnóstico/normas , Testes Sorológicos/métodos , Coqueluche/imunologia
4.
Vaccine ; 29(10): 1974-80, 2011 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-21216309

RESUMO

Bordetella pertussis pertactin (Prn) is an important attachment factor and protective immunogen, which serves as a component in most acellular pertussis vaccines (APVs). Here, we over-expressed recombinant Prn (r-Prn) without an affinity tag using an Escherichia coli expression system. Compared to the native Prn (n-Prn) from B. pertussis, the recombinant protein showed a comparable reactivity in Western blotting and ELISA as well as a similar structure as analyzed by circular dichroism, fluorescence spectroscopy and size-exclusion chromatography. Furthermore, parenteral immunization of mice with native or r-Prn produced a similar increase in serum anti-Prn antibodies and similar protective activity following aerosol challenge. Our results indicate that the recombinant protein approach may be useful for developing a potential component of APVs as well as an antigen material which can be used in both clinical epidemiological evaluation and laboratory vaccine evaluation studies. Moreover, these studies also provide further evidence for the role of Prn in pertussis immunity.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Bordetella pertussis/imunologia , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/imunologia , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Bordetella pertussis/genética , Cromatografia em Gel , Dicroísmo Circular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Expressão Gênica , Pulmão/microbiologia , Camundongos , Vacina contra Coqueluche/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectrometria de Fluorescência , Fatores de Virulência de Bordetella/genética , Coqueluche/prevenção & controle
5.
Hum Vaccin ; 5(3): 166-71, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18758243

RESUMO

All current acellular pertussis vaccines (ACVs) contain detoxified pertussis toxin (PT) as a major component. An essential part of the safety evaluation of these vaccines, required by regulatory authorities, is to monitor their active PT content and to check for reversion to toxicity of the detoxified PT. Although various in vitro tests are under investigation, the only practicable means for detecting active PT at present is the histamine sensitization test. The methods given in the European Pharmacopoeia and in the US Pharmacopoeia are based on recording a binary response to histamine challenge (using a lethal end point). A more sensitive method based on measurement of rectal temperature is given in the Japanese Minimum Requirements for Biological Products. More recently, a refinement of this method based on dermal temperature measurement has been developed for ACVs in combination with diphtheria and tetanus vaccines (DTaP). We show that this method also can be used for more complex combination vaccines and is readily transferable. Furthermore use of dermal temperature provides a more precise quantitative estimate of toxin activity than the binary response, leading to an increase in information from a specified number of animals, or allowing a reduction in the number of animals required. We suggest that, pending the development of an alternative in vitro replacement method, the temperature based method may serve as an intermediate solution to the estimation of PT activity giving a precise estimate with reduction in animal numbers.


Assuntos
Toxina Pertussis/toxicidade , Vacina contra Coqueluche/análise , Temperatura Cutânea , Testes de Toxicidade/métodos , Animais , Feminino , Humanos , Japão , Camundongos , Controle de Qualidade
6.
Cell Immunol ; 227(1): 51-8, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15051514

RESUMO

Recent clinical trials have shown that the new generation of acellular pertussis vaccines (Pa) can confer protection against whooping cough with negligible adverse reactions. We have compared the effects of pertussis whole cell and acellular vaccines on pulmonary immune responses after aerosol challenge in a murine model of infection. Mice were vaccinated with PBS, Pw or Pa and challenged with Bordetella pertussis by the aerosol route. Cytokine gene expression was analysed from lung tissue and cells; lung lymphocytes were re-stimulated in vitro and cytokines produced measured. The results obtained are consistent with the proposal that a strong Th-1 response is associated with bacterial clearance in both the non-vaccinated and Pw vaccinated mice. The acellular vaccine treated mice cleared the bacterial challenge (with an intermediate efficacy) in the presence of low levels of any of the cytokines assessed. This suggests that Pa protects via a Th-2 independent mechanism.


Assuntos
Vacina contra Coqueluche/imunologia , Sistema Respiratório/imunologia , Vacinas Acelulares/imunologia , Administração por Inalação , Aerossóis , Animais , Bordetella pertussis/imunologia , Complexo CD3/metabolismo , Células Cultivadas , Feminino , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Pulmão/citologia , Pulmão/microbiologia , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Células Th1/imunologia , Coqueluche/prevenção & controle
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...