Rapid enzyme immunoassays for the detection of three sulphonamides in milk

Two rapid competitive enzyme immunoassays, utilizing test strip and immunofiltration techniques, for the detection of three sulphonamides (sulphadiazine, sulphamethazine and sulphamethoxypyridazine) in milk were developed. In these direct immunoassays, free sulphonamide competed with the relevant sulphonamide—horseradish peroxidase conjugate for the binding sites of membrane‐bound polyclonal antibodies. Test strips were incubated in the sample solution containing the sulphonamide—enzyme conjugate, while, for the immunofiltration assays, sample and enzyme conjugate solutions were added consecutively. After incubation of enzyme substrate/chromogen solution, the colour reaction was evaluated visually by observing the difference to the negative control. The detection limits for each of the three compounds were between 5 and 10 ng ml‐1 of milk using the test strip assays and between 10 and 20 ng ml‐1 of milk using the immunofiltration assays.