Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in<i>Escherichia coli</i>

Strachan, Gillian; McElhiney, Jacqui; Drever, Matthew Ross; McIntosh, F.S.; Lawton, Linda A.; Porter, A.J.

doi:10.1111/j.1574-6968.2002.tb11190.x

Cited by 45 publications

(8 citation statements)

References 18 publications

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“…However, modified panning strategies used to increase the likelihood of isolating high-affinity hapten binders are: 1 to gradually decrease the coating conjugate concentration during successive rounds of panning [25]; 2 to use the free hapten to elute the hapten-specific antibodies; 3 to use analogues with which the antibodies are preincubated prior to their incubation with the target hapten conjugate; 4 to perform subtractive panning, where the antibodies are incubated with the carrier protein prior to incubation with the hapten–protein conjugate [26]. Subtractive panning should remove any antibody binding to the carrier protein, but not those antibodies binding to the bridging group linking the protein and the hapten.…”

Section: Discussionmentioning

confidence: 99%

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

et al. 2012

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Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured KD was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

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Section: Discussionmentioning

confidence: 99%

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

et al. 2012

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“…Recent studies have suggested that while shorter heavy-chain CDR3s tend to form planar binding sites suitable for binding large proteins, longer heavychain CDR3s are capable of forming anti-hapten binding pockets (5,36). The heavy-chain CDR3 in the Tomlinson I library is conserved at seven amino acids, while the Griffin.1 library incorporates variable heavy-chain CDR3 lengths.…”

Section: Discussionmentioning

confidence: 99%

“…The heavy-chain CDR3 in the Tomlinson I library is conserved at seven amino acids, while the Griffin.1 library incorporates variable heavy-chain CDR3 lengths. Panning of the Griffin.1 library against microcystin-LR conjugates enabled the isolation of a more sensitive anti-microcystin-LR clone (3A8), which was found to have 10 amino acid residues at this position (36).…”

Section: Discussionmentioning

confidence: 99%

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

McElhiney

Drever

Lawton

et al. 2002

Appl Environ Microbiol

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A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 g liter ؊1 ) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

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“…Alternatively, recombinant antibody libraries can be built using synthetic diversity (7–9) in which antibody complementarity determining region (CDR) containing gene fragments are generated with mixed-nucleotide synthesis (10). A semi-synthetic approach can also be used to further diversify native heavy and light chain genes by synthetically randomizing the CDRs (11). Disadvantages to these library types include variable biophysical properties and expression levels of heterogeneous frameworks, and stop codons in mixed-nucleotide sequences in synthetic and semi-synthetic approaches.…”

Section: Introductionmentioning

confidence: 99%

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

et al. 2016

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Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>1020), the practical diversity that can be achieved by transformation of E. coli is limited to about 1010. To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

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Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed inEscherichia coli

Cited by 45 publications

References 18 publications

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

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