Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism
| Autor: | Kunxia Zhao, Jiancheng Li, Xiaojuan Sun, Yingxian Chen, Jingjie Huang, Miao Li |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Lasalocid
Ionophore Enzyme-Linked Immunosorbent Assay Biochemistry Analytical Chemistry chemistry.chemical_compound Limit of Detection Antibodies Bispecific parasitic diseases Animals Homology modeling Overlap extension polymerase chain reaction IC50 Salinomycin Pyrans chemistry.chemical_classification biology Molecular biology Amino acid Liver chemistry biology.protein Antibody Chickens Single-Chain Antibodies |
| Zdroj: | Analytical and Bioanalytical Chemistry. 413:7031-7041 |
| ISSN: | 1618-2650 1618-2642 |
| DOI: | 10.1007/s00216-021-03666-0 |
| Popis: | Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|