RESUMO
Analytical cytometry has significant potential beyond cellular analysis. The inherent capability of flow cytometers to efficiently discriminate between uniformly sized particles based on their intrinsic properties provides the foundation for multiplex bead assays. The technology can be exploited in designing immunoassays, Western blot-like antibody assays, and nucleic acid hybridization assays. This chapter focuses on immunoassay applications. The multiplex bead assays have recently evolved as a new and increasingly popular area for flow cytometry, becoming a good alternative to enzyme-linked immunosorbent assay for efficient evaluation of panels of analytes. This chapter provides detailed information about two bead platforms, the BD Cytometric Bead Array kits and the BD Cytometric Bead Array Flex Set Assays.
Assuntos
Citometria de Fluxo/métodos , Anafilaxia/diagnóstico , Anafilaxia/imunologia , Western Blotting/métodos , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Humanos , Imunoensaio/métodos , Isotipos de Imunoglobulinas/análise , Inflamação/diagnóstico , Inflamação/imunologia , Kit de Reagentes para DiagnósticoRESUMO
The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.
