Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody
Abstract
:1. Introduction
2. Results
2.1. Recognition Features of the mAb Measured by Surface Plasmon Resonance (SPR)
2.1.1. The mAb Selectivity
2.1.2. Kinetics and Affinity of the mAb
2.2. Sequence Diversity and Abundance of VR genes
2.2.1. VR Gene Clone Types and Abundances
2.2.2. Accurate Full-Length Sequences of Interested VH and VL Clones
2.3. Determination of VR Sequences of the mAb
2.4. Performance of the Full-Length rAb and the Parental mAb
2.4.1. Selectivity and Sensitivity to Neonicotinoid Pesticides
2.4.2. Gold Immunochromatographic Strip (GICS) Application
2.5. The Key Amino Acids in the CDRs Responsible for Specific Binding of Thiacloprid
3. Discussion
4. Materials and Methods
4.1. SPR Evaluation of the mAb against Thiacloprid
4.2. Seeking of Multiple Accurate Sequences and Abundances of VR genes
4.2.1. 5′-RACE and NGS of Antibody Transcriptome of Hybridoma
4.2.2. Amplification of Accurate Full-Length Sequences of Related VRs by Sanger Sequencing
4.3. High-Resolution LC-MS/MS and Peptide Coverage Analysis
4.4. Performance Tests of the Expressed Full-Length rAb and the Parental mAb
4.4.1. IC-ELISA
4.4.2. GICS Test
4.5. Discovery of the Key Amino Acids in the CDRs by Silico Analysis and Site-Directed Mutagenesis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
mAb | monoclonal antibody | SPR | surface plasmon resonance |
rAb | recombinant antibody | LMW | low molecular weight |
Ig | immunoglobulin | RU | resonance unit |
CR | cross reactivity | UMB | unique molecular barcode |
NGS | next-generation sequencing | IC-ELISA | indirect competitive ELISA |
CDR | complementarity-determining region | κ | kappa |
LOD | limit of detection | λ | lambda |
VR, VH, VL | variable region, variable region of a heavy chain, and variable region of a light chain | GICS | gold immunochromatographic strip |
LC-MS/MS | liquid chromatography coupled with tandem mass spectrometry | Ka, Kd, KD | association rate, dissociation rate, dissociation equilibrium constant |
Fv | variable fragment | VDW | van der Waals |
BSA | bull serum albumin | OVA | ovalbumin |
CM7 | carboxymethyl dextran surface matrix 7 | Q score | quality score |
BCR | B-cell receptor |
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Type of Antibody | Detection Method | IC50 (μg/L) | Year Reference |
---|---|---|---|
pAb | IC-TRFIA a | 1.90 | 2013, [31] |
pAb | IC-ELISA | 10.00 | 2013, [32] |
pAb | CL-ELISA b | 30.90 | 2013, [33] |
mAb | IC-ELISA | 26.30 | 2015, [34] |
mAb | IC-ELISA | 2.31 | 2019, [35] |
mAb | IC-ELISA | 0.46 | this study |
full-length rAb | IC-ELISA | 0.73 | this study |
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Liu, P.; Guo, Y.; Jiao, S.; Chang, Y.; Liu, Y.; Zou, R.; Liu, Y.; Chen, M.; Guo, Y.; Zhu, G. Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody. Int. J. Mol. Sci. 2020, 21, 6857. https://doi.org/10.3390/ijms21186857
Liu P, Guo Y, Jiao S, Chang Y, Liu Y, Zou R, Liu Y, Chen M, Guo Y, Zhu G. Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody. International Journal of Molecular Sciences. 2020; 21(18):6857. https://doi.org/10.3390/ijms21186857
Chicago/Turabian StyleLiu, Pengyan, Yuanhao Guo, Shasha Jiao, Yunyun Chang, Ying Liu, Rubing Zou, Yihua Liu, Mengli Chen, Yirong Guo, and Guonian Zhu. 2020. "Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody" International Journal of Molecular Sciences 21, no. 18: 6857. https://doi.org/10.3390/ijms21186857