Abstract：
:We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Jennings NS,Smethurst PA,Knight CG,O'Connor MN,Joutsi-Korhonen L,Stafford P,Stephens J,Garner SF,Harmer IJ,Farndale RW,Watkins NA,Ouwehand WHdoi
10.1016/j.jim.2006.08.003subject
Has Abstractpub_date
2006-10-20 00:00:00pages
75-83issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(06)00226-2journal_volume
316pub_type
杂志文章abstract：:A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red bloo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90097-2
更新日期：1983-02-25 00:00:00
abstract：:The possibility that immortalisation of in vitro immunised splenocytes may result in hybridomas secreting polyreactive antibodies was investigated. A panel of nine murine hybridomas, secreting IgM(kappa) anti-goat immunoglobulin G (anti-GIgG), was produced by immortalising splenocytes that had been immunised in vitro ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00481-1
更新日期：2001-12-01 00:00:00
abstract：:Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, howe...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.02.006
更新日期：2015-04-01 00:00:00
abstract：:A method is described for isolation of antibody-forming cells with the help of cluster formation. This was achieved by incubating spleen cells from immunized mice with sheep erythrocytes at 37 degrees C. During incubation rabbit-anti-mouse immunoglobulin serum was present in order to facilitate the formation of cluste...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90103-4
更新日期：1976-01-01 00:00:00
abstract：:A proteinaceous inhibitor of alpha-amylase, Tendamistat, was evaluated as an immunogen to induce antibodies that mimic the enzyme activity and to investigate a new strategy to produce catalytic antibodies. Anti-Tendamistat polyclonal antibodies (pAbs) were shown to cross-react with acarbose, a strong carbohydrate inhi...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00238-7
更新日期：2002-11-01 00:00:00
abstract：:An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90240-9
更新日期：1980-01-01 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：:The Farr technique has been compared with equilibrium dialysis in regard to its ability to measure antibody concentration and affinity. An excellent qualitative agreement between the two techniques was obtained. Quantitatively, the Farr technique tended to underestimate antibody concentration and overestimate antibody...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(75)90005-8
更新日期：1975-02-01 00:00:00
abstract：:To develop a stable immuno-assay system for quantification of human hemoglobin (Hb), the interaction between various antibodies and Hb was studied using a surface plasmon resonance (SPR) biosensor in the BIAcore equipment (Amersham Pharmacia Biotech) with an immobilized anti-Hb antibody sensor chip. When polyclonal an...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(00)00144-7
更新日期：2000-04-21 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：:A simple immunoenzyme technique is described in which the enzyme beta-galactosidase of E. coli (Z) is used as a marker. The principle of the test is based on the ability of the cells to bind the antigen. Conditions of the assay using Z both as the antigen and marker are described. As a screening technique, conditions ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90268-9
更新日期：1980-01-01 00:00:00
abstract：:We describe here a lateral flow-based assay (LFA) for the detection of antibodies against immunodominant antigen Pgp3 from Chlamydia trachomatis, the causative agent of urogenital chlamydia infection and ocular trachoma. Optimal signal detection was achieved when the gold-conjugate and test line contained Pgp3, creati...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2016.05.008
更新日期：2016-08-01 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：:Antibodies directed against elliptinium acetate, a quaternary ammonium compound with antineoplastic activity in man, were obtained in rabbits, after conjugation of the drug with haemocyanin. These antibodies are specific for the quaternary ammonium structure. However, the recognition of the drug can be markedly decrea...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90124-4
更新日期：1987-05-20 00:00:00
abstract：:Historically, tumour markers are substances selectively released by tumour cells in the blood stream so that they can be detected in the serum or other body fluids for clinical monitoring of various malignancies. The term is now extended to cell or tissue characteristics, such as cytogenetic markers, oncogenes or abno...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/0022-1759(92)90072-2
更新日期：1992-06-24 00:00:00
abstract：:The level of CD4(+) T-lymphocytes represents a useful marker with which to monitor the progression of HIV infection. Sex and geographical differences in the reference values of lymphocyte subsets have been reported. We have compared two flow cytometric methods (MultiSET and SimulSET) for the quantification of lymphocy...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00174-1
更新日期：2003-06-01 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：:Although many different protocols for transfection of lymphoid cell lines exist, successful DNA transfer into primary B lymphocytes has, to date, not been demonstrated. We now describe a simple method for gene transfer into highly purified normal and malignant B lymphocytes by electroporation. Using a powerful express...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90321-l
更新日期：1990-10-04 00:00:00
abstract：:A system proposed for measurement of mutational risk consists in detection of hemoglobin mutations expressed in erythrocytes. For this detection the production of antibodies specific for Hb variants is essential. Recently we reported a sensitive solid-phase EIA for the production and selection of polyclonal and monocl...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90189-8
更新日期：1983-05-13 00:00:00
abstract：:Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00029-7
更新日期：2002-05-01 00:00:00
abstract：:Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2018.04.007
更新日期：2018-07-01 00:00:00
abstract：:Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and effic...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2018.05.007
更新日期：2018-10-01 00:00:00
abstract：:The common marmoset is a small New World Primate that has been used as an immunological model for a number of human diseases. Dendritic cells (DC) have not been extensively characterised in this species and in particular protocols to derive DC from living donors without the need for animal sacrifice are presently lack...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2009.11.008
更新日期：2010-01-31 00:00:00
abstract：:We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human heart type fatty acid-binding protein (H-FABP) in human plasma and urine using the combination of two distinct monoclonal antibodies (MAbs) directed against human H-FABP purified from human heart muscle. The total ass...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)00248-u
更新日期：1995-01-13 00:00:00
abstract：:Two methods for estimating IgA in body fluids of rats were devised and tested for their accuracy and reliability. Specifically purified monomeric and polymeric IgA was prepared so that results obtained could be properly assessed. The two systems tried were prepared so that results obtained could be properly assessed. ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90293-x
更新日期：1979-01-01 00:00:00
abstract：:Systems biology approaches that utilize large genomic data sets hold great potential for deciphering complex immunological process. In this paper, we propose such an approach to derive informative modules and networks from large gene expression data sets. Our approach starts with the clustering of such data sets to de...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.09.012
更新日期：2013-01-31 00:00:00
abstract：:Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2005.10.012
更新日期：2006-01-20 00:00:00
abstract：:A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90168-1
更新日期：1985-06-12 00:00:00
abstract：:Formation of rosettes with sheep RBC by blood lymphocytes from young pigs was increased from 24.4% +/- 2.2 (mean +/- S.E.) in PBS to 54.1% +/- 1.9 in the presence of dextran. This increase was achieved without inducing appreciable rosette formation with other RBC which do not form rosettes in PBS. Lymphocytes which r...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90146-1
更新日期：1978-01-01 00:00:00
abstract：:Foot and mouth disease is caused by a non-enveloped virus (FMDV), which disposes several antigenic sites at the surface of their capsid proteins. The most relevant and immunodominant antigenic site of FMDV (site A or AnSA) includes a key virus-cell interaction element (RGD motif) located in the Viral Protein 1 (VP1), ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.06.008
更新日期：2015-10-01 00:00:00
