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 cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell. We have developed a novel strategy to identify higher productivity cells at the FACS step by leveraging a simple viable cell staining method that detects mitochondrial membrane potential (Ψm), a key indicator of cellular metabolic activity. We chose a dual-emission dye (Mito-ID, Enzo Life Sciences) that fluoresces green and orange in living cells with the intensity of the orange fluorescence being dependent on the cells Ψm status. Using available clonal cell lines with known productivity, or stable transfectant pools, we evaluated Ψm of cell populations with Mito-ID dye. We determined that the intensity of the Ψm fluorescent signal correlates with the known fed-batch titers of the producer clones, and that cell sorting based on an optimal Ψm staining intensity selectively enriches for higher producing clones from nonclonal transfectant pools. These clones are phenotypically stable for recombinant protein production. Furthermore, the strategy has been successfully applied to identifying higher producing cell lines for a range of antibody molecular formats. Using this method, we can combine an enriching step with the cloning step for high producers, thereby saving time and resources in cell line development.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Chakrabarti L,Mathew A,Li L,Han S,Klover J,Albanetti T,Hawley-Nelson Pdoi
10.1016/j.jim.2018.10.007subject
Has Abstractpub_date
2019-01-01 00:00:00pages
31-39eissn
0022-1759issn
1872-7905pii
S0022-1759(18)30192-3journal_volume
464pub_type
杂志文章abstract：:Endogenous heterophilic antibodies in blood are known to interfere with two-site enzyme-linked immunosorbent assays (ELISAs) evoking false positive signals. In the present study, we describe an assay for the assessment of components of the plasminogen activation system (uPA, tPA and PAI-1, and their complexes) in bloo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00213-2
更新日期：2002-10-15 00:00:00
abstract：:There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibod...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00281-2
更新日期：1996-04-19 00:00:00
abstract：:Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the unde...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00124-5
更新日期：1998-03-01 00:00:00
abstract：:The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90323-l
更新日期：1992-08-30 00:00:00
abstract：:The presence of KIR4.1 antibodies has been proposed to be a characteristic of Multiple Sclerosis (MS). This could have a significant impact on disease management. However, the validation of the initial findings has failed till date. Conflicting results have been attributed to difficulties in isolating the lower-glycos...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2017.03.008
更新日期：2017-06-01 00:00:00
abstract：:The present article reports a multiparametric cytofluorimetric analysis of apoptosis in murine thymocytes and human PBMC from healthy donors or HIV-infected patients. We have evaluated four previously described cytofluorimetric methods of apoptosis quantification, each of them detecting distinct cellular alterations o...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(96)00148-2
更新日期：1996-10-30 00:00:00
abstract：:A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OK...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90482-5
更新日期：1986-07-24 00:00:00
abstract：:Nonspecific effects of reagents used to selectively isolate lymphocyte subpopulations are most often analyzed according to their influence on cell viability and proliferation. While characterizing monoclonal anti-human B cell antibodies, we detected consistent changes in B cell function between individual donors, no m...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90352-1
更新日期：1984-02-10 00:00:00
abstract：:The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for human blood group antigens extracted from blood stains with the surfactant, n-octyl-beta-D-glucopyranoside (OBG), at concentrations below the critical micelle concentration can be increased by the introduction of a single freeze-thaw step. The ELISA s...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90245-o
更新日期：1992-05-18 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2014.10.009
更新日期：2015-01-01 00:00:00
abstract：:As much as one percent of antibodies in human plasma are reported to be specific for the non-human disaccharide structure αGal. Various microbes express αGal. However, the implications of anti-αGal antibodies for the anti-microbial defenses are poorly established. With the perspective of studying the biological import...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2011.07.017
更新日期：2011-10-28 00:00:00
abstract：:Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generat...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.01.005
更新日期：2010-02-28 00:00:00
abstract：:A simple apparatus for measuring chemotaxis is described which contains thirty chambers per unit. Only very few cells and small volumes of chemotactic factor solution are required per chamber. For satisfactory results the apparatus must be centrifuged briefly prior to incubation. Under these conditions results can be ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)90008-9
更新日期：1977-01-01 00:00:00
abstract：:Several methods have been used to measure anti-double-stranded DNA auto-antibody (anti-dsDNA). Our aim was to determine the most efficient strategy to test anti-dsDNA in systemic lupus erythematosus (SLE). In this study, anti-dsDNA and anti-nuclear antibody (ANA) tests were requested for 644 patients. Anti-dsDNA was t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.09.003
更新日期：2015-12-01 00:00:00
abstract：:Hybridoma cells producing monoclonal antibodies against tumor-shed antigens were generated by fusing mouse myeloma cells with spleen cells from immunosuppressed mice bearing human melanoma xenografts. Thirty-eight fusion experiments were performed at different stages of tumor growth. Hybridomas producing anti-melanoma...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90074-1
更新日期：1983-02-25 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：: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：:Fungi represent emerging infectious threats to human populations worldwide. Mice and other laboratory animals have proved invaluable in modeling clinical syndromes associated with superficial and life-threatening invasive mycoses. This review outlines salient features of common vertebrate animal model systems to study...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/j.jim.2014.03.022
更新日期：2014-08-01 00:00:00
abstract：:A mixed hemadsorption (MH) test for naturally acquired rubella immunity and immune responses after rubella vaccination has been compared with the hemagglutination-inhibition (HI) test, the immunodiffusion (ID) test, the neutralization test (NT), the hemolysis-in-gel (HIG) test and the indirect immunofluorescence (IF) ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90282-3
更新日期：1980-01-01 00:00:00
abstract：:We describe an intracellular cytokine staining, flow cytometry based technique for quantitative and qualitative assessment of CD8+ T lymphocyte-mediated antigen specific immune responses. This adaptation of the technique first described by Suni et al. [J. Immunol. Methods, 212 (1998) 89; Cytometry, 40 (2000) 60] utili...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00025-x
更新日期：2002-04-01 00:00:00
abstract：:Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2014.01.010
更新日期：2014-03-01 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.10.005
更新日期：2013-01-31 00:00:00
abstract：:In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2009.09.003
更新日期：2009-12-31 00:00:00
abstract：:We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 X 10(6) mol.wt. were easily and rapidly focused. ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90130-3
更新日期：1979-01-01 00:00:00
abstract：:We found that some plasma proteins such as haptoglobin (Mr 200,000) and alpha 1-acid glycoprotein (Mr 40,000) do not bind at all to nitrocellulose membranes in classical transfer conditions. In contrast, these proteins can be clearly identified when a charged nylon membrane is disposed on the anodal side of the nitroc...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90226-8
更新日期：1988-03-16 00:00:00
abstract：:Signalling pathways modulated by the family of IL-1/TLR receptors are central to innate immune responses. Novel components playing key regulatory roles in these pathways continue to be isolated. Here we describe the use of autocatalytic vectors for identifying critical components of these signalling pathways. The meth...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.11.002
更新日期：2008-01-31 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：:An ELISA method for the detection of monoclonal antibodies (MAb) to the T3-T cell antigen receptor (TCR) complex was devised. The T3-TCR complex was solubilised using digitonin and a rat anti-T3 MAb (Campath 3) was used to bind it to an ELISA plate. Normal rat serum was used to block cross-reactivity between the rat M...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(88)90280-3
更新日期：1988-01-21 00:00:00
abstract：:Intracellular probes used for oxidative burst evaluation could be a substrate of the multidrug resistance proteins (MDR), which may cause misinterpretation of experimental data. We aimed to study the effect of the culture condition and of the MDR-interfering antioxidant quercetin on free radical measurement in serum d...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.12.009
更新日期：2014-03-01 00:00:00
abstract：:Immunoadsorbents of Sepharose-4B-conjugated antibodies were shown to be suitable for the characterization, by subsequent SDS-polyacrylamide gel electrophoresis, of splenocyte-membrane Immunoglobulin (Ig) solubilized by detergent lysis of surface 125I-labelled cells. However, high non-specific binding of 125I-labelled ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90053-3
更新日期：1976-01-01 00:00:00
