Martin Thomas French

Rapid fluorescence flow injection immunoassay using a novel perfusion chromatographic material

A rapid fluorescence flow injection immunoassay has been developed for a model analyte (testosterone) using a novel perfusion chromatographic matrix (POROS) coated with protein A. The fluorophore used was Texas Red. The perfusion matrix contains two classes of pores, which radically enhances mass transport and provides a very high capacity unaffected by flow rate. For each assay the antibody–protein A reaction takes place at nearly neutral pH, and the complexes are eluted at acidic pH. Both pre-incubation and on-line assay formats were investigated. The total time for the pre-incubation and on-line assays was less than 4 and 6 min, respectively. Serum testosterone was determined by this method and good relative standard deviations and recoveries were achieved. The use of an avidin–biotin system in the preparation of the testosterone–Texas Red conjugate is also described.

Flow injection immunoassay using a protein A immunoreactor

Competitive immunoassays have been developed for the immunosuppressant cyclosporin A and the anti-asthmatic drug theophylline utilizing identical controlled pore glass-protein A microcolumns and flow injection techniques. For cyclosporin A the assay was based on a monoclonal antibody with fluorescence detection whilst for theophylline sheep antiserum and electrochemical detection was used.

A new long-wavelength fluorigenic substrate for alkaline phosphatase: synthesis and characterisation

Naphthofluorescein diphosphate has been synthesised from the parent dye, and shown to be an attractive long-wavelength alternative to other fluorigenic substrates for the determination of alkaline phosphatase. Its application to the determination of theophylline, an inhibitor of this enzyme, has been demonstrated. The optimum excitation wavelength of the hydrolysis product naphthofluorescein has been found to depend on the presence of additives such as cyclodextrins and (3-[3-cholamidopropyl]-dimethylamino)-1-propane sulfonate (CHAPS): such effects can be used to raise the excitation wavelength to match the output of a 635 nm diode laser in a simple and sensitive fluorescence detector.

New fluorescent derivatives of cyclosporin for use in immunoassays

The synthesis of new fluorescent derivatives of cyclosporin is described and their affinity with the specific Sandoz monoclonal antibody investigated. Synthesis was carried out using cyclosporin-C-hemisuccinate as the starting material with monodansylcadaverine, 4-bromomethyl-7-methoxycoumarin, and 4-bromomethyl-6,7-dimethoxycoumarin as labels. After extraction the derivatives were purified by HPLC and their binding affinity with the monoclonal antibody evaluated by the Incstar Cyclo-Trac SP radioimmunoassay. All three derivatives showed good binding and it is suggested that they may be of use in an immunoassay for measuring cyclosporin.

Flow injection fluorescence immunoassay for serum phenytoin using perfusion chromatography

An immunoassay, has been developed for phenytoin in serum. The assay involves flow injection with fluorescence detection and use of Poros A material as the solid phase.