Three steps improving the sensitivity of sulfonamide immunodetection in milk

Three approaches improving the sensitivity of previously established immunoassays for broad detection of sulfonamides (SAs) in milk are described. A novel bis-SA hapten, P2S ( N , N ′-1,4-phenylenebis(4-aminobenzenesulfonamide)), was synthesized for development of a heterologous format of a direct competitive ELISA. Three types of conjugates were compared as heterologous coating antigens. Among them, glutaraldehyde-linked Gel–P2S was found to be the one providing a 2–4-fold increase in assay sensitivity. The second step of improvements was based on adaptation of analysis for a chemiluminescent detection system. The developed chemiluminescent immunoassay (CLIA) allowed one order of reduction of the coating antigen concentration that improved the sensitivity of detection of a broad range of SAs in PBST by two-fold. Application of a matrix-imitation approach was effective for analyzing whole-milk samples without complex pretreatment or dilution. Two simulators, skimmed milk powder reagent and casein, were selected as equivalents of the matrix effect of raw milk. Using standard curves based on these simulators, SA detection in whole milk using the CLIA had 3–6-fold higher sensitivity in comparison with ELISA, which was exemplified with sulfamethoxazole (SMX, IC 50 = 0.15–0.3 vs. 0.91–0.98 ng mL −1 ). The resultant sensitivity improvement of the developed CLIA permitted detectability of 19 SAs in milk at their maximum residue level (25 μg kg −1 ) and below. The recovery of 0.1–2.0 ng mL −1 of SMX in whole milk obtained using skimmed milk- and casein-based standard curves in ELISA was 86–108% and 102–133%, respectively; the analogic values for CLIA were 65–109% and 89–134%, respectively.