De novo assisted AFB1-Specific monoclonal antibody sequence assembly and comprehensive molecular characterization.
Anal Biochem
; 656: 114883, 2022 11 01.
Article
em En
| MEDLINE
| ID: mdl-36063915
ABSTRACT
Despite their widely used and access as biological reagents in analytical methods, the detailed structural features for most of the antibodies were rarely known. Here, a new antibody for AFB1 with high specificity in constructing ELISA was studied in detail. The molecular structure and modification were elucidated mainly by nano-electrospray ionization mass spectrometry. The mass experiments, including MALDI-TOF MS, revealed complete and specific fragments, including antibody molecular weight, peptides, glycopeptide, and N-glycoform. By proteolytic treatment of pepsin and trypsin and high-resolution tandem-MS, the primary structure of the newly developed anti-AFB1 antibody was assembled by several rounds of Database search process assisted with the de novo results. The antibody CDR annotation and constraint-based multiple alignment tool were used to differentiate and align the sequences. The method uses only two proteases to generate numerous peptides for de novo sequencing. This artificial assembled AFB1-specific monoclonal antibody sequence was validated by comparison with the sequencing results of the immunoglobulin gene. The results showed that this method achieves full sequence coverage of anti-AFB1 monoclonal antibody, with an accuracy of 100% in the CDR regions of light chain and four amino acid mismatch in heavy chain. This simple and low-cost method was confirmed by treating a public dataset. The secondary structure information of intact antibody was also elucidated from the results of circular dichroism spectrum.
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Pepsina A
/
Anticorpos Monoclonais
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2022
Tipo de documento:
Article