CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100.

Autor: Sritrakarn T; Biochemistry-Electrochemistry Research Unit, School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand., Lowhalidanon K; Biochemistry-Electrochemistry Research Unit, School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Institute of Research and Development, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand., Khunkaewla P; Biochemistry-Electrochemistry Research Unit, School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Electronic address: kpanida@sut.ac.th.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Proteins and proteomics [Biochim Biophys Acta Proteins Proteom] 2025 Jan 01; Vol. 1873 (1), pp. 141058. Date of Electronic Publication: 2024 Oct 23.
DOI: 10.1016/j.bbapap.2024.141058
Abstrakt: In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297-3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1-1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (K aff ) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 10 9 Mol -1 , 7.25 ± 3.56 × 10 8 Mol -1 and 4.39 ± 2.63 × 10 6 Mol -1 , respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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