The effects of the water-miscible organic solvent acetonitrile on the enzymatic activity of horseradish peroxidase (HRP) and on HRP-anti-HRP binding have been investigated. Results showed that both the catalytic activity of HRP and the binding ability of the antibody were affected on increasing the concentration of the organic solvent. The activity of HRP varied with the organic composition of the solvent, indicating that the conformation of the enzyme was affected. The binding ability of the antibody also decreased significantly with an increase of the organic composition of the solvent, and in absolute acetonitrile, the activity of the antibody is about 500 times lower than that in aqueous medium. Binding reversibility experiments indicated that the antibody was not irreversibly damaged in solutions with acetonitrile composition greater than 80% and below 40%; however, an irreversible decrease in the binding was observed in solutions with an acetonitrile composition between 40 and 80%. The reduction in the binding ability is probably due to the irreversible conformation changes in the antibody.

An amperometric immunosensor, based on a non-competitive sandwich assay and flow injection analysis (FIA), was developed for the detection of human red blood cells (RBCs). A dual working electrode, on which specific IgM and nonspecific IgM were chemically immobilised to form sensing and blank electrodes, respectively, was employed to determine the binding of specific blood cells and non-specific adsorption in one determination. Horseradish peroxidase (HRP)-labelled antiblood group A IgM was used in the assay. Sensor preparation involved chemical immobilisation of the IgMs on glassy carbon electrodes using l-ethyl-3(3-dimethyl aminopropyl)carbodiimide (EDC) as a coupling reagent in the presence of N-hydroxysuccinimide (NHS). The interference contributions, such as the non-specific adsorption of the enzyme conjugate and the blood cells, were determined and removed. A quantitative relationship between the cell binding response and its concentration was obtained in the region 1 − 30 × 108 cells ml−1.

An amperometric immunosensor containing a non-diffusional redox polymer to transfer electrons between the electrode surface and the antigen (horseradish peroxidase; HRP) bound to its specific antibody on the sensing surface was developed. The redox polymer [Os(bpy)(2)(PVP)(10)Cl]Cl (bpy = bipyridyl, PVP = poly-4-vinylpyridine) was co-immobilised with the antibody on the electrode surface by crosslinking between the antibody with glutaraldehyde to form a combined sensing and electron transfer system. When the antigen (HRP) was bound to the antibody on the surface of the sensing film, an 'electrical wiring' occurred between the electrode and the redox centres in the bound HRP. The immunosensor showed greatly enhanced performance in terms of the magnitude of the response and the detection limit than that for a similar sensor employing a diffusional mediator.

An amperometric immunosensor was developed based on a non-diffusional redox polymer used for transferring electrons between the electrode surface and the antigen (horseradish peroxidase; HRP) bound to the anti-HRP antibody on the sensing surface. The redox polymer [Os(bpy)2(PVP)10Cl]Cl (bpy = bipyridyl, PVP = poly-4-vinylpyridine) was co-immobilised with the antibody on the electrode surface by crosslinking the antibody with glutaraldehyde to form a combined sensing and electron transfer system. The resulting sensing film exhibits the classical features of a kinetically fast redox couple strongly bound to an electrode surface. The properties of the sensor depend on the content of osmium polymer, the extent of crosslinking and the loading of the antibody in the film. The detection limit for HRP of the sensor was found to be 0.01 μg ml−1, which is one order of magnitude lower than that for a traditional ELISA experiment. By employing an antibiotin antibody with an immuno-competitive approach, the sensor exhibited excellent performance for the detection of the hapten biotin.

A regenerable immunosensor, based on a competitive assay principle and utilizing electrochemical detection, was developed for the determination of 7-hydroxycoumarin. Horseradish peroxidase (HRP) labeled anti-7-hydroxycoumarin antibody was employed in the determination, and the enzyme activity was measured as a function of current due to the reduction of hydrogen peroxide in the presence of a mediator (potassium ferrocyanide). Sensor preparation involved chemical immobilization of 7-hydroxy-coumarin-BSA conjugate on a glassy carbon electrode surface using a water soluble carbodiimide, 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide, as coupling reagent in the presence of N-hydroxysuccinimide. The competition between the free and immobilized forms of the hapten for their HRP labeled antibody in solution allows for the quantitation of free 7-hydroxycoumarin. The sensor can be regenerated by simply immersing the electrode tip in acidic media. Under these conditions the hapten-antibody complex was selectively disrupted without adversely affecting the sensor performance. The conditions for removing the non-specific adsorption of the enzyme conjugate were also studied. The results showed that the detection range obtained for the hapten was physiologically relevant.

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

The immunological activities of an IgG antibody bound on Fc receptors, such as protein A and protein G, pre-coated on a glassy carbon electrode surface have been studied. In order to discriminate the effects of molecular orientation and the conformation retention of the antibody on a protein sublayer, the activities of the antibody chemically coupled to both bare and bovine serum albumin (BSA)-pre-coated electrode surfaces were also determined. A non-competitive sandwich electrochemical enzyme immunoassay was applied to determine the relative surface activities. It was shown that the general antigen binding ability of the antibody on the protein A surface was the highest among the four immobilisation techniques used, whereas the activity on the BSA-modified surface was the lowest. Although the antibody coupled on the protein G surface also offers a certain degree of orientation, its general activity showed no large increase compared to that of antibody bound to the bare electrode surface, since the concentration of the antibody on its surface was lower than that of the bare surface.

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Fab' fragments of rabbit anti-human IgG were immobilized in oriented form on derivatized silica surfaces containing pyridyl disulfide groups. The fragments were also covalently coupled in random form to glutaraldehyde-bound silanized silica surfaces, The surfaces were characterized by X-ray photoelectron spectroscopy and ellipsometry. Antigen binding activities of the immobilized Fab' fragments were determined by solid-phase enzyme immunoassay. The results showed that the changes in orientation of the immobilized fragments drastically influence the antigen binding activity of the fragments, and the antigen binding activity of the fragments in oriented form is 2.7 times higher than that in random form. Such immobilized fragments could find extensive applicability in the design of immunosensors for monitoring of soluble antigens.