Ranald M. Sutherland

Opto-electronic immunosensors: a review of optical immunoassay at continuous surfaces.

Optical techniques for monitoring immunological reactions on continuous surfaces are reviewed. Initially Langmuir-Blodgett film techniques and ellipsometry are discussed, followed by internal reflection spectroscopy (IRS) systems. The latter includes attenuated total reflection (ATR) and total internal reflection fluorescence (TIRF). Finally, light scattering and surface plasmon resonance methods are presented. Overall, it was considered that the IRS systems and ellipsometric approaches offered the most promise for the design of a specific immunosensor device. Of these two, the ellipsometric methods are the most sensitive but also the most vulnerable to non-specific signal interference. Although lacking in extreme sensitivity, the IRS approaches reviewed were more specific in signal generation and were considered to have considerable potential for the future.

Immunoassays at a quartz-liquid interface: Theory, instrumentation and preliminary application to the fluorescent immunoassay of human immunoglobulin G

The theoretical basis and instrumental requirements of an optical detection technique for monitoring antibody-antigen reactions at a quartz-liquid interface are described. The antibody is covalently immobilized on the optical surface of a planar, fused-quartz waveguide and reacted with antigen solution. A light beam is internally reflected within the waveguide and penetrates into the solution only a fraction of the wavelength of the incident light. This is the evanescent wave which interacts optically with the growing number of antigen-antibody complexes but minimally with the bulk solution. A two-site immunofluorescent assay for human IgG measurement is described using fluorescein as the label. The assay detection limit is approximately 0.8 micrograms/ml and individual fluorescence measurements are completed within 10 min. It is expected that this evanescent wave immunoassay should have wide applicability in both routine and research fields.

Preliminary results obtained with a No-Label, Homogeneous, Optical Immunoassay for Human Immunoglobulin G

Abstract A novel optical immunoassay system was developed and tested using human IgG as a model antigen. Following adsorption of antiserum to the surface of an optical waveguide, the immobilised antibody was then reacted with a solution containing antigen. The reaction was detected utilising the evanescent wave component of a light beam totally internally reflected within the waveguide. The growing antigeh-antibody layer resulted in an increase of scattered light which was monitored kinetically. Individual measurements were completed within 5 minutes and the resultant dose response curve had a sensitivity limit of approximately 10 nmol/L.

Immunoassay Kinetics at Continuous Surfaces

The key to immunoassay success is analytical sensitivity and specificity, which in turn lead to clinical utility. The immunoassay reagent, the antibody, has the characteristic of being able to bind tightly and relatively specifically to the invading agent (the antigen) and thereafter initiate a series of events terminating in the biological neutralization of the invading agent. It is this binding event that gives immunoassays their importance in clinical medicine. The reaction between the selected antibody and its target antigen is generally highly specific, rapid, and effectively irreversible. Thus, using antibodies as reagents in a testing system should allow the specific detection of relatively small amounts of antigen from mixtures of generically similar materials. For example, thyroid stimulating hormone can be determined at 10-12mol/L in the presence of greater than approx 108-fold other proteins that are essentially physically identical(1)

Interface Immunoassays Using the Evanescent Wave

The use of the evanescent wave to detect fluoro-immunoassay reactions at an interface is reviewed. Two systems are discussed in detail as practical examples of applying the evanescent wave detection of immunoassays. These systems are based on light guides of different geometry, a planar quartz slide and a quartz optical fibre. Both systems are based on attaching antibodies covalently to the waveguide surface and using fluoresceine as the label. Examples are given of measuring human IgG and human albumin by two-site immunofluorometric assay with the slide waveguide, and ferritin using the fibre optic. An example is also given of measuring digoxin by a limited reagent assay using the optical fibre. The future application of evanescent wave immunoassay is analyzed in the discussion.

Detection Of Antibody-Antigen Reactions At A Glass-Liquid Interface: A Novel Fibre-Optic Sensor Concept

The reaction of antibody molecules immobilized onto the sur-face of fused silica fibre optic or planar waveguides with antigens in solution was detected by interaction with the evanescent wave. By detecting in-line fluorescence, the measurement of human IgG is described.