Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water
Lei, Xingmei, Li, Peipei, El-Aty, A. M. Abd, Zhao, Jing, Xu, Lingyuan, Gao, Song, Li, Jia, Zhao, Yun, She, Yongxin, Jin, Fen, Wang, Jing, Zheng, Lufei, Hammock, Bruce D., Jin, Maojun
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 IC 50 value of 1.35 mu g/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 80 - 100%, 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 rAb14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a K D of 5.71 x 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.
- Dergi
- JOURNAL OF HAZARDOUS MATERIALS
- Yıl
- 2024
- Cilt
- 469
- ISSN
- 0304-3894
- eISSN
- 1873-3336
- DOI
- 10.1016/j.jhazmat.2024.134067
- WoS UID
- WOS:001217379200001