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Abstract: A novel fluorimetric assay for dopamine using calcein blue (CB) complexed with Fe2+ ion as a chemical sensor is described. The fluorescence arising from CB of the CB–Fe2+ complex is quenched by the Fe2+ ion. When dopamine is added to a solution of the CB–Fe2+ complex, a dopamine–Fe2+ complex is formed as the result of a ligand exchange reaction between CB and dopamine which permits the fluorescence from CB to be recovered. The fluorescence intensity at the wavelength of 440nm (at the excitation wavelength of 340nm) was found to be proportional to the concentration of the dopamine added to the CB–Fe2+ complex solution, which permits dopamine to be quantitatively determined. The selectivity for dopamine in the presence of other catecholamines and related compounds was good. The calibration curve for dopamine, determined using experimental data was successfully simulated based on the equilibrium of the ligand exchange reaction between CB and dopamine. The working range is from 50μM to 1mM and the limit of detection and limit of quantization are ca 10μM and 50μM, respectively. The assay is simple and economical, compared with conventional methods such as an enzyme-linked immunosorbent assay (ELISA). [Copyright &y& Elsevier]

Abstract: A simple and sensitive method for the determination of a specific IgG antibody against ovalbumin (anti-OVA IgG antibody) for diagnosis of egg allergy by use of a surface plasmon resonance (SPR) immunosensor in a flow system is described. An OVA conjugate was immobilized on a sensor chip via a self-assembled monolayer of 11-mercaptoundecanoic acid through an amino coupling method. The determination of the anti-OVA IgG antibody was based on a kind of sandwich immunoassay using an anti-IgG secondary antibody in order to enhance the sensitivity of the SPR immunosensor. The sensitivity and detection limit of the present method for the anti-OVA IgG antibody were 7mdeg/ppm and 300ppb, respectively. The present method showed an enhanced sensitivity and detection limit for the determination of the anti-OVA antibody, compared with a direct immunoassay, by which the sensitivity and detection limit were 3mdeg/ppm and 1ppm, respectively. By assuming a Langmuir type of adsorption isotherm, the affinity constants of an anti-OVA IgG antibody immunocomplex with the anti-IgG secondary antibody and of the anti-OVA antibody to the OVA conjugate immobilized on the sensor chip were calculated to be 2.1×106 M−1 and 2.0×106 M−1, respectively. The present flow immunoassay for the anti-OVA IgG antibody by an SPR sensor has potential applications in both research and diagnosis of egg allergy. [Copyright &y& Elsevier]

Abstract: A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0–500ppb) under optimum conditions. The time required for analysis is less than 15min. [Copyright &y& Elsevier]

Abstract: A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25ppt to 25ppb, and the coefficient of variation of the SPR signals for the 25ppb TNP solution was determined to be 13% (n =4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations. [Copyright &y& Elsevier]