Abstract:
:One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Greenman J,Jones E,Wright MD,Barclay ANdoi
10.1016/0022-1759(96)00074-9subject
Has Abstractpub_date
1996-08-14 00:00:00pages
169-80issue
2eissn
0022-1759issn
1872-7905pii
0022-1759(96)00074-9journal_volume
194pub_type
杂志文章abstract::Chemokines, a large family of inflammatory cytokines, have been shown to play a critical role in the regulation of angiogenesis during several pathophysiologic processes, such as tumor growth, wound healing and ischemia. Semiquantitative or quantitative angiogenesis assays are commonly utilized to screen the angiogeni...
journal_title:Journal of immunological methods
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abstract::Relatively purified populations of helper T cells free of active PFC precursors can be prepared from primed splenocytes by irradiation, hypotonic shock, and filtration over glass wool. These purified populations can be stored and helper activity recovered undiminished after up to 6 months of cryopreservation. ...
journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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abstract:BACKGROUND:Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) and monocyte subsets as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative inte...
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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更新日期:1999-12-17 00:00:00
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90419-9
更新日期:1986-12-24 00:00:00
abstract::Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the fir...
journal_title:Journal of immunological methods
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更新日期:2017-11-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
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更新日期:2011-01-05 00:00:00
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journal_title:Journal of immunological methods
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更新日期:2004-05-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90481-3
更新日期:1986-07-24 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1979-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90180-5
更新日期:1980-01-01 00:00:00
abstract::Use of whole blood for lymphocyte proliferation experiments have advantages over the usual isolated lymphocyte procedure. We have systematically optimized methodology for whole blood lymphocyte proliferation in male Sprague-Dawley rats. Both phytohemagglutinin (PHA) and concanavalin A (ConA) stimulate lymphocyte blast...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00084-n
更新日期:1995-08-18 00:00:00
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journal_title:Journal of immunological methods
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doi:10.1016/0022-1759(90)90044-v
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2004.05.008
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journal_title:Journal of immunological methods
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更新日期:1993-03-15 00:00:00
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journal_title:Journal of immunological methods
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更新日期:2012-12-14 00:00:00
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
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更新日期:1994-03-29 00:00:00
abstract::Human primary resting CD4+ T cells are difficult to transfect while preserving viability. The present study evaluated gymnotic delivery and RNase H1-dependent gene expression knockdown mediated by antisense oligonucleotides, called GapmeRs. Exposure of primary resting CD4+ T cells to GapmeRs did not cause cell activat...
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