Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody
| العنوان: | Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody |
|---|---|
| المؤلفون: | Yirong Guo, Shasha Jiao, Yuanhao Guo, Yunyun Chang, Guonian Zhu, Mengli Chen, Liu Pengyan, Ying Liu, Rubing Zou, Yihua Liu |
| المصدر: | International Journal of Molecular Sciences, Vol 21, Iss 6857, p 6857 (2020) International Journal of Molecular Sciences Volume 21 Issue 18 |
| بيانات النشر: | MDPI AG, 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | 0301 basic medicine, Insecticides, Immunoglobulin Variable Region, Thiazines, Complementarity determining region, 01 natural sciences, law.invention, lcsh:Chemistry, Mice, Neonicotinoids, law, Tandem Mass Spectrometry, lcsh:QH301-705.5, Spectroscopy, Mice, Inbred BALB C, sequences of variable region genes, biology, medicine.diagnostic_test, Antibodies, Monoclonal, High-Throughput Nucleotide Sequencing, General Medicine, Computer Science Applications, Molecular Docking Simulation, recognition sites, Recombinant DNA, Female, Antibody, Immunoglobulin Heavy Chains, medicine.drug_class, Computational biology, Monoclonal antibody, Catalysis, Article, Inorganic Chemistry, 03 medical and health sciences, Inhibitory Concentration 50, medicine, Animals, Humans, recombinant full-length IgG, Amino Acid Sequence, immunoassay, Physical and Theoretical Chemistry, LC-MS/MS, Molecular Biology, Gene, Hybridomas, 010401 analytical chemistry, Organic Chemistry, Antibody Diversity, Hydrogen Bonding, Complementarity Determining Regions, 0104 chemical sciences, Kinetics, 030104 developmental biology, HEK293 Cells, lcsh:Biology (General), lcsh:QD1-999, Immunoassay, biology.protein, Mutagenesis, Site-Directed, next-generation sequencing, Rab, thiacloprid, Chromatography, Liquid |
| الوصف: | Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 &mu g/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH&ndash &pi interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1661-6596 1422-0067 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a6cf3ff277944130cb0641902f5169d1 https://www.mdpi.com/1422-0067/21/18/6857 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....a6cf3ff277944130cb0641902f5169d1 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 16616596 14220067 |
|---|