Abstract：
:Among the many methods currently available for quantifying mRNA transcript abundance, reverse transcription-polymerase chain reaction (RT-PCR) has proved to be the most sensitive. Recently, several protocols for real-time relative RT-PCR using the reporter dye SYBR Green I have appeared in the literature. In these methods, sample and control mRNA abundance is quantified relative to an internal reference RNA whose abundance is known not to change under the differing experimental conditions. We have developed new data analysis procedures for the two most promising of these methodologies and generated data appropriate to assess both the accuracy and precision of the two protocols. We demonstrate that while both methods produce results that are precise when 18S rRNA is used as an internal reference, only one of these methods produces consistently accurate results. We have used this latter system to show that mRNA abundances can be accurately measured and strongly correlate with cell surface protein and carbohydrate expression as assessed by flow cytometry under different conditions of B cell activation.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Marino JH,Cook P,Miller KSdoi
10.1016/s0022-1759(03)00103-0keywords:
subject
Has Abstractpub_date
2003-12-01 00:00:00pages
291-306issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175903001030journal_volume
283pub_type
杂志文章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：:A sensitive procedure based on the use of iodinated antibody allows the detection of antigenic material by double-diffusion analysis. An autoradiographed immunodiffusion pattern reveals distinct radio-labeled precipitation lines for an extract of yeast with wild type HIS4 protein and for extracts of mutants containin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90048-0
更新日期：1978-01-01 00:00:00
abstract：:The U937 cell line has been shown to generate tumour necrosis factor alpha (TNF-alpha) in response to soluble stimuli such as PMA and LPS, but only after treatment with GM-CSF. We report here the generation of TNF-alpha from U937 cells following phagocytosis of opsonised zymozan particles without the need for pre-trea...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90141-f
更新日期：1992-08-10 00:00:00
abstract：:Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the ex...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2008.04.014
更新日期：2008-07-31 00:00:00
abstract：:We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG....
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(82)90033-3
更新日期：1982-12-17 00:00:00
abstract：:We have evaluated two novel enzyme-linked immunosorbent assays (ELISAs) used to quantitate cyclic AMP. In one assay ELISA plates are coated with antigen consisting of a cyclic AMP-polylysine conjugate. Cyclic AMP samples added to plates are quantified by their ability to decrease the binding and anti-cyclic AMP antibo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90119-e
更新日期：1992-07-06 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：:We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichi...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00321-0
更新日期：2001-05-01 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：:A micro-assay has been developed for the separate evaluation of phagocytosis and intracellular killing of Staphylococcus aureus by human monocytes and granulocytes. The minimal number of phagocytes required for the investigation of these functional activities of phagocytic cells has been established by performing phag...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90095-9
更新日期：1983-02-25 00:00:00
abstract：:We examined two ELISA methods for measuring antibodies to Streptococcus pneumoniae using tyraminated S. pneumoniae polysaccharide types 3, 7N, 9F and 14 as antigens. The ELISA has the usual format with a relatively long incubation time whereas the FAST-ELISA has a short incubation time and employs a different solid-ph...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90238-x
更新日期：1989-06-21 00:00:00
abstract：:The attachment of cells to plastic tissue culture plates is a widely used criterion of cell viability in microcytotoxicity assays. When cell suspensions of primary human colon carcinoma and melanoma cells which were of low initial viability (assessed by trypan blue exclusion) were allowed to adhere to tissue culture p...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(81)90039-9
更新日期：1981-01-01 00:00:00
abstract：:Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional ma...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.09.034
更新日期：2011-03-31 00:00:00
abstract：:A solid-phase chemiluminescence immunoassay for determining immunoglobulin G (IgG) in eluates of psoriatic scales is described. Polystyrene balls coated with protein A bound the IgG present in the eluates and in human sera. The bound IgG was then detected by a luminol-coupled goat anti-human IgG conjugate, characteriz...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90184-4
更新日期：1984-05-25 00:00:00
abstract：:A solid-phase immunofluorescence assay (SIFA) is described for screening of monoclonal hybridoma antibodies against soluble antigens. The test is simple, needs a small amount of material (20-100 ng of labeled antigen per sample) and allows a large number of samples (500-1 000) to be tested in 2 days. The assay is perf...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(81)90330-6
更新日期：1981-01-01 00:00:00
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：:Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diag...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.02.017
更新日期：2013-05-31 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：:Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recom...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(99)00073-3
更新日期：1999-07-30 00:00:00
abstract：:A method is presented for measuring the hemolytic activity of undiluted guinea pig serum. Undiluted serum is placed in contact with a column of sensitized sheep red blood cells in an agar matrix in a capillary tube. After incubation, the length of the zone of lysis is measured. Comparison with a standard curve prepare...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90212-6
更新日期：1979-01-01 00:00:00
abstract：:A method is described for the simultaneous in situ detection of surface receptors and cytoplasmic granules in mast cells of frozen sections of mouse skin. Surface IgE receptors are detected after saturation of the receptors with a murine monoclonal antibody of IgE isotype. The latter is subsequently detected by monosp...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90121-9
更新日期：1987-05-20 00:00:00
abstract：:The length distributions of the third complementarity determining region (CDR3) of the T cell receptor (TCR) beta chain were determined quantitatively from peripheral blood lymphocytes of healthy donors. RT-PCR products of 26 V beta families and subfamilies were analyzed by an A.L.F. DNA sequencer and Fragment Manager...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00178-d
更新日期：1995-11-16 00:00:00
abstract：:γδ T cells function as sentinels in early host response to infections and malignancies. Although γδ T cells are regarded as innate immune cells and recognize antigens in a non-MHC restricted manner, they possess a huge diversity of complementary determinant region 3 (CDR3) of T cell receptor (TCR) generated by the rea...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2017.01.009
更新日期：2017-04-01 00:00:00
abstract：:Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2017.03.011
更新日期：2019-12-01 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
abstract：:In the guinea pig system, there are no T lymphocyte tumors available. In addition, in this species the production of T cell lines in vitro for analysis of T cell markers or function proved to be difficult. As an alternative approach, guinea pig X mouse and guinea pig X rat T cell hybridomas were obtained by fusion of ...
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：:This paper describes a method for processing fresh tissue that allows immunohistological analysis on paraffin sections. The method is based on the use of periodate-lysine-paraformaldehyde fixation. The effects of variation in fixation time, concentration of paraformaldehyde, dehydration, clearing, wax embedding and en...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90106-6
更新日期：1984-12-31 00:00:00
abstract：:The light and heavy chain cDNA of a murine monoclonal antibody (MoAb) with specificity for human colorectal carcinoma cells have been expressed separately, together, and as a dual construct in insect cells infected with recombinant baculoviruses. High levels of the MoAb were expressed under the control of the polyhedr...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90118-d
更新日期：1992-07-06 00:00:00
abstract：:An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coa...
journal_title：Journal of immunological methods
pub_type： 临床试验,杂志文章,多中心研究
doi：10.1016/0022-1759(92)90163-n
更新日期：1992-04-08 00:00:00
abstract：:A polymorphism at position 559 in the cDNA of the FCGR3A gene encoding the FcgammaRIIIa generates two allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158. This polymorphism is of major importance in immunopathology and in pharmacogenetics, especially monoclonal antibody treatments....
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00123-6
更新日期：2003-06-01 00:00:00
