Development of a fluoroimmunosensor for theophylline using immobilised antibody.

A flowthrough theophylline fluoroimmunosensor with an antibody covalently immobilised on a solid support has been developed. The immobilisation technique proposed in this paper used Protein-A on control pore glass (Protein A-CPG) in an immunoreactor and dimethylsuberimidate as a cross-linking agent. Several supports and cross-linking reagents were tested in order to obtain oriented immobilisation and thus efficiency of the immunological reaction and reusability of the immunosensor. The immunosensor performance characteristics were established. The precision expressed as RSD, was 1.6%; the detection limit was 3 mug l(-1); the immunoreactor lifetime was established in 80 assays and there were no interferences with structurally similar compounds such as aminophylline, dihydroxypropyltheophylline and caffeine in the determination of the analyte. This fluoroimmunosensor was applied to determine theophylline in human serum samples from patients of the Puerta de Hierro Hospital in Madrid. The results obtained show that there are no significant differences between the proposed immunosensor and the high-pressure liquid chromatographic method with UV detection used by the Hospital, thus demonstrating the validity of the method.