Abstract：
:We describe a stable chelating linkage for the reversible immobilization of oligohistidine tagged proteins in the flow cell of the 'BIAcore' surface plasmon resonance (SPR) biosensor. The carboxymethylated dextran surface of the flow cell was covalently derivatized with N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single primary amino group, and the derivatized surface charged with Ni2+. 6His-VP55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to this surface in a manner that was dependent upon the immobilized NTA ligand, the prior injection of Ni2+ at a concentration of > 10(-5) M and the 6His tag, and which was reversible upon injection of EDTA. The stability of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containing NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His-VP55 remained stably immobilized during 60 min of buffer flow at moderate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) polymerase, interacted with the immobilized 6His-VP55. Approximately 99% of immobilized 6His-VP55 molecules were available for VP39 binding, in contrast to the approximately 40% availability for 6His-VP55 molecules immobilized covalently, via primary amino groups. Three additional proteins, tagged at either the N- or C-terminus with oligohistidine, were shown to be stably immobilized via the chelating linkage. This simple method permits immobilization of proteins in the BIAcore biosensor via a commonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generation of immobilized protein surfaces.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Gershon PD,Khilko Sdoi
10.1016/0022-1759(95)00032-6subject
Has Abstractpub_date
1995-06-14 00:00:00pages
65-76issue
1eissn
0022-1759issn
1872-7905pii
0022-1759(95)00032-6journal_volume
183pub_type
杂志文章abstract：:Household contacts of tuberculosis patients are at high risk of infection and development of active disease. In this study we evaluated the cytokine production and mRNA expression of IFN-γ, TNF-α, IL-10&IL-6 stimulated with r32kDa M. bovis BCGAg in active pulmonary tuberculosis patients (APTB), household contacts (HHC...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2016.02.012
更新日期：2016-05-01 00:00:00
abstract：:Covalently cross-linked large and intermediate-sized IgA oligomers, prepared with IgA anti-dinitrophenyl (DNP) and bis-DNP-pimelic acid ester, were used to examine the ability of different concentrations (3.5%, 5% and 7%, w/v) of polyethylene glycol (PEG) to precipitate IgA immune complexes (IgA-IC). The size of the I...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90295-x
更新日期：1987-11-05 00:00:00
abstract：:To date, phase I/II dendritic cell (DC)-based cancer vaccine trials have required repeated venesection or leukapheresis to generate the DCs. Previous studies have suggested that DCs may be cryopreserved and revived for clinical use as sequential immunisations. We have developed a method of cryopreserving monocyte-deri...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00430-1
更新日期：2003-01-15 00:00:00
abstract：:A competitive RT-PCR method that permits reliable quantification of minute amounts of reverse-transcribed mouse lymph node mRNA is described. Using this technique, an absolute number of cDNA copies ranging from 10(3) to 10(5) can be determined, with a precision superior to 25%. The standard templates described in the ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00186-5
更新日期：1997-12-29 00:00:00
abstract：:In this report the use of surface-linkage to expand the potential experimental and therapeutic applications of single chain antibody (scFv) constructs is reviewed. A strategy for the generation and functional characterization of surface-linked scFvs that bind selectively to the T-cell proteins CD3epsilon, CD28, and CD...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/s0022-1759(00)00344-6
更新日期：2001-02-01 00:00:00
abstract：:We have developed an antigen presenting system based on the capsid protein of the Flock House virus (FHV) and used it to display, in different positions on its external surface, two neutralizing epitopes found at residues 735-752 of HIV-1 gp160. We have compared the immunoreactivity of these FHV chimeric proteins and ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(96)00097-x
更新日期：1996-10-16 00:00:00
abstract：:Most current methods for measuring cell death are based on plasma membrane disintegration and the consequent release of cytoplasm. The relevant cells are usually loaded with a label (usually 51Cr or 125I), the release of which is measured. I describe here a method, based on the recent evidence that dying cells often d...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90325-a
更新日期：1991-12-15 00:00:00
abstract：:A unique method for assessing the in vivo recovery of cellular immunity following sublethal irradiation and syngeneic bone marrow transplantation of CBA/J mice is described. This method employs the serial surgical implantation of a urethane sponge matrix followed by impregnation of the sponge matrix with alloantigen. ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90283-2
更新日期：1985-12-17 00:00:00
abstract：:Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against pe...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)00249-v
更新日期：1995-01-13 00:00:00
abstract：:Immune complexes have been implicated in the pathogenesis of a number of infectious diseases. The predominant immunoglobulin class associated with circulating immune complexes is IgG, although immune complexes containing IgM have been described. The role of IgM immune complexes in disease pathogenesis has been difficu...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(87)80005-4
更新日期：1987-08-24 00:00:00
abstract：:A novel rigid and renewable transducing material for electrochemical immunosensing, based on Protein A bulk-modified graphite-epoxy biocomposite (ProtA-GEB) is reported. Protein A is able to bind to the Fc region of antibodies and provide an affinity matrix for antibody immobilisation onto the transducer. The rigid co...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2003.11.014
更新日期：2004-03-01 00:00:00
abstract：:The detection of specifically internal immunoglobulin by staining of fixed preparations of lymphoid cells with appropriate fluorescein-conjugated antisera can be hampered by binding of the conjugates to membrane-bound immunoglobulin. Without preventing staining through membrane-bound immunoglobulin, it is impossible ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90209-6
更新日期：1979-01-01 00:00:00
abstract：:A simple apparatus for measuring chemotaxis is described which contains thirty chambers per unit. Only very few cells and small volumes of chemotactic factor solution are required per chamber. For satisfactory results the apparatus must be centrifuged briefly prior to incubation. Under these conditions results can be ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)90008-9
更新日期：1977-01-01 00:00:00
abstract：:Phage-displayed random peptide libraries are important tools in identifying novel epitopes/mimotopes that may lead to the determination of antigen specificity. In this approach, high-affinity phage peptides are enriched by affinity selection (panning) on a monoclonal antibody. To facilitate identification of all poten...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.08.006
更新日期：2006-10-20 00:00:00
abstract：:In this study we describe a fast, simple, and objective flow cytometry method to quantify simultaneously phagocytosis and Fc gamma R-mediated oxidative burst in human monocytes. Peripheral blood monocytes isolated by elutriation were sequentially incubated with 2',7'-dichlorofluorescein-diacetate (DCFH-DA) and sheep e...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90155-z
更新日期：1993-02-26 00:00:00
abstract：:Leucocyte procoagulant activity (PCA) has previously been advocated as a useful measurement of cell-mediated immune reactivity. The assay is, however, susceptible to inter- and intra-experimental variation. This investigation identifies several factors which influence the stability and reproducibility of the test. Maj...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90151-6
更新日期：1985-12-27 00:00:00
abstract：:The production and characterisation of 17 monoclonal antibodies to human parathyroid hormone-related protein (PTH-rP) 1-34 is described. Five of the antibodies were shown to be of high avidity (Ka 4 X 10(10)-1.9 X 10(11) L/M) and able to detect 15-100 pg PTH-rP 1-34 per tube by RIA. None cross-reacted with PTH 1-34, a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90346-w
更新日期：1990-02-20 00:00:00
abstract：:Dendritic cells are a specialized but trace population of antigen presenting cells that always have been enriched by multi-step procedures over a period of 1 or more days in tissue culture. Here we describe the isolation of dendritic cells from fresh mouse spleen suspensions using the FACS and a monoclonal antibody, N...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90318-p
更新日期：1990-10-04 00:00:00
abstract：:The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.06.002
更新日期：2013-08-30 00:00:00
abstract：:A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect lo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.02.011
更新日期：2006-05-30 00:00:00
abstract：:Using in vitro established tumour cell lines attempts were made to assess the suitability of tetrazolium salt reduction (MTT) assay to replace the conventional radioactive base techniques for measuring cell proliferation and cell killing. The optimum conditions for MTT loading time, concentration of MTT and the time f...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90012-v
更新日期：1993-03-15 00:00:00
abstract：:ELISAs for urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have shown that tumor levels of these molecules are prognostic parameters in breast cancer as well as other types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inac...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00008-2
更新日期：1997-04-11 00:00:00
abstract：:The antigen capture capacity of antibodies covalently immobilized on injection-molded polystyrene beads was evaluated. Bromoacetyl groups on the bead surfaces rendered them reactive to protein nucleophilic groups. The bromoacetyl surface exhibited up to a ten-fold greater capacity for protein compared to unmodified po...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90136-6
更新日期：1988-07-22 00:00:00
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 paylo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2004.03.011
更新日期：2004-06-01 00:00:00
abstract：:The simian immunodeficiency virus (SIV) model of AIDS is widely used for the development of human immunodeficiency virus (HIV) vaccine strategies, particularly for the analysis of correlates of protective immunity. As it is not always possible to establish autologous B-lymphoblastoid cell lines (B-LCL) for use as targ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00480-x
更新日期：2001-12-01 00:00:00
abstract：:We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partner...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2008.04.008
更新日期：2008-07-31 00:00:00
abstract：:Polymorphisms of immune defence genes may act as disease modifiers and are studied by many researchers. A conclusive analysis of the impact of genetic variations typically requires a large number of sample specimens, and in retrospective studies this may include samples of reduced quality, e.g. formalin-fixed paraffin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.05.006
更新日期：2007-07-31 00:00:00
abstract：UNLABELLED:In this study the flow-cytometric crossmatch results were compared between fresh cells and cells processed by various cryopreservation and storage methods. Platelets from healthy donors were incubated with 12 sera containing platelet reactive antibodies as well as with 62 control sera from blood donors. Dire...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2004.12.002
更新日期：2005-02-01 00:00:00
abstract：:Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90020-v
更新日期：1990-01-24 00:00:00
abstract：:A method for synthesising omega-stearoyldextran for the detection of plaque forming cells specific for the alpha-(1 replaces 6) determinant is described. The omega-stearoyl derivative is obtained by reacting dextran with phenylboronic acid to give a 2,4-phenylboronate, which can be subsequently esterified in a pyridin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90206-4
更新日期：1976-01-01 00:00:00
