This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infec...This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infectious bursal disease virus （IBDV）. McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.展开更多
Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA d...Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical patholog36 to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods （transmission electron microscopy, X-ray fluorescence microscop~ etc.） have low throughput and technical and operational complications. Herein, we describe two in situ post- treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne- conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy （XFM） at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.展开更多
基金supported by the National Natural Science Foundation of China (30671561)the National Key Technology R&D Program of China (2006BAD06A04-6)the National Basic Research Program of China(2005CB523203)
文摘This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infectious bursal disease virus （IBDV）. McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.
文摘Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical patholog36 to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods （transmission electron microscopy, X-ray fluorescence microscop~ etc.） have low throughput and technical and operational complications. Herein, we describe two in situ post- treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne- conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy （XFM） at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.
