Journal of Immunological Methods, 43 (1981) 313--317 313 Elsevier/North-Holland Biomedical Press MEASUREMENT OF ANTIGEN CONCENTRATIONS WITH A ONE-STEP INHIBITION ASSAY CHARLES R. KEESE I and IVAR GIAEVER General Electric Research and Development Center, Schenectady, NY 12301, U.S.A. (Received 19 September 1980, accepted 21 January 1981) A device is described which is used to measure the concentration of antigen in solu- tion. A wedge-shaped gap containing the antigen to be detected is bounded on one side by a reservoir containing specific antiserum and on the opposite side by a detection slide which has been previously coated with a layer of adsorbed antigen. As the antibodies dif- fuse through the gap, they complex with the soluble antigen in the wedge until these become saturated leaving additional antibody free to complex with antigen on the detec- tion slide. By allowing diffusion to continue for an appropriate time, it is possible to relate the distance along the gap where this has occurred to the concentration of antigen within the gap. Using human IgG as a model antigen, we have been able to measure con- centrations down to approximately 10 pg/ml with this test. INTRODUCTION We wish to report on the design and use of a novel device capable of mea- suring the concentration of antigenic substances in solution. The measure- ment made in the test is basically a variation of an inhibition assay (Cebra, 1971). Such a test normally consists of two distinct steps. Firstly, a known amount of specific antibody is incubated with an unknown quantity of anti- gen; then, the amount of uncombined antibody is measured in various ways, and this value is used to infer the antigen concentration. In the test described in this paper, the inhibition assay is performed in a single step. The basic principle of the test is depicted in Fig. 1. A reservoir containing antiserum specific for the antigen whose concentration is to be measured is separated by a wedge-shaped gap containing the solution of unknown anti- gen concentration from a detection slide, which has a layer of pure antigen adsorbed to its surface. As antibodies diffuse from the reservoir into the gap, they complex with antigen molecules in solution and are unable to attach to the antigen coating on the detection slide. In time, however, the soluble anti- gen molecules in the narrow portion of the gap are saturated with antibody, and now, free antibodies diffuse across the gap and complex with the detec- tion slide. This is not true for regions where the slide is more distant from z Supported by an NSF. Science Faculty Professional Development Grant while on leave from State University of New York at Cobleskill, CobleskiU, NY 12032, U.S.A. 0 022-1759/81/0000---0000/$02.50 © Elsevier/North-Holland Biomedical Press