Dataset Information

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

ABSTRACT: Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

SUBMITTER: Wang B

PROVIDER: S-EPMC6888773 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

Wang Bei B Wang Fei F Huang He H Zhao Zhendong Z

Virologica Sinica 20190625 6

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the D ...[more]

PMID: 31240617

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Project description:We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1?. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.

Dataset Information

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

Publications

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

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