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 AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10(6) cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Goujon L,Brossette T,Dereudre-Bosquet N,Creminon C,Clayette P,Dormont D,Mioskowski C,Lebeau L,Grassi Jdoi
10.1016/s0022-1759(98)00101-xsubject
Has Abstractpub_date
1998-09-01 00:00:00pages
19-30issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(98)00101-Xjournal_volume
218pub_type
杂志文章abstract：:Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously ad...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/0022-1759(92)90066-3
更新日期：1992-06-24 00:00:00
abstract：:Regulatory T cells (Treg) have recently come to the fore in studies of immune regulation, particularly in autoimmune disease and cancer. While there appear to be several distinct subsets of T cells with regulatory function, a population described as natural Treg and characterized by expression of the transcription fac...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.06.006
更新日期：2007-10-31 00:00:00
abstract：:A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antig...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90356-3
更新日期：1983-03-25 00:00:00
abstract：:A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. P...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90032-b
更新日期：1991-03-21 00:00:00
abstract：:We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2004.10.006
更新日期：2004-12-01 00:00:00
abstract：:The rise in the analytical speed of mutiparameter flow cytometers made possible by the introduction of digital instruments, has brought up the possibility to manage progressively higher number of parameters simultaneously on significantly greater numbers of individual cells. This has led to an exponential increase in ...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/j.jim.2019.07.003
更新日期：2019-12-01 00:00:00
abstract：:An immunomagnetic technique to detect and identify Salmonella serogroup C1 has been developed. Monoclonal antibodies (MAbs) specific for the O antigen 6,7 of Salmonella lipopolysaccharide (LPS) coupled to magnetic beads were used to isolate the salmonellae. Captured bacteria were easily identified by acridine orange s...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90387-u
更新日期：1991-03-01 00:00:00
abstract：:The sensitivity of any immunoassay is a complex function of the underlying physico-chemical basis of the technique and the size and source of 'experimental' errors. Analysis of this relationship emphasizes the distinction between competitive labelled analyte methods, e.g., RIA, and the non-competitive labelled antibod...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90338-8
更新日期：1986-02-27 00:00:00
abstract：:A procedure to enhance incorporation of radioactive iodine into cell-surface proteins is described. The effects of chemical modification of cells with the iodinatible haptens N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) and methyl-p-hydroxybenzimidate HC1 (Wood reagent) are compared. The Wood ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90177-2
更新日期：1985-02-28 00:00:00
abstract：:A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90068-7
更新日期：1985-10-24 00:00:00
abstract：:Staphylococcus aureus causes a variety of infections. Knowledge about the physiological role of most S. aureus antigens in colonization and infection is only limited. This can be studied by measuring antigen-specific antibody responses. In this study, we optimized the multiplex microsphere bead-based flow cytometry te...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.12.013
更新日期：2011-02-28 00:00:00
abstract：:A recombinant retroviral vector with an inserted bacterial neomycin resistance (neo) gene was used to transfer in vitro neomycin resistance (neoR) into murine cytotoxic lymphocyte precursors (CLP). The infection protocol involved co-cultivation of mitogen-activated splenic T-blasts with irradiated cells that produced ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90036-0
更新日期：1986-05-01 00:00:00
abstract：:Using a solid-phase non-radioisotopic (non-RI) reverse transcriptase (RT) assay, antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) RT activity (RTI antibody) were investigated for their ability to inhibit binding of RT to a template-primer and DNA polymerization. The RTI antibody inhibited the binding ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(96)00180-9
更新日期：1996-12-15 00:00:00
abstract：:A cell ELISA for human leukocytes has been established and standardized. The assay was based on the use of air-dried and methanol-fixed cells which were attached to microplate wells. Cellular antigens were measured through the subsequent binding of monoclonal antibody (MoAb), biotinylated sheep Ig against mouse IgG an...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90219-x
更新日期：1986-11-20 00:00:00
abstract：:The use of colloidal gold and colloidal gold followed by silver enhancement as marker for dot or blot immune overlays is described. Colloidal gold probes concentrating at the sites of immune reaction gradually develop a pinkish colour during incubation that can be seen with the naked eye. With a physical developer, ve...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90303-x
更新日期：1984-11-30 00:00:00
abstract：:Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragm...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2003.12.001
更新日期：2004-02-15 00:00:00
abstract：:The length of the heavy chain complementarity-determining region two (HCDR2) of the unmutated anti-p-azophenylarsonate (Ars) monoclonal antibody (36-65 mAb) was extended by three residues in order to test whether this insertion can provide additional contacts between the Ab and the antigen. Two libraries were generate...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00488-4
更新日期：2002-01-01 00:00:00
abstract：:Previous studies on lymphocyte cytotoxicity against autologous human hepatocytes had been studied using Terasaki plates, in which dead hepatocytes after incubation with lymphocytes were counted visually. No studies with 51Cr-labeled human hepatocytes as targets have been reported, although it can give us more objectiv...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(93)90277-e
更新日期：1993-08-26 00:00:00
abstract：:Antibody avidity, defined as the strength of binding between antibody and antigen, represents a functional measure of affinity maturation of antibodies. Determination of the antibody avidity is usually performed separating high and low avidity antibodies by dissociating agents, but measurement of the antibody avidity ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.09.008
更新日期：2013-01-31 00:00:00
abstract：:A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90317-6
更新日期：1987-02-11 00:00:00
abstract：:We wished to determine whether injection of mice with anti-isotype antibody would be a means to regulate in vivo isotype expression and to obtain hybridomas secreting monoclonal antibodies (mAbs) of desired antigen specificity and isotype. Treatment with a rat mAb (7D2) reactive with both the IgG2a and IgG2b isotypes ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90007-5
更新日期：1988-02-24 00:00:00
abstract：OBJECTIVE:The central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type. METHOD...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2019.02.001
更新日期：2019-04-01 00:00:00
abstract：:This paper shows how to obtain accuracy and efficiency in an ELISA analysis by allocating the wells on a 96-well microplate between calibration and determination of unknowns, and by choosing the known concentrations for calibration. The method also can determine how much is lost in precision by using a convenient but ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90036-u
更新日期：1990-09-14 00:00:00
abstract：:The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90067-1
更新日期：1988-06-28 00:00:00
abstract：:Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purp...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.09.001
更新日期：2011-01-05 00:00:00
abstract：:An enzyme linked immunosorbent assay (ELISA) method for the detection of antibodies to platelets and leucocytes is presented. The method can be used for large numbers of samples. The method is objective when photometers are used. Approximately 50% of all cases of possible autoimmune thrombocytopenic purpura (A.T.P.) a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(81)90116-2
更新日期：1981-01-01 00:00:00
abstract：:The detection of site-directed anti-idiotypic antibodies is usually based on their ability to inhibit the binding of antigen to idiotype, in either solid- or fluid-phase radioimmunoassays. Passage of serum over antigen-coupled immunosorbents for the purpose of removing the idiotypes from complexes with putative anti-i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90172-4
更新日期：1987-06-26 00:00:00
abstract：:Effector-target conjugation between different cell populations of human NK cells and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios. Non-linear and linear ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00153-1
更新日期：1997-12-01 00:00:00
abstract：:Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.09.022
更新日期：2011-01-05 00:00:00
abstract：:The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibiti...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90202-7
更新日期：1976-01-01 00:00:00
