Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split-Mix-Split Method and Its Application

Yin, Chang-Cheng; Ren, Lili; Zhu, Liping; Wang, Xiang-Bin; Zhang, Zhong; Huang, Hua-Liang; Yan, Xiyun

doi:10.1093/jb/mvn103

Cited by 10 publications

(7 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Owing to the technical limitations, the maximum size of naïve or synthetic libraries is 8 × 10 9 CFU/mL [ 32 - 35 ]. Based on a split-mix-split method, a synthetic human scFv antibody library with randomized CDR3 was successfully constructed and applied for isolation of a scFv specific to antigen BHL (anti-CD3 × anti-ovarian carcinoma bispecific antibody) [ 36 ]. Naïve phage display Nb libraries have also been reported in several studies [ 15 , 37 ].…”

Section: Discussionmentioning

confidence: 99%

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications

Yan

et al. 2014

J Transl Med

View full text Add to dashboard Cite

BackgroundNanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.MethodsWe constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.ResultsA large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 109 CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL.ConclusionThis proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.

show abstract

Section: Discussionmentioning

confidence: 99%

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications

Yan

et al. 2014

J Transl Med

View full text Add to dashboard Cite

show abstract

“…Libraries encoding proteins with mutations at a variety of positions have so far been constructed using methods that require PCR amplification (Cobaugh et al, 2008;Feldhaus et al, 2003;Hoet et al, 2005;Knappik et al, 2000;Orlandi et al, 1989;Rajpal et al, 2005;Rothe et al, 2008;Silacci et al, 2005;Soderlind et al, 2000;Yin et al, 2008). However, PCR amplification can introduce a significant number of inadvertent mutations in constant regions since any errors in early amplification cycles are amplified during subsequent rounds (Cline et al, 1996;Pienaar et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Rapid construction and characterization of synthetic antibody libraries without DNA amplification

Mazor

Hunicke-Smith

et al. 2010

Biotech & Bioengineering

View full text Add to dashboard Cite

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.

show abstract

“…21 Therefore, a fixed CDR length was designed to incorporate the NNK degenerate trinucleotide for each of the CDRs with two complementary overlaps of 18 bp at the 5′ and 3′ ends. The trinucleotides in general encode all 20 amino acids and eliminate two out of three stop codons.…”

Section: Vh Domain Antibody Gene Designmentioning

confidence: 99%

Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

et al. 2016

View full text Add to dashboard Cite

The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

show abstract

Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split-Mix-Split Method and Its Application

Cited by 10 publications

References 22 publications

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications

Rapid construction and characterization of synthetic antibody libraries without DNA amplification

Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

Contact Info

Product

Resources

About