A silicon oxynitride integrated optical waveguide was used to evanescently excite fluorescence from a multianalyte sensor surface in a rapid, sandwich immunoassay format. Multiple analyte immunoassay (MAIA) results for two sets of three different analytes, one employing polyclonal and the other monoclonal capture antibodies, were compared with results for identical analytes performed in a single-analyte immunoassay (SAIA) format. The MAIA protocol was applied in both phosphate-buffered saline and simulated serum solutions. Point-to-point correlation values between the MAIA and SAIA results varied widely for the polyclonal antibodies (R2 = 0.42-0.98) and were acceptable for the monoclonal antibodies (R2 = 0.93-0.99). Differences in calculated receptor affinities were also evident with polyclonal antibodies, but not so with monoclonal antibodies. Polyclonal antibody capture layers tended to demonstrate departure from ideal receptor-ligand binding while monoclonal antibodies generally displayed monovalent binding. A third set of three antibodies, specific for three cardiac proteins routinely used to categorize myocardial infarction, were also evaluated with the two assay protocols. MAIA responses, over clinically significant ranges for creatin kinase MB, cardiac troponin I, and myoglobin agreed well with responses generated with SAIA protocols (R2 = 0.97-0.99).

Antifluorescyl IgG antibody and Fab binding to two fluorescein-conjugated lipids was measured using the quartz crystal microbalance methodology. By use of the Langmuir-Blodgett technique, the fluorescein lipids, which were diluted to 5% in a L-alpha-dipalmitoyl phosphatidylethanolamine (DPPE) matrix, were deposited directly onto one gold electrode of the quartz crystal. Binding to films containing the fluorescein hapten was significantly enhanced compared to films of the pure DPPE matrix lipid, indicating that binding occurred primarily through a specific interaction. Association constants were 40-300 times less than for binding to haptens free in solution. Binding of IgG to the lipid in which the hydrocarbon chains and the fluorescein hapten were linked via a hydrophilic spacer was approximately 7 times as great as to the lipid containing no spacer. IgG binding to the lipid containing the spacer was increased 1.5-4.4 times compared to Fab binding for the same lipid. Equilibrium binding curves and kinetic measurements are analyzed quantitatively and compared.