Abstract:
:The majority of immunological processes are mediated by cell-to-cell contact or receptor-ligand interactions that transmit intracellular signals and affect the regulation of transcription in the nucleus. As a consequence, precursor cells develop into their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cells or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method, differential display (DDRT-PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and denaturing polyacrylamide gel electrophoresis. This technique is applicable to multiple samples of clonal or purified cell populations as well as to complex tissues and can be used to provide mRNA fingerprints. However, the main purpose of DDRT-PCR is to isolate differentially regulated genes in biological systems. The method is carried out without prior hypothesis as to which genes should be examined and so increases the possibility of identifying completely novel and unexpected changes in transcription. A major drawback has been the isolation of false positive clones and the need to confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have been recommended to reduce these problems, including reverse-northern analysis as a confirmatory step for screening putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a cell, several focused approaches have been suggested. These include targeted differential display for the isolation of multigene families that have conserved protein domains or gene signatures and subtractive differential display whereby one population is subtracted from the other prior to screening. The purpose of this review is to provide some guidance to the immunologist who might wish to apply DDRT-PCR in their research. A number of examples where DDRT-PCR has been used successfully in immunological research are included.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Ali M,Markham AF,Isaacs JDdoi
10.1016/s0022-1759(01)00304-0keywords:
subject
Has Abstractpub_date
2001-04-01 00:00:00pages
29-43issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175901003040journal_volume
250pub_type
杂志文章,评审abstract::Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biol...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00237-9
更新日期:2000-03-06 00:00:00
abstract::Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) w...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(98)00167-7
更新日期:1998-12-01 00:00:00
abstract::We have used the ELISPOT method employing plastic ELISA plates without substrate in agar for the detection of single cells producing interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). When using PBMC directly stimulated in the assay wells with T cell mitogens it was possible to measure production of human IFN-gamm...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(96)00170-6
更新日期:1997-01-15 00:00:00
abstract::No standard procedure is available for the purification of human monoclonal antibodies for human i.v. administration. Here we describe the procedure developed for pilot scale purification of the human IgM monoclonal antibody COU-1 directed against a cancer-associated antigen. The hybridoma cells were grown in protein-...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00051-3
更新日期:1997-06-23 00:00:00
abstract::Rabbit antiserum, specific for protein A from Staphylococcus aureus, was conjugated to alkaline phosphatase and used in a double antibody solid-phase enzyme immunoassay. The assay was developed to monitor eluate from a large-scale protein A-Sepharose affinity column used to purify monoclonal antibodies for human clini...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90070-5
更新日期:1985-10-24 00:00:00
abstract::To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/horizontal apparatus in which a concentration gradient is established reproducibly for a given stimulus. The observed linear-mode trajectory pattern of neutrophils toward...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2013.12.005
更新日期:2014-02-01 00:00:00
abstract::Antibody to the human nicotinic acetylcholine receptor has been demonstrated in the disorder myasthenia gravis and is the pathologic factor producing the characteristic symptoms of the disorder. A rapid, quantitative and sensitive radioimmunoassay using human acetylcholine receptor, affinity labeled with iodinated alp...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(81)90089-2
更新日期:1981-01-01 00:00:00
abstract::LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2009.11.016
更新日期:2010-01-31 00:00:00
abstract::Synovial fluid (SF) is a difficult biological matrix to analyse due to its complex non-Newtonian nature. This can result in poor assay repeatability and potentially inefficient use of precious samples. This study assessed the impact of SF treatment by hyaluronidase and/or dilution on intra-assay precision using the Lu...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2012.08.012
更新日期:2012-12-14 00:00:00
abstract::IL-2 activity is commonly estimated in cell culture supernatants by an IL-2-dependent cell proliferation assay. This method is both reproducible and sensitive. However, it often appears from the literature that statistical analysis of the titration data either is disregarded or, when performed, is based on statistical...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90463-1
更新日期:1986-02-12 00:00:00
abstract::The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2007.09.010
更新日期:2008-01-01 00:00:00
abstract::A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current systems are not sufficiently sensitive at detecting and quantifying ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2019.02.003
更新日期:2019-04-01 00:00:00
abstract::We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90397-x
更新日期:1991-03-01 00:00:00
abstract::Flow cytometric methods for the evaluation of the cell surface binding properties of monoclonal antibody (MoAb)-drug/toxin conjugates defining tumor-associated antigens are presented. In these techniques, suspension cultures of solid human tumor cell lines are incubated with either varying dilutions of MoAb or MoAb-dr...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90310-3
更新日期:1987-02-11 00:00:00
abstract::Affinity chromatography and subsequent ion-exchange chromatography of pooled anti-benzylpenicilloyl (anti-BPO) hyperimmune sera separated 5 different anti-BPO IgG fractions as described in the preceding paper. These fractions were tested for activities in passive hemagglutination (PHA), passive immune hemolysis (PIH),...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90163-1
更新日期:1978-01-01 00:00:00
abstract::A fluorometric enzyme immunoassay using an alkaline phosphatase-conjugated monoclonal antibody was developed to quantitate feline leukemia virus (FeLV) infection. Monoclonal antibodies, directed against the FeLV structural protein p27, were conjugated with alkaline phosphatase using a modified maleimide method. The en...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90181-0
更新日期:1988-11-10 00:00:00
abstract::A new rapid staining and measuring method has been developed for the quantification of migrated cells in a microchemotaxis chamber. The migrated cells were, after staining, evaluated by a transmission densitometer. The method introduced here is more accurate and faster than those described previously. In addition the ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90051-3
更新日期:1989-03-10 00:00:00
abstract::An enzyme-linked immunosorbent assay (ELISA) for the quantitation of diphtheria toxin antibodies is described. When the ELISA technique was compared to a single radial immunodiffusion assay the results correlated well but the ELISA technique was ten thousand times more sensitive. It was also at least ten times as sens...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(77)90107-7
更新日期:1977-01-01 00:00:00
abstract::The therapeutic use of dendritic cells (DC) in antigen-specific anti-tumor vaccines, requires sufficient numbers of functional DC, the preparation of which should comply with the code of Good Manufacturing Practice. In addition, the expression of tumor specific antigen should be possible in these DC. As a preclinical ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(00)00214-3
更新日期:2000-07-31 00:00:00
abstract::Bovine serum albumin (BSA) was used as a non-amphipathic model protein to be included into iscoms. Pretreatment at an acidic pH (2.5) was used to reveal hydrophobic regions, after which BSA could be integrated. In immunization experiments in mice the BSA iscoms induced long lasting and considerably higher serum antibo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90208-d
更新日期:1990-04-17 00:00:00
abstract::Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2018.10.007
更新日期:2019-01-01 00:00:00
abstract::A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mix...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90450-2
更新日期:1984-08-03 00:00:00
abstract::TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00239-2
更新日期:2000-04-03 00:00:00
abstract::A method is described for the study of lymphokine-mediated cellular interactions using triple wells, which permits co-culture of cell subpopulations without direct physical contact. The triple wells are constructed by slitting the walls to half height between three adjacent wells of a 96-well microtiter plate. The cel...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90279-8
更新日期:1994-07-12 00:00:00
abstract::A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of effic...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2020.112794
更新日期:2020-08-01 00:00:00
abstract::The application of freeze-dried paraffin sections for immunohistology eliminates many of the problems associated with the cryostat technique: the sections are thin sufficiently, the cell morphology is improved, there are no diffusion artifacts and the intensity of immunohistological reactions is superior to that obtai...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90365-9
更新日期:1982-01-01 00:00:00
abstract::Serum factors which interact with human peripheral blood lymphocyte Fc gamma receptors (Fc gamma Rs) may be detected in vitro by the EA rosette inhibition assay (EARIA). This assay has been used to detect circulating immune complexes and certain alloantibodies directed against cell surface antigens situated in close p...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90222-s
更新日期:1993-02-03 00:00:00
abstract::The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2014.01.012
更新日期:2014-03-01 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::Four alleles are currently recognised at the HLA-DRB3 locus (DRB3*0101, DRB3*0201, DRB3*0202 and DRB3*0301). We studied whether 271 bp fragments of the polymorphic second exon, which were prepared using the polymerase chain reaction, could be typed using temperature gradient gel electrophoresis. Thermal stability curv...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90063-9
更新日期:1994-02-10 00:00:00