Development of a flow fluoroimmunosensor for determination of theophylline

Abstract

A new fluorescence immunosensor for theophylline with on-line incubation and in situ fluorescence detection was developed to overcome interference problems and speed up sample throughput. Protein A on controlled-pore glass acts as a solid-phase immunoreactor sited in the flow cell of the spectrofluorimeter. The immunosensor has a limit of detection of 0.3 ng ml-1, a throughput of 10-12 samples per hour and high reproducibility (4.4% at 2.5 ng ml-1 and 3.3% at 3.75 ng mk.1) and selectivity. Possible interferents such as 7-(2,3-dihydroxypropyl)theophylline, caffeine and aminophylline do not interfere up to concentrations SO-fold higher than that of the theophylline present. This immunosensor was successfully applied to the determination of theophylline in serum samples.

Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats

“…The second assay format that was evaluated for use with the protein G microcolumns was a column-based simultaneous injection immunoassay [8–11,22,23,35–37]. This method is illustrated in Fig.…”

“…There are several parameters which can be varied to adjust the response in a column-based simultaneous injection immunoassay [10,22,23,35–37,45,46]. These factors include the relative amounts of the label and antibodies that are used, the binding capacity of the column, the rate of the antibody-target/label interaction, and the injection flow rate [10,45,46].…”

“…This type of work has included the utilization of protein G, or the related agent protein A, in such techniques [17,19–23,32–37]. One approach for creating small-scale chromatographic immunoassays is to use affinity microcolumns, which are columns that contain biologically-related binding agents (e.g., immobilized antibodies) and which have volumes in the low microliter range [29,38].…”

A simple and rapid HPLC/UV method for the simultaneous quantification of theophylline and etofylline in human plasma

“…For the analysis of theophylline and etofylline in plasma samples, spectrophotometry [7,8], immunoassays [9][10][11], capillary electrophoresis (CE) [12][13][14][15] and chromatographic methods [16][17][18][19][20][21][22][23][24] have been introduced. The immunological and spectrophotometric methods would be inappropriate for determination of the combination of different methylxanthines.…”

Use of a Disposable Modified Carbon Paste Electrode for Liquid Chromatography‐amperometric Detection of Theophylline and Three Metabolites in Human Serum

“…A number of studies have been published during the past 15 years, which have been directed toward the characterization of the cytochrome p 450 (cyp) isoforms involved in the different pathways. Reported analytical procedures for theophylline metabolites in body fluids have been based on the methods of liquid chromatography with ultraviolet, [1][2][3][4][5][6][7][8][9] electrochemical, [10][11][12] immunoassay [13][14] and capillary electrophoresis detection. [15][16] In a previous investigation the advantage of using a solvent program 1,2,6,8 and ternary solvent 4,5,7,9 for LC-UV determination of theophylline and its metabolites in serum and urine samples was established.…”