Clinical Chemistry, Vol 30, 1533-1538, Copyright © 1984 by American Association for Clinical Chemistry

Optical detection of antibody-antigen reactions at a glass-liquid interface

RM Sutherland, C Dahne, JF Place and AS Ringrose

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.

The following articles in journals at HighWire Press have cited this article:

				R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]

				A.J. Guthrie, R. Narayanaswamy, and D.A. Russell Optical fibres in chemical sensing - a review Transactions of the Institute of Measurement and Control, April 1, 1987; 9(2): 71 - 80. [Abstract] [PDF]