Abstract：
:We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Hu GR,Harrop P,Warlow RS,Gacis ML,Walls RSdoi
10.1016/s0022-1759(96)00235-9subject
Has Abstractpub_date
1997-03-28 00:00:00pages
113-21issue
2eissn
0022-1759issn
1872-7905pii
S0022-1759(96)00235-9journal_volume
202pub_type
杂志文章abstract：:In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2017.04.005
更新日期：2017-08-01 00:00:00
abstract：:A simplified technique for the production of chimeric thymus completely in vitro from the third pharyngeal pouch (TPP) of 10-day mouse embryos has been examined. The results are similar to those obtained by a more cumbersome technique published by others. This modified technique permits the production of large numbers...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90279-x
更新日期：1983-03-11 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 optimal conditions for the detection of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immunoblotting were evaluated. The variables examined included the equilibration time of the gels before transfer, composition of the transfer buffer, type of blotting membrane, blockin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90072-8
更新日期：1994-01-03 00:00:00
abstract：:A rapid, direct method for the purification of sheep red cell rosetting lymphocytes (ERFC) was developed. The whole procedure, including rosette formation, density separation and hemolysis could be completed within 10 min. A mixture of human peripheral blood mononuclear cells (PBMC) and 2-aminoethylisothiouronium brom...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90217-7
更新日期：1988-03-16 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：: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：: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：:As interest in the development of oral vaccines continues to rise, alternative animal models for studies of mucosal immunity are needed. The present study examines a simplified procedure for delivering antigen to rabbit Peyer's patches via an indwelling cannula. The cannula was placed 3-4 cm proximal to the Peyer's pa...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(96)00055-5
更新日期：1996-07-17 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.12.007
更新日期：2011-02-28 00:00:00
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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：:Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00036-4
更新日期：2002-05-01 00:00:00
abstract：:The performance of immunoassays for the detection of autoantibodies is of critical importance to the diagnosis and assessment of patients with systemic lupus erythematosus (SLE). Our objective was to compare 3 multiplexed assays for measurement of multiple autoantibodies and their association with global disease activ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.04.005
更新日期：2010-06-30 00:00:00
abstract：:Immunoabsorbents for the removal of antibodies against cell membrane antigens have been prepared by a variety of techniques in common use. Cells were surface-labelled by lactoperoxidase-catalysed radioiodination, and the different immunoabsorbent preparations were compared in terms of efficiency of incorporation and s...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90222-3
更新日期：1978-01-01 00:00:00
abstract：:Recent data generated in our laboratory indicates that human lymphokine-activated killer (LAK) cells can be induced in several commercially available serum-free medium formulations. Interestingly, LAK cells were also generated in RPMI 1640 medium plus 1000 U/ml IL-2 without addition of human serum. However, higher lev...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90239-6
更新日期：1988-05-09 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90100-8
更新日期：1992-07-06 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90401-0
更新日期：1984-10-26 00:00:00
abstract：:We describe the use of 7-amino-actinomycin D (7AAD) to measure phagocytosis and the opsonizing capacity of serum. Heat-inactivated Candida albicans was previously stained with 7AAD and incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytic cells were identified by th...
journal_title：Journal of immunological methods
pub_type：
doi：10.1016/s0022-1759(01)00358-1
更新日期：2001-07-01 00:00:00
abstract：:A new strategy has been developed for enzyme immunoassay of small molecules. The method is based on the formation and estimation of the complex Ab.H-H.Ab* where Ab and Ab* are immobilised and labeled antibodies respectively and H-H is a synthetic bis-analogue of the hapten H. The amount of the complex formed is invers...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(12)80005-6
更新日期：1992-03-04 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90021-7
更新日期：1991-03-21 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90388-6
更新日期：1985-05-10 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2009.06.010
更新日期：2009-08-31 00:00:00
abstract：:Conditions for the reproducible measurement of thymic hormonal effect on the functional activity of human peripheral blood lymphocytes (PBL) were determined using a mixed lymphocyte-tumor culture assay. Three thymic hormonal preparations (thymopoietin, thymic humoral factor and TP-1) were tested by this assay and foun...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90312-9
更新日期：1980-01-01 00:00:00
abstract：:AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to the depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help in diagnosi...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2009.10.001
更新日期：2009-12-31 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90159-1
更新日期：1987-08-03 00:00:00
abstract：:Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK-STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the rece...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00177-7
更新日期：2003-07-01 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：:A 51Cr-release assay and terminal 51Cr-labeling assay for measuring cell-mediated immunity to adherent target cells is described. Both techniques utilize small 10 microliter-per well microtiter plates, require low numbers of target cells (50-500 per well), and consequently, relatively small numbers of effector cells p...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90054-6
更新日期：1978-01-01 00:00:00
abstract：:A new quantitative assay for migration inhibitory factor (MIF) employs 3H-labelled cultured mouse or human lymphoid cells migrating from capillary tubes. Capillaries filled with labelled cells are placed in liquid scintillation counting vials, along with the MIF-containing sample and are removed at the end of a five-h...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90258-2
更新日期：1978-01-01 00:00:00
abstract：:A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, suppl...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90487-4
更新日期：1986-07-24 00:00:00
