A new system for the expression of recombinant antibody in mammalian cells

Iba, Yoshitaka; Kaneko, Takashi; Ekida, Teiji; Miyata, Kenji; Inouye, Kuniyo; Kurosawa, Yoshikazu; Yasukawa, Kiyoshi

doi:10.1007/bf00127976

Cited by 6 publications

(1 citation statement)

References 10 publications

(9 reference statements)

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“…Some efforts have been paid in order to overcome the difficulty in application of IgM. Screening class switch variants producing IgG mAbs from IgM-producing cells (Steplewski et al, 1985) and preparing single-chain antibodies by gene engineering (Winter and Milstein, 1991;Iba et al, 1995) are the examples. We have recently developed a convenient method for the preparation of F(ab') 2 fragments of 145 kDa from pepsin digests of mouse and rat IgM mAbs (Inouye and Morimoto, 1993a, 1993b, 1994 and suggested that the term F(ab') 2 should be used instead of F(ab') 2 to indicate that the fragments are prepared from IgM by cleaving the heavy chains.…”

Section: Introductionmentioning

confidence: 99%

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Morimoto¹,

Inouye²

1997

Cytotechnology

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F(ab')2 fragments, herein designated as F(ab')2μ fragments, were prepared from a mouse IgM monoclonal antibody specific to sialyl Lewis A antigen. The fragments were applied to flow cytometry to analyze the antigen on human cancer cells. The binding of the fragments to the antigen-positive cells was stronger than that of the original IgM. The non-specific binding of the IgM antibody to the antigen-negative cells was much decreased by using the F(ab')2μ fragments. These results indicate that the F(ab')2μ fragments are more suitable than the original IgM monoclonal antibody in flow cytometric analysis.

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Section: Introductionmentioning

confidence: 99%

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Morimoto¹,

Inouye²

1997

Cytotechnology

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Converting Monoclonal Antibodies into Fab Fragments for Transient Expression in Mammalian Cells

Nettleship

Flanagan

Rahman-Huq

et al. 2011

Methods in Molecular Biology

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In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

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Genetically engineered antibodies which have reduced binding affinity for thyroxine but retain the same affinity for tri-iodothyronine

et al. 1996

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Based on a three-dimensional molecular model of the variable region of a monoclonal antibody (Ab) TT1, in which the complementarity determining regions (CDRs) associate to form a cavity large enough to accommodate a single molecule of tri-iodothyronine (T3) or thyroxine (T4), we designed TT1 mutants with one amino acid substitution as candidates which have their binding affinity for T4 reduced but retain the same affinity for T3. Each candidate was subsequently tested by site-directed mutagenesis, transient expression in COS cells, and surface plasmon resonance (SPR) analysis for its binding ability for T3-or T4-conjugates with alkaline phosphatase (AP). Of the candidates, the Ab with serine in place of glycine at position 92 of the light chain (L;G92S) and the Ab with alanine in place of leucine at position 47 of the heavy chain (H;L47A) had the association constant (KA = kass / kdiss) for binding to T4-AP decreased by 5-fold, but retained the same KA for T3-AP.

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A new system for the expression of recombinant antibody in mammalian cells

Cited by 6 publications

References 10 publications

Untitled

Untitled

Converting Monoclonal Antibodies into Fab Fragments for Transient Expression in Mammalian Cells

Genetically engineered antibodies which have reduced binding affinity for thyroxine but retain the same affinity for tri-iodothyronine

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