Abstract：
:Rapid development in design and production of recombinant antibodies and antibody fragments specific for cell surface markers opens new opportunities for targeted delivery of therapeutic or imaging agents. However, the progress in this field is slowed by inactivation of many antibodies by chemical conjugation of payloads and by lack of internalization of complexes formed on the cell surface. Here, we describe conversion of a non-internalizing single chain Fv (scFv) antibody P4G7 specific for vascular endothelial growth factor receptor 2 (VEGFR-2) into a targeting protein (Hu-P4G7) for assembly of a novel type of targeting complexes. Hu-P4G7 contains an N-terminal "docking" Hu-tag, a 15-aa fragment of human RNase I, capable of high affinity binding of S-protein fragment of human RNase I or bovine RNase A. Purified Hu-P4G7 and complexes of Hu-P4G7 with S-protein bind both soluble and full-length cellular VEGFR-2. To assemble targeted DNA delivery complexes, S-protein modified with a DNA condensing agent was "docked" to Hu-P4G7, and then loaded with luciferase plasmid DNA. As expected for a non-internalizing targeting protein, Hu-P4G7-based complexes did not deliver DNA in VEGFR-2 expressing cells. However, in the presence of vascular endothelial growth factor (VEGF), these complexes selectively delivered DNA into the cells overexpressing VEGFR-2 suggesting that even a non-internalizing scFv antibody can be used for targeted intracellular drug delivery.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Backer MV,Elliot J,Gaynutdinov TI,Backer JMdoi
10.1016/j.jim.2004.03.011keywords:
subject
Has Abstractpub_date
2004-06-01 00:00:00pages
37-45issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175904001188journal_volume
289pub_type
杂志文章abstract：:Here we extensively describe a FACS-based protocol for isolating intact non-stained human eosinophils from peripheral blood; a stop forward from our recently published initial study. This method of purification could be accomplished in <3 h with only small volumes of whole blood necessary, even in healthy subjects gen...
journal_title：Journal of immunological methods
pub_type： 杂志文章
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更新日期：2018-12-01 00:00:00
abstract：:An enzyme-linked immunosorbent assay (ELISA) was developed for screening production of monoclonal antibodies with specificity for HBsAg. Mouse hybridoma IgG were firstly extracted from assay medium with goat anti-mouse IgG adsorbed on polystyrene beads. The specific antibody was revealed by saturation with HBs antigen...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(82)90361-1
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abstract：:We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5'-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an A...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(98)00101-x
更新日期：1998-09-01 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90003-3
更新日期：1989-03-31 00:00:00
abstract：:Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking ag...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90259-a
更新日期：1993-01-14 00:00:00
abstract：:We have used a surface plasmon resonance biosensor (BIACORE 3000) to detect serum antibodies in chickens having current or recent infections. Three well-defined Salmonella flagellar recombinant DNA antigens reflecting Salmonella enteritidis (H:g,m flagellin) and Salmonella typhimurium (H:i and H:1,2 flagellins) expres...
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pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00102-3
更新日期：2002-08-01 00:00:00
abstract：:Multiplex bead-based assays have many advantages over ELISA, particularly for the analyses of large quantities of samples and/or precious samples of limited volume. Although many commercial arrays covering multitudes of biologically significant analytes are available, occasionally the development of custom arrays is n...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2016.02.018
更新日期：2016-06-01 00:00:00
abstract：:We describe an intracellular cytokine staining, flow cytometry based technique for quantitative and qualitative assessment of CD8+ T lymphocyte-mediated antigen specific immune responses. This adaptation of the technique first described by Suni et al. [J. Immunol. Methods, 212 (1998) 89; Cytometry, 40 (2000) 60] utili...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00025-x
更新日期：2002-04-01 00:00:00
abstract：:A scheme for quantitation of serum and urinary paraproteins is described using isoelectric focusing and scanning densitometry. Paraproteins could be quantified using this system when present at concentrations ranging from 1 mg/ml to 26 mg/ml, depending on the immunoglobulin class. The relevance of these results to mon...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90082-7
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90177-2
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abstract：:Monoclonal antibodies (Mab) are the fastest growing group of biopharmaceuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been deve...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.10.010
更新日期：2007-01-10 00:00:00
abstract：:A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90355-2
更新日期：1985-10-10 00:00:00
abstract：:Pretreatment of polystyrene microplate wells with certain doses of UV light enhances their capacity for binding to single-stranded DNA, double stranded DNA and various synthetic polynucleotides. The use of UV-irradiated plates to immobilize nucleic acid antigens provides a simple, rapid, and specific ELISA for measuri...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90390-x
更新日期：1986-06-10 00:00:00
abstract：:Elucidation of local immune response at the cervix is important for understanding and evaluating STD vaccine approaches currently being proposed. However, no well-validated method exists for the collection of cervical secretions for evaluation of cervical immune response. The purpose of this study was to determine the...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(99)00037-x
更新日期：1999-05-27 00:00:00
abstract：:There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibod...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00281-2
更新日期：1996-04-19 00:00:00
abstract：:We have studied the primate cellular requirements in the antibody forming cell (AFC) assay, using a 185,000 molecular mass streptococcal antigen. Cultures of B cells alone stimulated with the antigen were unable to develop into AFC. Reconstitution of B cells with T cells resulted in a significant increase in the numbe...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90026-r
更新日期：1990-09-14 00:00:00
abstract：:Immunochemical quantitative determination of a lipid antigen, ganglioside GM2, has been developed, based on the inhibition of the immune lysis of liposomes containing the antigen in their lipid bilayer. It has been shown that the full expression of the antigenicity of the competing lipid requires its dispersion in acc...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(77)90071-0
更新日期：1977-01-01 00:00:00
abstract：:Procedures to reproducibly obtain pure preparations of murine Kupffer cells are described. Pure Kupffer cell preparations obtained following collagenase digestion, metrizamide separation and centrifugal elutriation remained viable and maintained their phagocytic functions for at least 4-5 days in vitro. Furthermore, w...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90281-b
更新日期：1993-08-26 00:00:00
abstract：:The differences between murine and human neutrophils mean that findings in mice may not translate to humans, and therefore an in vivo model with human neutrophils would be an important methodological advance. We generated humanised mice by injecting human cord blood derived CD34+ stem cells into irradiated NOD-scid-γc...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.08.005
更新日期：2012-11-30 00:00:00
abstract：:Immunological techniques have been used to identify and quantitate rabbit antibody light chains in the urine of rabbits. The influence of these light chains on the radioallergosorbent test (RAST) used for the diagnosis of laboratory animal allergy to rabbit urinary protein allergens is described. The presence of IgG a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90009-1
更新日期：1983-06-24 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2011.10.010
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abstract：:Visceral leishmaniasis, which is also known as Kala-Azar, is one of the most severely neglected tropical diseases recognized by the World Health Organization (WHO). The threat of this debilitating disease continues due to unavailability of promising drug therapy or human vaccine. An extensive research is undergoing to...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
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更新日期：2015-07-01 00:00:00
abstract：:A new adhesion assay was developed that utilizes buoyancy, rather than washing or centrifugation, to remove non-adherent cells. Biotinylated cells were added to wells containing cell monolayers or purified protein substrates. Non-adherent cells were then removed by floatation on a dense Percoll solution. The adherent ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00187-6
更新日期：1995-12-01 00:00:00
abstract：:Interleukin 2 (IL-2) is a class of T cell growth factors produced by T cells of a number of species. The unique growth-promoting properties of these molecules allow the development of antigen-specific effector T cell lines which can be used to analyze the molecular basis of lymphocyte interactions. A murine T cell tum...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90055-1
更新日期：1980-01-01 00:00:00
abstract：:In transplantation techniques previously described it was impossible to separate the effects of immunosuppressive treatments on the host from the effects directed at the allograft itself. The skin allograft retransplantation technique described here allows one to assess changes in the immunogenicity of the allograft g...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90005-x
更新日期：1984-09-04 00:00:00
abstract：:The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of th...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00395-7
更新日期：2001-08-01 00:00:00
abstract：:A simple roller bottle was constructed to house three dialysis tubes, each with a capacity of 75 ml. Cells were grown inside the dialysis tubing, which was immersed in ordinary DMEM medium without serum supplement. Cultures of hybridomas at medium or low density (2 X 10(5) cells/ml) could be expanded directly in the d...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90046-v
更新日期：1992-01-21 00:00:00
abstract：OBJECTIVES:To report on a novel immunofluorescence pattern specifically associated with antibodies to SS-A/Ro. METHODS:A novel immunofluorescence pattern, herein designated SS-A/Ro pattern, was characterized as myriad discrete fine speckles throughout the nucleus. Eighty-six sequential samples presenting the SS-A/Ro p...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.01.006
更新日期：2013-04-30 00:00:00
abstract：:Foot and mouth disease is caused by a non-enveloped virus (FMDV), which disposes several antigenic sites at the surface of their capsid proteins. The most relevant and immunodominant antigenic site of FMDV (site A or AnSA) includes a key virus-cell interaction element (RGD motif) located in the Viral Protein 1 (VP1), ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.06.008
更新日期：2015-10-01 00:00:00
abstract：:Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90330-2
更新日期：1979-01-01 00:00:00
