An ELISA for sulfonamide detection using affinity-purified polyclonal antibodies

An ELISA was developed to detect larger sulfonamides. Affinity purification of polyclonal serum resulted in up to 100-fold increase in sensitivity over the original serum. Rabbit antibodies produced by immunization with N1-[4-methyl-5-[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulfanilamide (1) linked to Limulus polyphemus hemolymph (LPH), were affinity purified by immobilizing N1-[4-(carboxymethyl)-2-thiazolyl]sulfanilamide linked to ovalbumin (OVA-TS) on a sepharose support. Desired antibodies were eluted from the affinity column with 500 ppm of N1-2-thiazolylsulfanilamide (sulfathiazole). After regeneration, the column could be reused. Affinity-purified antibodies, representing only about 0·2% of the total serum antibodies, were dialyzed and used in an indirect competition ELISA with OVA-TS as coating conjugate. In this ELISA procedure a reduction in absorbance of 50% could be obtained using concentrations of 0·032 ppm sulfaquinoxaline, 0·58 ppm sulfadimethoxine, 0·87 ppm sulfachloropyridazine, 4·8 ppm sulfathiazole, 5·2 ppm sulfamoxole, 10 ppm sulfamethazine and 10 ppm sulfapyridine.