Dataset Information

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

ABSTRACT: We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

SUBMITTER: Ding L

PROVIDER: S-EPMC2998729 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

Ding Ling L Azam Mark M Lin Yu-Huei YH Sheridan James J Wei Shuanghong S Gupta Gigi G Singh Rakesh K RK Pauling Michelle H MH Chu Waihei W Tran Antares A Yu Nai-Xuan NX Hu Jiefeng J Wang Wei W Long Hao H Xiang Dong D Zhu Li L Hua Shao-Bing SB

Protein science : a publication of the Protein Society 20101001 10

We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One ...[more]

PMID: 20718039

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Project description:ObjectiveRheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA.MethodsBlood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell.ResultsBioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated.ConclusionThese findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Dataset Information

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

Publications

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

Similar Datasets

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