Development of a tetramethylrhodamine-labeled probe for a capillary electrophoresis-based competitive immunoassay of staphylococcal enterotoxin B Original Research Article

Staphylococcal enterotoxin B (SEB) was labeled with tetramethylrhodamine isothiocyanate (TRITC) and used as a probe for a competitive immunoassay. Labeling conditions such as solution pH and time were varied to observe the effect on the fluorescent product. It was found that solution pH of the labeling reaction had little effect on the fluorescence signal of the resulting products. However, labeling at pH 7.0 produced a probe that had a higher affinity for the antibody used in this study than the probes produced at pH 8.0 and 9.0. The fluorescent probes were used to perform a competitive assay for SEB in model skim milk samples. Detection limit was approximately 300 fg of SEB. Quantitation was achieved by curve fitting of fluorescent signals for bound/free probe versus log[SEB] with logarithmic functions. Accuracy in the model skim milk samples was acceptable for 3 and 5 nM SEB, but decreased considerably for a concentration of less than 1 nM SEB. The error was attributed to deviation in linearity in the standard curve at lower concentrations. Reproducibility for the analysis of both standard solutions used for the calibration curves and the model skim milk samples was excellent, with standard deviations of approximately 10% from data collected over a 3-week period. No cross-reactivity was found when the assay was tested with a 700 nM sample of staphylococcal enterotoxin A. Although competitive immunoassays are usually used for small molecules, such as therapeutic drugs, the results demonstrate that relatively large molecules (SEB, 27 kDa) can also be assayed with the technique.