Development of a natural dihydrofolate reductase receptor binding assay for detecting the sulfonamide synergists...

The monitoring of sulfonamide synergists (SSGs) in animal-derived food is crucial for ensuring food safety and public health. Dihydrofolate reductase (DHFR) based receptor binding assays provided the promising ability to detect a class of SSGs with uniform affinity compared to antibody. The receptor was generally expressed as a His-tagged fusion proteins to facilitate affinity purification and to obtain sufficient material for assay establishment. However, how His-tag location on recombinant DHFR shapes assay format and analytical sensitivity remains poorly understood. In this study, we assessed the impact of affinity-tag location (N- vs C-terminal) on the receptor's binding capacity. Two formats were compared: direct competitive receptor binding assay (DCRBA) and indirect competitive receptor binding assay (ICRBA), in which DHFR function as the coated or detecting reagent. Results showed that the N-terminally tagged DHFR receptor performed better in the DCRBA format. The N-terminally tagged DHFR based assay exhibited high sensitivity and broad specificity, with IC₅₀ values for five major SSGs, trimethoprim, diaveridine, ormetoprim, baquiloprim, and brodimoprim ranging from 8.82 to 38.53 μg/L. The method was further validated in real samples, including milk, skimmed milk, fish, pork, and chicken meat, affording satisfactory average recoveries of 71.30-118.56% and coefficients of variation below 19.27%. Overall, we explored how tags location modulate receptor affinity and suitable detection formats, and ultimately developed an N-terminal DHFR based receptor binding assay for simple, sensitive, high-throughput screening of SSG residues in animal-derived products.