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Direct competitive enzyme-linked immunosorbent assays (ELISAs) were developed for sulfathiazole (ST) analysis. Polyclonal antibody rabbit serum adsorption to polystyrene microtiter plates was enhanced using a 1 l.tg/mL Protein A precoating solution. Detection limits (20% reduction in maximum enzyme signal) for ST in milk and honey were 12.0 and 34.7 ppb (ng/L), respectively. ST-horseradish peroxidase (HRP) conjugates prepared by linking the N4-amino group of ST to periodate-treated HRP were more efficient than those prepared using a more conventional two-step glutaraldehyde method. This represents a new method of preparing sulfonamide conjugates for use in ELlSAs.

Fluorescence polarization immunoassays (FPIAs) were developed for potato glycoalkaloids (GAs) using a polyclonal antiserum and a monoclonal antibody (MAb). Fluorescently labeled solanidine (AMF-SOL) was synthesized by coupling 4'-(aminomethyl)fluorescein (4'-AMF) to the hemisuccinate derivative of solanidine using an active ester method. Both polyclonal antibody (PAb) and MAb could quantify the major potato GAs in the 20-100 nM range; however, the affinities of PAb and MAb differed slightly among the individual GAs. PAb displayed the greatest affinity for a-chaconine, while MAb was more sensitive to changes in -solanine levels. Affinity constants (ifaff) of PAb and MAb for AMF-SOL were estimated at 4.2 x 108 and 4.7 x 107 M-1, respectively. Potato samples containing low, medium, and high levels of GAs were successfully analyzed by the PAb FPIA. Equilibrium in the FPIA reaction was reached within 1-2 min, and standard curves were stable for at least 14 days.

Some basic components of an indirect competitive enzyme-linked immunosorbent assay (ELISA) were manipulated to reduce sulfathiazole (ST) detection limits. 3.3'.5.5'-Tetramethvlbenzidine fTMBj was more kffeitive than several aliemative chromogens tested fdr the hetection of horseradish peroxidase activity. Color development was maximized at pH 4.0. Using TMB and a 2M sulfuric acid stopping reagent, an assay was develoued using highlv diluted (l:SOO.OOO~ rabbit serum and low levels (o.oji3 pg/mY.,) 07 coating col;juga&, with a C value (inflection point of sigmoid standard curve) of 4.4 ppb ST. Urease was unsuitable as a marker for indirect competitive ELISAs.

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