Development of a monospecific monoclonal antibody and a homogeneous chemiluminescence immunoassay for selective detection of "Остановлено" lomefloxacin.

BACKGROUND

Lomefloxacin (LOM) has been stopped from use in food-producing animals in China and is regulated at lower maximum residue limits than permitted fluoroquinolones (FQs). However, the highly conserved FQ scaffold makes conventional broad-specificity antibodies unable to discriminate LOM from related analogues, leading to frequent false-positive results in immunoassays. Therefore, the development of monospecific antibodies that selectively recognize LOM and high-throughput screening methods for monitoring LOM residues remains urgently needed.

RESULTS

Here, a unique epitope-exposed antigen design was combined with specificity-oriented hybridoma screening to direct antibody generation toward the characteristic structural features of LOM. The obtained mAb17D9 was the first antibody with monospecificity toward LOM, exhibiting an icELISA IC50 of 0.164 μg/L and cross-reactivities below 1% against 16 structurally related FQs. SPR analysis further confirmed its preferential binding to LOM, with an affinity 27.3- to 15024.0-fold higher than that for FQ analogues. Structural feature analysis and molecular interaction analysis indicated that this monospecific recognition mainly originated from preferential interactions between mAb17D9 and the characteristic groups of LOM at the N1, C7, and C8 positions. Based on mAb17D9, a homogeneous chemiluminescence immunoassay (HCLIA) was established for the rapid screening of LOM residues, achieving limits of detection of 0.077-0.136 μg/kg in various animal-derived food matrices.

SIGNIFICANCE: This study offers a viable solution for the selective detection of a target analyte among structurally similar drugs by providing a broadly applicable strategy for generating monospecific antibodies. The established HCLIA provides a robust tool for monitoring LOM residues in animal-derived foods, enhancing surveillance efficiency and strengthening food safety assurance.