Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping.
Commun Biol
; 7(1): 805, 2024 Jul 03.
Article
em En
| MEDLINE
| ID: mdl-38961245
ABSTRACT
Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Mapeamento de Epitopos
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Glicoproteína da Espícula de Coronavírus
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SARS-CoV-2
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COVID-19
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Epitopos
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article