Continuous cultures of fused cells secreting antibody of predefined specificity

Köhler, Georges; Milstein, C.

doi:10.1038/256495a0

Cited by 15,664 publications

(5,303 citation statements)

References 12 publications

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“…The hybridoma approach, pioneered by Köhler and Milstein in 1975, was the most used technology to isolate and produce antibodies for many years 2 . More recently, in vitro production of mAbs revolutionized the way to isolate and produce mAbs and, to date, these methods represent the default way to perform screenings against new targets 3 .…”

Section: Introductionmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

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Section: Introductionmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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show abstract

“…This procedure results in antibodies with higher specificity than conventional polyclonal antibodies, still retaining the ability to recognize different epitopes on the same antigen (Lindskog et al., 2005). A monoclonal antibody is generated by selection of one single B cell from spleen or bone marrow of the immunized animal and fusing this cell with immortal myeloma cells to produce hybridoma cells (Kohler and Milstein, 1975). As such, the culture supernatant contains only one type of antibody specific for a single epitope of the immunizing peptide.…”

Section: Review Of Factors Influencing the Ihc Processmentioning

confidence: 99%

Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers

et al. 2014

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The use of immunohistochemistry (IHC) in clinical cohorts is of paramount importance in determining the utility of a biomarker in clinical practice. A major bottleneck in translating a biomarker from bench‐to‐bedside is the lack of well characterized, specific antibodies suitable for IHC. Despite the widespread use of IHC as a biomarker validation tool, no universally accepted standardization guidelines have been developed to determine the applicability of particular antibodies for IHC prior to its use. In this review, we discuss the technical challenges faced by the use of immunohistochemical biomarkers and rigorously explore classical and emerging antibody validation technologies. Based on our review of these technologies, we provide strict criteria for the pragmatic validation of antibodies for use in immunohistochemical assays.

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“…mAbs feature the recognition of a single antigenic determinant, i.e. a specific epitope [35]. Therefore the application of mAbs provides a novel approach to monitoring protein conformation, structural dynamics and folding [43].…”

Section: Antibody Epitope Analysis To Monitor Calponin Conformationalmentioning

confidence: 99%

“…Antigen-antibody interaction is a protein-protein interaction depending on the conformational fits between the antigenic epitope and the specific antibody paratope. Using a panel of monoclonal antibodies (mAbs) [35] against multiple specific epitopes on calponin, we investigated in the present study the role of serine-175 in the modulation of calponin molecular conformation. Quantification of the interactions between the mAbs and calponin demonstrated that the conformation of calponin was allosterically modulated by phosphorylation of serine-175, supporting the regulatory role of this site.…”

Section: Introductionmentioning

confidence: 99%

A role for serine-175 in modulating the molecular conformation of calponin

Jin

Walsh

Sutherland

et al. 2000

Biochemical Journal

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Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175 alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects : (a) structures of calponin are reconfigured by serine-175 modification, supporting

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Continuous cultures of fused cells secreting antibody of predefined specificity

Cited by 15,664 publications

References 12 publications

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers

A role for serine-175 in modulating the molecular conformation of calponin

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