Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework

Jirholt, Pernilla; Ohlin, Mats; Borrebaeck, Carl; Söderlind, Eskil

doi:10.1016/s0378-1119(98)00317-5

Cited by 80 publications

(50 citation statements)

References 19 publications

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“…20) Assembly and Expression of the Wild-Type scFv Gene The V H and V L DNA fragments for Ab-CS#3, which were cloned in pBluescript II plasmid (Agilent Technologies; Hachioji, Tokyo, Japan) previously, 20) were amplified separately to add restriction sites for subcloning into the phagemid vector pEXmide 7, a variant of pEXmide 5 21) having cloning sites for V H and V L separately, each of which is directly adjacent (upstream and downstream) to a linker sequence present in the vector ( Fig. 2; preparation not published previously).…”

Section: Methodsmentioning

confidence: 99%

A Single-Step “Breeding” Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity

Oyama

Morita

Kiguchi

et al. 2017

Biological & Pharmaceutical Bulletin

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Cortisol levels in bodily fluids represent a useful index for pituitary adrenal function, and thus practical anti-cortisol antibodies are required. We have studied "antibody-breeding" approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A "wild-type" single-chain Fv fragment (wt-scFv; K a , 3.4 10 8 M 1 ) was prepared by bacterial expression of a fusion gene combining the V H and V L genes for this antibody. Then, random point mutations were generated separately in V H or V L by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity (K a 1.2 10 10 M 1 ). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its V L sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated doseresponse curves with measuring range ca. 0.03-0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.Key words antibody; single-chain Fv fragment; cortisol; in vitro evolution; affinity maturation; enzymelinked immunosorbent assay Immunoassays are essential tools for monitoring various biomarkers in bodily fluids because of the excellent specificity conferred by antigen−antibody reactions. 1) Currently, most diagnostic antibodies are produced by B-cell hybridoma technology, 2,3) which generates "native" antibodies (in vivo antibodies) induced in animals by immunization as cloned products ensuring constant binding abilities. However, the limited B-cell clone repertoire in mammals often prevents the generation of antibodies with practical performance. In particular, the equilibrium affinity constant (K a ) of antibodies against small biomarkers (i.e., haptenic compounds) rarely exceeds the 10 10 M −1 range. 4,5)We have employed an "antibody-breeding" approach to overcome this limitation inherent to native antibodies and to enable subfemtomole detection of small molecules. 5) Using this approach, the antigen-binding affinity can be enhanced by in vitro mutagenesis and subsequent selection of improved species (i.e., in vitro affinity maturation).3,5-9) Typically, the antibody of interest is converted to the corresponding singlechain Fv fragment (scFv) [10][11][12] or Fab fragment (Fab) by ...

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Section: Methodsmentioning

confidence: 99%

A Single-Step “Breeding” Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity

Oyama

Morita

Kiguchi

et al. 2017

Biological & Pharmaceutical Bulletin

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“…14,16) The primers used were THC#33V H -Rev and THC#33V H -For (for amplifying V H ) and THC#33V L -Rev and THC#33V L -For (for amplifying V L ), respectively ( Table 1). Amplification products were gel-purified and spliced by overlap extension PCR 13,14) to generate a gene encoding "wild-type" scFv (wt-scFv), which was then subcloned into the NcoI-SalI site in pEXmide 5, 25) and propagated in Escherichia coli (E. coli) XL1-Blue cells (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…The mutated V H and V L genes that were amplified using the same unbalanced dNTPs (the products obtained with 0 and 0.10 mM MnCl 2 were combined) were spliced to produce scFv genes. 13,14) The two gene libraries of the mutated scFv ("dATP-diminished V H /V L " and "dGTPdiminished V H /V L " combinations) were separately ligated into the pEXmide 5, 25) and transformed into XL1-Blue cells. The resulting bacterial libraries were used to rescue phage particles, which were partially purified and examined as follows.…”

Section: Methodsmentioning

confidence: 99%

Antibody Fragments for On-Site Testing of Cannabinoids Generated <i>via in Vitro</i> Affinity Maturation

Morita

Oyama

Yasuo

et al. 2017

Biological & Pharmaceutical Bulletin

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Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ 9 -tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with K a 6.2 10 7 M 1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with K a , 1.1 10 7 M 1 was prepared by bacterial expression of a fusion gene combining the V H and V L genes for Ab-THC#33. Subsequently, random point mutations in V H and V L were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (K a 1.1 10 8 M 1 ) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the V H -complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

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“…One is the shuffling of gene segments, where VH and VL populations, for example, can be randomly recombined with each other (Figini et al, 1994;Schier et al, 1996) or be performed with CDRs (Jirholt et al, 1998;Knappik et al, 2000). An alternative approach is the possibility that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type.…”

Section: Affinity Increase By Random Mutationsmentioning

confidence: 99%

“…Naive libraries from non-immunized donors have been generated by PCR-cloning Ig repertoires from various B-cell sources (Marks et al, 1991;Vaughan et al, 1996;Sheets et al, 1998;de Haard et al, 1999)) derived from human or camel germ line genes and randomized only in the CDR3 regions (Hoogenboom & Winter, 1992;Nissim et al, 1994;de Kruif et al, 1995). Synthetic libraries have been generated from a repertoire of 49 human germline VH genes segments rearranged in vitro to create a synthetic CDR3 region (Hoogenboom & Winter, 1992) or derived from a single V-gene with complete randomization of all CDRs (Jirholt et al, 1998;Soderlind et al, 2000) (Table 1).…”

Section: Introduction On the Natural Diversity Of Immunoglobulinsmentioning

confidence: 99%

Parallel Processing of Complex Biomolecular Information: Combining Experimental and Computational Approaches

Jean-Luc¹,

Pierre²

2011

Systems and Computational Biology - Bioinformatics and Computational Modeling

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Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework

Cited by 80 publications

References 19 publications

A Single-Step “Breeding” Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity

A Single-Step “Breeding” Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity

Antibody Fragments for On-Site Testing of Cannabinoids Generated <i>via in Vitro</i> Affinity Maturation

Parallel Processing of Complex Biomolecular Information: Combining Experimental and Computational Approaches

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