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We describe an optical technique for detecting and monitoring antibody-antigen reactions at a solid-liquid interface. The antibody is covalently immobilized onto the surface of either a planar (microscope slide) or cylindrical (fibre optic) waveguide made of fused quartz. The reaction of immobilized antibody with antigen in solution is detected through use of the evanescent wave component of a light beam, which has a characteristic depth of penetration of a fraction of a wavelength into the aqueous phase, thus optically interacting primarily with substances bound (or located very close) to the interface and only minimally with the bulk solution. This resulting in-situ spatial separation of the antibody-bound from free antigen precludes a formal separation step and allows the reaction to be monitored kinetically. An immunoassay for methotrexate by absorption spectrometry achieved a detection limit of about 270 nmol/L; binding of methotrexate by immobilized antibody was monitored by the decrease in transmittance at 310 nm. A two-site immunofluorometric assay for human IgG could detect as little as 30 nmol/L; binding of fluorescein-labeled antibody was monitored by the increase in signal above 520 nm (lambda ex = 495 nm). With both immunoassays the signal-generating phase was monitored kinetically and was completed within 15 min.

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Affinity capillary electrophoresis (ACE) is a robust tool for the study of noncovalent biomolecular interactions and to determine the binding constants. It is advantageous due to the speed of analysis, the high and reproducible separation efficiencies, the low consumption of analytes, the ability to study several interactions at the same time, and to cover a wide range of affinity. The use of an ion trap-mass spectrometer as a sensitive and specific detector, coupled on-line with a classical UV detector, permits extracting simultaneously the electropherograms corresponding to each ionic species. The mass spectra, acquired by scanning the results of a first separation due to ACE, were assimilated into a virtual two-dimensional (2-D) gel. We developed a software application, which was designed to create and analyze these virtual 2-D gels. The validity of this new analytical tool for probing biomolecular interactions has been demonstrated on mixtures of antibiotics of the vancomycin group and several dipeptide substrates. Using the dynamic equilibrium affinity electrophoresis approach, we have shown that molecular components interacting with a low affinity are easily located on the virtual 2-D gels, and that binding constants and stoichiometry of the interactions can be assessed. As the binding constants derived from ACE-electrospray ionization-mass spectrometry (ESI-MS) are unreliable, they must only be determined with the UV detector.

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