Derek A. Palmer

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate–p-aminophenol as the detection system

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.

Thiophilic gels: applications in flow-injection immunoassays for macromolecules and haptens

Abstract Simple fluorescence flow-injection immunoassays have been developed for theophylline (a hapten) and albumin (a macromolecule) using a thiophilic affinity matrix (T-gel), This gel is far superior to the previously used bacterial immunoglobulin binding proteins (e.g., proteins A and G) in terms of cost, elution conditions, non-specific binding and specificity for immunoglobulins regardless of type or subclass. Both preincubation and on-line immunoassay formats were investigated. Human serum albumin and theophylline were determined by this method and good R.S.D. and %recoveries were achieved.

A model on-line flow injection fluorescence immunoassay using a protein a immunoreactor and lucifer yellow

Abstract A heterogeneous on-line fluorescence immunoassay has been developed for a model analyte (transferrin) using a flow injection analysis system containing a controlled pore glass protein A immunoreactor. Lucifer yellow VS (LYVS) a 4-aminonaphthalimide with a large stokes shift and pH independence was the fluorophore. For each assay the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. Transferrin levels in human serum has been determined by this method, and good within assay variations have been achieved.

Flow Injection Immunosensor for Theophylline

Abstract A heterogeneous on line electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline using a flow injection analysis system containing a protein A immunoreactor to separate antibody bound and unbound theophylline-alkaline phosphatase. In this assay the antibody, label and sample are sequentially immobilised onto the protein A immunoreactor. The antibody-protein A and antibody-protein A label/sample reaction takes place at near neutral pH, and the complexes are eluted at acid pH. p-Aminophenyl phosphate (PAPP) and p-aminophenol (PAP) have been used as the detection system. Experimental variations such as flow rate and reactant concentration have been studied and serum theophylline has been determined by this method and good r.s.d values and % recoveries have been achieved.

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.

Use of protein A in a liposome-enhanced flow-injection immunoassay

A flow-injection immunoassay for the herbicide alachlor has been developed in a sequential format using both a protein A matrix and liposomes. The protein A matrix is a commercially available controlled-pore glass that serves as the support for antibodies. The liposomes are tagged with a phospholipid–analyte conjugate and encapsulate a fluorescent dye that generates the signal. This report is the first demonstrated combination of these two technologies.

Dual analyte flow injection fluorescence immunoassays using thiophilic gel reactors and synchronous scanning detection

Heterogeneous fluorescence immunoassays have been automated using flow injection manifolds incorporating thiophilic gel solid phase reactors to separate antibody-bound and unbound analyte molecules. Antibody elution is achieved by changes in ionic strength, thus allowing the use of pH sensitive fluorescent labels. This facilitates the development of dual analyte systems, in which two competitive immunoassays with separate labels are monitored in parallel. Detection of the fluorophores by high speed synchronous fluorescence scanning while the flow is briefly stopped utilises either one synchronous interval which detects both fluorophores, or two separate scans at different wavelength intervals, one for each fluorophore. Simultaneous analyses of serum albumin and transferrin exemplify these novel approaches. Spectroscopic interferences are very small, analyte recoveries are close to 100%, with a relative standard deviation of 5-6% and a sampling rate of 20 h-1.

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.