Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water
Copyright © 2024 Elsevier B.V. All rights reserved..
High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:469 |
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| Enthalten in: |
Journal of hazardous materials - 469(2024) vom: 05. Apr., Seite 134067 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lei, Xingmei [VerfasserIn] |
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| Links: |
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| Themen: |
059QF0KO0R |
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| Anmerkungen: |
Date Completed 08.04.2024 Date Revised 08.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jhazmat.2024.134067 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM370030141 |
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| 245 | 1 | 0 | |a Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water |
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| 500 | |a Date Revised 08.04.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2024 Elsevier B.V. All rights reserved. | ||
| 520 | |a High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Ethirimol | |
| 650 | 4 | |a Ic-ELISA | |
| 650 | 4 | |a Monoclonal antibody | |
| 650 | 4 | |a Recognition mechanism | |
| 650 | 4 | |a Recombinant full-length antibody | |
| 650 | 7 | |a ethirimol |2 NLM | |
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| 650 | 7 | |a Antibodies, Monoclonal |2 NLM | |
| 650 | 7 | |a Water |2 NLM | |
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| 650 | 7 | |a Recombinant Proteins |2 NLM | |
| 650 | 7 | |a Pyrimidinones |2 NLM | |
| 700 | 1 | |a Li, Peipei |e verfasserin |4 aut | |
| 700 | 1 | |a Abd El-Aty, A M |e verfasserin |4 aut | |
| 700 | 1 | |a Zhao, Jing |e verfasserin |4 aut | |
| 700 | 1 | |a Xu, Lingyuan |e verfasserin |4 aut | |
| 700 | 1 | |a Gao, Song |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Jia |e verfasserin |4 aut | |
| 700 | 1 | |a Zhao, Yun |e verfasserin |4 aut | |
| 700 | 1 | |a She, Yongxin |e verfasserin |4 aut | |
| 700 | 1 | |a Jin, Fen |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Jing |e verfasserin |4 aut | |
| 700 | 1 | |a Zheng, Lufei |e verfasserin |4 aut | |
| 700 | 1 | |a Hammock, Bruce D |e verfasserin |4 aut | |
| 700 | 1 | |a Jin, Maojun |e verfasserin |4 aut | |
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