Cloning, Expression, and Purification of an Anti‐Desipramine Single Chain Antibody in NS/O Myeloma Cells †

Kitchin, Kirsten; Lin, Gaofeng; Shelver, Weilin L.; Murtaugh, Michael P.; Pentel, Paul R.; Pond, Susan M.; Oberst, Janelle C.; Humphrey, J. A. C.; Smith, John L.; Flickinger, Michael C.

doi:10.1002/jps.2600841009

Cited by 9 publications

(2 citation statements)

References 45 publications

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“…Generally the secreted protein is then correctly folded and has a homogeneous N‐terminus [1]; nevertheless, the yield is often low and periplasmic inclusion bodies and dimers or higher polymers can occur [20]. Alternative expression systems have been used for scFv, including yeast [21–23], mammalian cells [24], plants [25, 26], and insect cells with the baculovirus vector [27, 28]. The latter offers theoretically a strong advantage over the other systems for therapeutic purposes.…”

Section: Introductionmentioning

confidence: 99%

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Lemeulle¹,

Chardès²,

Montavon³

et al. 1998

FEBS Letters

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A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 F ab , whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.z 1998 Federation of European Biochemical Societies.

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Section: Introductionmentioning

confidence: 99%

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Lemeulle¹,

Chardès²,

Montavon³

et al. 1998

FEBS Letters

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“…Due to the absence of intermolecular disulfide bonds, these fragments easily dissociate (Glockshuber et al, 1992); however, this instability can be removed by producing recombinant Fv fragments that have the heavy and light variable regions linked by a synthetic peptide linker. This recombinant fragment is a single-chain Fv fragment (scFv) and has a molecular weight of 27000 (Bird et al, 1988;Huston et al, 1988;Bird and Walker, 1991;Kitchin et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Immunoassay for Wheat Processing Quality: Utilization of a Sandwich Assay Incorporating an Immobilized Single-Chain Fragment

Hill

Giersch

Loh

et al. 1999

J. Agric. Food Chem.

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A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

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Effects of recombinant drug-specific single chain antibody Fv fragment on [3H]-desipramine distribution in rats

Shelver

Keyler

Lin

et al. 1996

Biochemical Pharmacology

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Cloning, Expression, and Purification of an Anti‐Desipramine Single Chain Antibody in NS/O Myeloma Cells †

Cited by 9 publications

References 45 publications

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Immunoassay for Wheat Processing Quality: Utilization of a Sandwich Assay Incorporating an Immobilized Single-Chain Fragment

Effects of recombinant drug-specific single chain antibody Fv fragment on [3H]-desipramine distribution in rats

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